ANTIBODIES AGAINST MAC-1

Abstract

The present invention provides an isolated monoclonal antibody or an antigen-binding portion thereof which a) binds to Mac-1, b) specifically inhibits the interaction of CD40L with activated Mac-1 and c) does not induce integrin outside-in signaling.

Claims

1. An isolated monoclonal antibody or an antigen-binding portion thereof which a) binds to Mac-1, b) specifically inhibits the interaction of CD40L with activated Mac-1 and c) does not induce integrin outside-in signaling, characterized in that the antibody or antigen-binding portion thereof comprises at least three CDRs selected from the group consisting of SEQ ID NOs:2-4 and SEQ ID NOs:6-8.

2. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the antibody or an antigen-binding portion thereof does not bind to non-activated Mac-1.

3. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the antibody or an antigen-binding portion thereof limits the expression of inflammatory cytokines.

4. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the antibody or an antigen-binding portion thereof blocks leukocyte recruitment in vitro and in vivo in intravital microscopy.

5. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the antibody or an antigen-binding portion thereof does not affect thrombotic and hemostatic functions of Mac-1.

6. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the antibody or an antigen-binding portion thereof binds specifically to a peptide having the sequence SEQ ID NO: 9

7. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the antibody or an antigen-binding portion thereof comprises at least four CDRs selected from the group consisting of SEQ ID NOs:2-4 and SEQ ID NOs:6-8.

8. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the antibody or an antigen-binding portion thereof comprises at least five CDRs selected from the group consisting of SEQ ID NOs:2-4 and SEQ ID NOs:6-8.

9. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the antibody or an antigen-binding portion thereof comprises six CDRs selected from the group consisting of SEQ ID NOs:2-4 and SEQ ID NOs:6-8.

10. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the light chain has an identity of at least 80% to the amino acid sequence of SEQ ID NO:1 and that the heavy chain has at least 80% identity to the amino acid sequence of SEQ ID NO:5.

11. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the light chain has the amino acid sequence of SEQ ID NO:1.

12. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the amino acid sequence of the heavy chain corresponds to SEQ ID NO:5.

13. The antibody or antigen-binding portion thereof according to claim 1 characterized in that the antibody or an antigen-binding portion thereof is selected from the group comprising F.sub.ab fragments, single chain antibodies, diabodies and/or nanobodies.

14. A pharmaceutical composition characterized in that it comprises a pharmaceutically active amount of an antibody or antigen-binding portion thereof according to claim 1.

15. The pharmaceutical composition according to claim 14 for the treatment of inflammation.

Description

BRIEF DESCRIPTION TO DRAWINGS

[0037] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0038] The preferred embodiments of the invention are shown in the Figures and in the Examples:

[0039] FIG. 1 shows that A mouse monoclonal antibody raised against the CD40L binding site within human Mac-1, anti-M7, is effective in targeting the human integrin. The peptide sequence M7 within the Mac-1, required for binding of CD40L, is a highly conserved binding motif between the human (SEQ ID NO:9) and murine (SEQ ID NO:10) integrin (FIG. 1A).

[0040] Furthermore, FIG. 1A shows the peptide M1 of human origin (SEQ ID NO:14) and the corresponding peptide M1 derived from Mus musculus having SEQ ID NO:15. The human peptide having the designation M8 corresponds to SEQ ID NO:13 and the peptide M8 derived from Mus musculus has SEQ ID NO:16.

[0041] Antibody anti-M7 generated by immunization of mice with the binding peptide VMEQLKKAKTLMQ (SEQ ID NO:11) coupled to diphtheria toxoid bound to a CHO cell line over-expressing native (WT) and permanently activated Mac-1 (del), but not to control CHO cells in western blot (FIG. 1B).

[0042] Specific binding of the antibody anti-M7 to the immobilized peptides M7 (EQLKKSKTL) (SEQ ID NO:9), sM7 (KLSLEKQTK) (SEQ ID NO:12), and M8 (EEFRIHFT) (SEQ ID NO:13) was tested in a solid phase binding with immobilized peptides (FIG. 1C).

[0043] Binding was quantified by binding of biotinylated anti-mouse IgG and color reaction after incubation with HRP-coupled streptavidin. Specific binding was calculated by subtraction of binding of mouse IgG to the peptides. Anti-M7 was coupled with the fluorochrome Alexa647 and binding to human leukocyte subsets was quantified in FACS. Alexa647 Isotype antibody served as control (FIG. 1D).

[0044] FIG. 2 shows that Anti-M7 selectively blocks the interaction of permanently activated Mac-1 with CD40L, but not of the native integrin or to alternative Mac-1 ligands. CHO-cells over-expressing the permanently activated Mac-1 mutant (Mac-1-del) adhered to immobilized CD40L in a static adhesion assay (FIG. 2A, 2B).

[0045] Cells were incubated with anti-M7 or the human pan-I-Domain blocking reference clone 2LPM19c 15 min prior to adhesion. Alternatively, adhesion of the native, non-activated Mac-1 integrin was tested (FIG. 2C). To exclude unspecific Fc-mediated interaction, F.sub.ab-fragment preparation of anti-M7 or anti-Mac-1 were used as inhibitor (FIG. 2D).

[0046] To test whether anti-M7 is specific for CD40L, a panel of classical Mac-1 ligands were separately immobilized and adhesion of permanently activated Mac-1 CHO cells was quantified in the presence of anti-M7 or pan I-Domain blocking anti-Mac-1 (FIG. 2E).

[0047] FIG. 3 shows that Anti-M7 does not induce integrin outside-in signaling, while conventional anti-Mac-1 antibodies induce activation of MAP-kinases and inflammatory cytokine expression in vitro and in vivo.

[0048] Murine macrophages were isolated by injection of 4% thioglycollate in the peritoneum of C57Bl/6 mice and incubation for 72 hours. Peritoneal cells were collected by peritoneal lavage, FACS analysis confirmed purity of >90 percent F4/80.sup.+ macrophages. Macrophages were cultured in 5% FCS RPMI overnight and stimulated with 10 μg/ml of mouse IgG, anti-human Mac-1 (clone 2LPM19c), anti-mouse Mac-1 (clone M1/70) or anti-M7 for 30 min. Cells were lysed and phosphorylated ERK1/2, NfκB and p38 were visualized by western blot (FIG. 3A), and the ratio of phosphorylated fractions was calculated (FIG. 3B). Values were calculated as relative arbitrary units (AU) normalized to signal of cells stimulated with saline alone. Mac-1 antibody clones were injected i.p. in mice and serum concentration of IL-6, TNFα, and MCP-1 was measured by cytometric bead array 4 hours after injection (FIG. 3C). Anti-Mac-1 clone 1/70 was used as control.

[0049] FIG. 4 shows that treatment with anti-M7 prevents inflammatory leukocyte recruitment in vitro and in vivo and decreases inflammatory cytokine expression. Murine RAW-cells were allowed to adhere on isolated and TNFα-primed murine endothelial cells in vitro in a flow chamber assay. Number of adhering cells was quantified in the presence of an anti-mouse IgG or anti-M7 antibody (FIG. 4A). C57Bl/6 mice were injected with 200 ng TNFα i.p. to induce peritoneal and mesenteric inflammation. Simultaneously, either IgG isotype control or anti-mouse anti-Mac-1 (clone M1/70) F.sub.ab-fragment preparations were injected. Leukocyte recruitment to inflamed mesenteric venules was monitored by intravital microscopy 4 hours after injection (FIG. 4B). Number of adhering and rolling leukocytes were quantified, as well as leukocyte rolling velocity, displayed as cumulative frequency (FIG. 4C-E). Mice expressing GFP in monocytes (CX3CR1-GFP) were subjected to intravital microscopy in the presence of IgG or anti-M7 F.sub.ab preparations (FIG. 4F). Migrated monocytes (white arrows) were quantified in the para-vascular space in the viewing field (FIG. 4G). Plasma cytokine levels in mice subjected to intravital microscopy after IgG or anti-M7 F.sub.ab treatment were assessed by CBA bead array (FIG. 4H).

[0050] FIG. 5 shows that Anti-M7 does not affect venous thrombosis and platelet effector function in vivo. Venous thrombosis was induced in mesenteric venules of C57Bl/6 mice by ferric chloride. Thrombus formation was visualized by in vivo rhodamine staining in intravital microscopy (FIG. 5A). Time to thrombus-occlusion of the vessel and rate of emboli (/min) was monitored and quantified (FIG. 5B, 5C). Mice were treated with either F.sub.ab-preparation of mouse IgG, anti-M7 or anti-Mac-1 (50 μg) by intraperitoneal injection 15 min prior to thrombus induction. Formation of platelet-monocyte aggregates was quantified by detection of CD41.sup.+ monocytes in flow cytometry after treatment with anti-Mac-1 antibody clones (FIG. 5D).

[0051] FIG. 6 shows that specific inhibition of Mac-1's interaction to CD40L, but not to other ligands, improves skin wound healing. Aseptic skin wounds were induced by a 4-mm biopsy punch after injection of anti-Mac-1 or anti-M7 F.sub.ab preparations. After 6 days skin wounds were photographed (FIG. 6A) and wound area was calculated (FIG. 6B).

[0052] FIG. 7 shows that Anti-M7 improves host defense, bacterial clearance, and inflammation during bacterial sepsis, while unspecific blockade of Mac-1 potentiates bacteremia in mice. To test whether blockade of Mac-1 or specifically of the CD40L binding site affects host defense and inflammation during bacterial sepsis, coecal-ligation and puncture sepsis (CLP) was induced. 20 hours after CLP procedure inflammatory and patrolling monocytes circulating in blood were quantified by flow cytometry (FIG. 7A). Granulocytes (F4/80.sup.−Gr-1.sup.+) invading into the peritoneal cavity were identified by flow cytometry (FIG. 7B) and total numbers were calculated (FIG. 7C). Levels of the acute phase protein SAA (FIG. 7D) and of bacterial LPS titers (FIG. 7E) were quantified in plasma. Accumulation of granulocytes in kidney parenchyma was determined by staining against DAP and Ly6G (FIG. 7F) and quantified as ratio of granulocytes/total cell nuclei (FIG. 7G).

[0053] FIG. 8 shows that Anti-M7 improves, while anti-Mac-1 decreases, survival during CLP-sepsis. Coecal-ligation and puncture sepsis (CLP) was induced. To assess if treatment with Mac-1 antibody clones affects survival, mice were treated by intraperitoneal injection with either anti-Mac-1 or anti-M7 F.sub.ab preparations at 0, 48, and 96 hours after induction of CLP sepsis. Relative survival was calculated and displayed as Kaplan-Maier survival cure.

[0054] FIG. 9 shows that treatment with Anti-M7 blocks inflammatory leukocyte infiltration in the injured myocardium following myocardial infarction. Myocardial infarction was induced by a surgical ligation of the left anterior descending coronary artery (LAD). Leukocytes infiltrating the infarcted myocardium were quantified by flow cytometry in digested hearts after myocardial infarction. Anti-M7 decreased the infiltration with monocytes and neutrophils and attenuated heart failure as assessed by echocardiography.

[0055] The results summarized in the Figure were obtained in the following examples:

EXAMPLE 1

[0056] Male mice on a C57BL/6N background received a standard chow diet. All mice were maintained under standardized conditions (12-hour light, 12-hour dark cycle) and had access to food and water ad libidum. At the age of 8 weeks, mice were subjected to intravital microscopy, wound healing or CLP sepsis as indicated. Treatment with antibodies was performed by intraperitoneal injection in the indicated concentration at a volume of 100 uL per injection. In some intravital experiments, GFP-transgene animals under the control of CXCR3-promoter (CXCR3-GFP) were used to track leukocytes. All experimental protocols were approved by the animal ethics committee of the Alfred Medical Research and Education Precinct (AMREP), Melbourne, Australia and the local animal ethics committee at the University of Freiburg. All procedures were carried out in accordance with institutional guidelines.

[0057] An antibody specific for a peptide corresponding to Mac-1 I-domain sequence V160-S172 was obtained by immunizing mice with the peptide C-VMEQLKKSKTLFS-NH2 (SEQ ID NO:17) coupled to diphtheria toxoid (Monash Antibody Technologies Facility, Monash University, Melbourne, Australia). Solid phase binding assays was employed to screen binding of sera to the immobilized peptide M7. Among different clones binding with high affinity to M7, the preferred clone RC3 (termed anti-M7) was further characterized.

EXAMPLE 2

[0058] A mouse monoclonal antibody raised against the CD40L binding site within human Mac-1, anti-M7, is effective in targeting the human integrin.

[0059] It has previously been shown that CD40L selectively binds to the EQLKKSKTL (SEQ ID NO:9) motif within the major Mac-1 ligand-binding domain. To obtain a specific inhibitor of the human binding site, mice were immunized with the human peptide V160-S172 containing the binding peptide M7. Interestingly, the M7 sequence was highly conserved between the human and murine protein sequence (FIG. 1A). Among several hybridoma clones with high-affinity binding of the according supernatant to the immobilized peptide M7 in a solid-phase binding assay, clone RC3 (mouse IgG2bκ) showed specific inhibition of Mac-1-CD40L binding, but not of the interaction to other ligands. This antibody clone, subsequently termed anti-M7, bound to a CHO cell line over-expressing native (WT) and permanently activated Mac-1 (del), but not to control CHO cells in western blot (FIG. 1B), confirming successful binding to the target protein.

[0060] Moreover, anti-M7 bound to the immobilized peptides M7 (EQLKKSKTL) (SEQ ID NO:9), but not to the control peptides scrambled sM7 (KLSLEKQTK) (SEQ ID NO:12) or the peptide M8 (EEFRIHFT) (SEQ ID NO:13) in a solid phase binding (FIG. 1C), indicating that anti-M7 specifically binds to the immunized peptide. To test binding of anti-M7 to Mac-1 expressing human cells, we coupled the antibody with the fluorochrome Alexa647 and quantified binding to human leukocyte subsets in flow cytometry. Interestingly anti-M7 showed concentration-dependent binding to human leukocytes expressing Mac-1, such as monocytes and neutrophils, but not to lymphocytes as expected (FIG. 1D). Binding of anti-Mac-1 clone M1/70 served as control and showed the same binding properties with highest binding to myeloid cells. These findings demonstrate that the binding sequence M7 within the human Mac-1 I-domain is accessible to binding with the monoclonal antibody anti-M7. Further DNA sequencing revealed CDRs and exact protein sequence of anti-M7 variable regions of heavy and light chain. This is shown in Table 1:

TABLE-US-00001 TABLE 1 Protein sequence of anti-M7 variable regions Light chain DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWHQQKPDGTIKRLLYS TSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYAISPPTFGG GTKLEIK (SEQ ID NO: 1) Heavy chain QVTLKESGPGILQTSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWL AHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTTDTATYYCALN YYNSTYNFDFWGQGTTLTVSS (SEQ ID NO: 5) Position of CDR 1, 2, 3 is underlined

EXAMPLE 3

[0061] Specific binding of the antibody anti-M7 to the immobilized peptides M7 (EQLKKSKTL) (SEQ ID NO:9), sM7 (KLSLEKQTK) (SEQ ID NO:12), and M8 (EEFRIHFT) (SEQ ID NO:13) was tested in a solid phase binding with immobilized peptides in 96-well ELISA plates (Nunc). Binding of anti-M7 was detected by addition of biotinylated anti-mouse IgG and subsequent color reaction after incubation with HRP-coupled streptavidin and TMB-substrate. Specific binding was calculated by subtraction of binding of mouse IgG to the peptides. To test binding of the antibody anti-M7 to human leukocytes, anti-M7 was labeled with Alexa Fluor 647 according to the manufacturers protocols (Monoclonal Antibody Labeling Kit, Life Technologies). Human leukocytes were isolated from healthy donors by centrifugation and Red Blood Cell lysis-Leukocytes were stimulated with PMA (200 ng/ml), incubated with anti-M7-Alexa 647 (1 μg and 5 μg) and antibody binding was quantified by flow cytometry.

[0062] It was found that anti-M7 is a ligand- and activation specific inhibitor of Mac-1's interaction with CD40L.

[0063] To test whether anti-M7 is able to functionally block the interaction of Mac-1 and CD40L, the adhesion of CHO-cells over-expressing a permanently activated Mac-1 mutant (Mac-1-del) to immobilized CD40L was tested in a static adhesion assay. Interestingly, anti-M7 blocked the cell adhesion by 65.6±7.2%, an effect nearly as strong as the anti-human pan-I-Domain blocking reference clone 2LPM19c (inhibition by 92.7±2.0%, FIG. 2A, B). In the experiment a concentration of 10 μg/ml was used. It can be concluded therefrom that in general concentrations of the antibody ranging from 1 to 50 μg/ml and preferably from 5 to 20 μg/ml are used. Most interestingly, in contrast to the reference anti-Mac-1 antibody, anti-M7 did not block adhesion of CHO cells expressing the native, non-activated Mac-1 integrin (FIG. 2C), indicating that blockade by anti-M7 was specific to high-affinity conformation of the integrin. Moreover, inhibition by anti-M7 was not restricted to human proteins, since interaction of murine macrophages and murine CD40L was significantly blocked by anti-M7. Furthermore, blocking by anti-M7 was not unspecifically caused by the F.sub.e-fragments of the antibody, since F.sub.ab-fragment preparations of anti-M7 or anti-Mac-1 were as effective as the whole antibody preparation (FIG. 2D). Different ligands can bind to separate or overlapping binding regions within the Mac-1 I-domain. To test whether anti-M7 is specific for the CD40L binding epitope, a panel of classical Mac-1 ligands, such as Fibrinogen, ICAM-1, NIF, heparin, and RAGE was separately immobilized and binding of Mac-1-del cells was tested in the presence of anti-M7 and anti-Mac-1 (FIG. 2E). Notably, anti-Mac-1 blocked each of the interactions, while blocking capacity of anti-M7 was restricted to CD40L. These data unveil that anti-M7 is an effective and specific inhibitor of the CD40L/Mac-1 interaction.

EXAMPLE 4

[0064] Murine peritoneal macrophages were obtained as described above. Flow cytometry revealed that the majority (>90%) of PECs were positive for the macrophage marker F4/80. After overnight starvation macrophages were stimulated with the indicated antibodies against Mac-1 in a concentration of 10 μg/ml for 30 minutes. After the indicated time points, cells were lysed, proteins were separated by SDS-PAGE and blotted to polyvinylidene difluoride membranes. Total protein and the phosphorylated fraction of NFκB, ERK1/2 and p38 were detected by specific antibody binding in western blot (Cell Signaling). The ratio of phosphorylated fractions was calculated and expressed as relative arbitrary unit (AU) normalized to signal of cells stimulated with saline alone.

[0065] The test results show that anti-M7 does not induce integrin outside-in signaling, while conventional anti-Mac-1 antibodies induce activation of MAP-kinases and inflammatory cytokine expression in vitro and in vivo.

[0066] Conventional anti-Mac-1 antibodies induce activation of the integrin, termed outside-in-signaling mediated by downstream activation of MAP-kinases, such as ERK and p38 upon ligand and antibody binding. It has previously been shown that CD40L is a biased agonist not inducing outside-in signaling events upon binding. To test whether anti-M7 would induce cell activation, thioglycollate-elicited peritoneal macrophages from male, 8 week old C57Bl/6 mice were collected. After overnight starvation in 5% FCS containing RPMI, the macrophages were stimulated with 10 μg/ml of either mouse IgG, anti-human Mac-1 (clone 2LPM19c), anti-mouse Mac-1 (clone M1/70) or anti-M7 for 30 min. Anti-Mac-1 treatment induced phosphorylation of ERK and p38 as quantified by an elevated ratio of the phosphorylated epitopes in western blot (FIG. 3A), while anti-M7 had no effects, indicating that the binding epitope targeted by anti-M7 is not involved in outside-in signaling (FIG. 3B). To assess whether this effect is relevant for an in vivo treatment, Mac-1 antibody clones were injected i.p. in mice and serum concentration of IL-6, TNFα, and MCP-1 were quantified 4 hours after injection. Surprisingly, the Mac-1 reference clone M1/70 (control) strongly elevated cytokine levels, while anti-M7 did not (FIG. 3C). In accordance, levels of pro-inflammatory cytokines increased in in vitro culture of macrophages after antibody stimulation. These findings indicate that anti-M7 is targeting an epitope not causing unwanted outside-in signaling during integrin blockade.

EXAMPLE 5

[0067] Before enzymatic digestion, the antibody was dialyzed in a SnakeSkin Dialysis Tubing 10k MWCO against PBS overnight at 4° C. Immobilized papain was used to prepare F.sub.ab fragments from anti-M7, anti-Mac-1 (clone M1/70) and an IgG isotype control as indicated according to the manufacturer's instructions (Pierce F.sub.ab Preparation Kit, Thermo Scientific). Briefly, F.sub.ab-fragments were generated in the presence of 25 mM cysteine for 3 h at 37° C., followed by purification on NAb Protein A Spin Columns. Purity of F.sub.ab-fragments was evaluated on SDS-PAGE.

[0068] 96-well plates (Nunc) were coated with sCD40L (10 μg/ml) and incubated with CHO-cells expressing constitutively activated Mac-1. Cells were pre-incubated with blocking antibodies (10 μg/mL) as indicated and allowed to adhere for 50 minutes. Adhering cells were counted after repeated washing with PBS. For dynamic adhesion assays, human umbilical endothelial cells (HUVECs) were grown to confluency in 35 mm cell culture dishes, stimulated with TNFα overnight and placed in a parallel flow chamber system (Glycotech). Number of adhering cells was quantified at the indicated shear rate in the presence of the indicated antibodies (10 μg/mL).

[0069] For intravital microscopy mice received an intraperitoneal injection of 100 μg of antibodies or 50 μg of F.sub.ab-fragments i.p. After 15 minutes mice were injected i.p. with 200 ng murine TNFα (R&D Systems). Surgery started 4 hours after TNFα administration. Briefly, mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (Essex) and xylazin (Bayer, Leverkusen, Germany). The mesentery was exteriorized and placed under an upright intravital microscope (AxioVision, Carl Zeiss). Videos of rolling and adhering in mesenteric venules were taken after retro-orbital injection of rhodamine. Rolling leukocyte flux was defined as the number of leukocytes moving at a velocity less than erythrocytes. Adherent leukocytes were defined as cells that remained stationary for at least 30 seconds.

[0070] Flow cytometry: Peritoneal exudate cells (PECs) and blood leukocytes were obtained as described below. Remaining red blood cells were removed by incubation with a red blood cell lysing buffer (155 mM NH4Cl, 5.7 mM K2HPO4, 0.1 mM EDTA, pH7.3). Cells were washed in PBS, and Fc-Receptors were blocked by anti-CD16/CD32 (eBioscience) for 10 minutes on ice. Cells were then labeled with the indicated antibodies before quantification with a flow cytometer (FACS Calibur, BD Biosciences). All antibodies were obtained from eBioscience. Distinct leukocyte populations were identified upon cell surface expression of the indicated antigens: granulocytes (Gr-1.sup.+F4/80.sup.−CD11b.sup.+CD115.sup.−), macrophages (F4/80.sup.+CD11b.sup.+CD115.sup.−), inflammatory monocytes (CD11b.sup.+CD115.sup.+Gr-1+F4/80.sup.−), non-inflammatory monocytes (CD11b.sup.+CD115.sup.+Gr-1.sup.−F4/80.sup.−).

[0071] Isolation and cultivation of murine peritoneal macrophages: Antibodies were injected i.p. 30 min before WT mice received an injection of 2 mL of 4% thioglycollate broth (Sigma). A peritoneal lavage was performed after 72 hours. Peritoneal exudate cells (PECs) were quantified and characterized by FACS as described above. In CLP experiments a peritoneal lavage was performed 20 hours after surgery.

[0072] It could be shown that treatment with anti-M7 prevents inflammatory leukocyte recruitment in vitro and in vivo and decreases inflammatory cytokine expression.

[0073] Mac-1 is a powerful adhesion factor, likely mediating its adhesive function through interaction with different ligands expressed at the endothelium, including ICAM-1, RAGE, and CD40L. To test if anti-M7 blocks cellular adhesion, murine monocyte-like RAW-cells were allowed to adhere on isolated and TNFα-primed murine endothelial cells in vitro in a flow chamber assay. Number of adhering cells decreased after incubation with anti-M7, indicating that CD40L/Mac-1 interaction is required for leukocyte arrest (FIG. 3A). To test for relevance of these findings in vivo, F.sub.ab-fragment preparation of anti-M7 and an according isotype were injected i.p. prior to intravital microscopy (FIG. 4B). Leukocyte recruitment to inflamed mesenteric venules was monitored after simultaneous stimulation with TNFα for 4 hours to induce inflammatory leukocyte recruitment. Consistently with our in vitro results, we observed that the number of adhering (FIG. 4C), but not of rolling leukocytes (FIG. 4D) was reduced after anti-M7 injection. In accord, leukocyte rolling velocity, displayed as cumulative frequency, was not changed (FIG. 4E), indicating that firm adhesion, but not rolling properties of leukocyte is blocked by anti-M7. To exclude that anti-M7 induces leukocyte depletion we injected anti-M7 or an according isotype control i.p., and quantified leukocyte populations. Of note, no changes were observed in both groups. To test if impaired monocyte arrest would affect down-stream effects, such as transmigration, mice expressing GFP in monocytes (CX3CR1-GFP) were subjected to intravital microscopy in the presence of IgG or anti-M7 F.sub.ab preparations after a TNFα challenge for 4 hours (FIG. 4F). In accordance, we observed that anti-M7 treated animals showed lower numbers of monocytes migrated to the perivascular space (FIG. 4G). Finally, we observed that plasma levels of the pro-inflammatory cytokines TNFα, IL-6, and MCP-1 were significantly reduced in mice subjected to intravital microscopy after anti-M7 F.sub.ab treatment compared with IgG F.sub.ab treated control animals (FIG. 4H). These results clearly indicate that leukocyte adhesion proceeds by the interaction of CD40L and Mac-1 and that this interaction can be functionally blocked by anti-M7 antibody.

EXAMPLE 6

[0074] It has also been shown that anti-M7 does not affect venous thrombosis and platelet effector function in vivo.

[0075] Mac-1 participates in haemostasis and thrombus formation, presumably by its interaction to the platelet glycoprotein GP1bα. Also, CD40L stabilizes thrombi and its therapeutic inhibition raises thromboembolic complications. To exclude that an antibody according to the invention would induce unwanted thrombus destabilization, venous thrombosis was induced in mesenteric venules of C57Bl/6 mice by ferric chloride. Thrombus formation was visualized by in vivo rhodamine staining in intravital microscopy (FIG. 5A). As described previously, inhibition of Mac-1 by an i.p. injected F.sub.ab-fragment prolonged vessel occlusion time and increased the release of thrombotic emboli (FIG. 5B, C), confirming that Mac-1 is needed to stabilize thrombi. However, inhibition by anti-M7 did not cause significant changes in vessel occlusion time or release of thrombotic emboli, proposing that participating pathways were not affected. Accordingly, formation of leukocyte-platelet aggregates was diminished by unspecific blockade of Mac-1, but not by specific inhibition of the CD40L/Mac-1 interaction (FIG. 5D). These data propose that anti-M7 is likely not inducing unwanted effects on the haemostatic system.

EXAMPLE 7

[0076] Interaction of Mac-1 to CD40L, but not to other ligands, improves skin wound healing. Leukocyte engagement is a critically step in wound healing and delayed wound healing has been reported in Mac-1 null mice. To test whether these effects are mediated by Mac-1's interaction to CD40L, we treated C57Bl/6 mice with i.p.-injections of F.sub.ab-fragments of either anti-M7, anti-Mac-1 or an according isotype control directly after induction of 4 mm dorsal skin wounds. Interestingly, during the time course of the experiment delayed wound healing in anti-Mac-1 treated mice was not detected. However, skin wounds tent to close faster in Kaplan-Maier wound closure analysis in anti-M7 treated mice and demonstrated a smaller wound surface 6 days after wound induction (FIG. 6A,B). This indicates that specific inhibition of the CD40L/Mac-1 interaction does not affect, but instead seems to exhibit protective effects on skin wound healing.

EXAMPLE 8

[0077] Unselective inhibition of Mac-1 aggravates, while specific blockade of its interaction to CD40L improves bacterial clearance, inflammation, and survival during bacterial sepsis.

[0078] It has recently been shown that mice with a genetic deficiency of Mac-1 demonstrated decreased survival during bacterial sepsis, highlighting the potential role of the leukocyte integrin in host defense and clearance of bacteria. To elucidate whether ligand-specific blockade of Mac-1 and CD40L is rather beneficial during bacterial sepsis, a model of coecal-ligation and puncture sepsis (CLP) was performed. 20 hours after CLP procedure inflammatory and patrolling monocytes circulating in blood and basic inflammatory parameters were quantified. Interestingly, CLP induced a strong mobilization of inflammatory Gr-1.sup.+ monocytes to the circulation, reaching a percentage of the inflammatory subset of about 82.4±4.6% of all monocytes in IgG F.sub.ab-fragment treated mice. This response was not affected by F.sub.ab anti-Mac-1 treatment (77.4±6.0%), but nearly reversed by F.sub.ab anti-M7 treatment (56.8±3.7%, FIG. 7A). During CLP, myeloid cells populate the peritoneal cavity. Granulocytes (F4/80.sup.−Gr-1.sup.+) invading the peritoneal cavity were identified by flow cytometry (FIG. 7B). Both, anti-Mac-1 and anti-M7, strongly reduced granulocyte accumulation by 59.9±12.2% and 73.8±7.1% for anti-Mac-1 and anti-M7, respectively (FIG. 7C). The anti-inflammatory effect of anti-M7 treatment was further reflected by a strong decrease of the acute-phase protein SAA by 63.4±19.7% (FIG. 7D). Notably, anti-M7 improved bacterial clearance in the plasma, while anti-Mac-1 worsened bacterial load in both, plasma and the peritoneal cavity (FIG. 7E). During CLP, accumulation of neutrophils is observed in the periphery, such as the kidney and lung. To quantify granulocyte trafficking to the spleen, ICH was performed against the granulocyte marker Ly6G in kidney sections (FIG. 7F). Notably, both anti-integrin therapies prevented neutrophil accumulation with a stronger effect in anti-Mac-1 treated animals (FIG. 7F). Finally, it was assessed if the new ligand-specific approach according to the invention is beneficial in surviving sepsis. Therefore, CLP was induced and animals were subsequently treated with F.sub.ab-preparations of IgG, anti-Mac-1 and anti-M7 at 0, 48, and 96 hours after induction of CLP operation. Survival rate was calculated employing Kaplan-Maier analysis and log-rank testing. Animals treated with anti-Mac-1 showed significantly decreased mean survival compared to IgG-control treated animals (0% vs. 6.7% after 169 hours after CLP-induction for anti-Mac-1 and IgG, respectively). Notably, anti-M7 treated showed a survival rate of 40.0% at the end of the study (FIG. 8), demonstrating that ligand-directed therapy is superior to unspecific inhibition.

EXAMPLE 9

[0079] Treatment with anti-M7 improves the infiltration with inflammatory leukocytes in the injured myocardium following myocardial infarction. Accumulation of inflammatory leukocyte occurs after myocardial infarction within days. Inflammatory leukocyte recruited to the infarcted heart cause an inflammatory response that aggravates wound healing and drives heart failure after myocardial infarction. Inhibition of leukocyte infiltration has been proposed to represent a therapeutic strategy, but not such strategy is available. After induction of myocardial infarction in mice by a surgical ligation of the left anterior descending coronary artery (LAD) and treatment with anti-M7 less infiltrating monocytes and neutrophils, a subclass of inflammatory leukocytes that express Mac-1, were found in the injured myocardium. As a result, anti-M7 attenuated heart failure.