WO1992011870A1

WO1992011870A1 - Method of preventing inflammatory damage

Info

Publication number: WO1992011870A1
Application number: PCT/US1992/000178
Authority: WO
Inventors: James R. Rusche; Daniel P. Witt
Original assignee: Repligen Corporation
Priority date: 1991-01-11
Filing date: 1992-01-10
Publication date: 1992-07-23
Also published as: JPH06505969A; EP0565642A1; CA2100084A1

Abstract

A method of inhibiting activated neutrophil-mediated damage to the gut, liver, brain, skin, kidneys, or lungs of a mammal resulting from blood loss followed by transfusion, the method involving administering to the mammal, at the time of or following said transfusion, an organ damage-inhibiting amount of an antibody capable of binding to the CD11b subunit of the neutrophil integrin Mo1, and being substantially incapable of binding to the CD18 subunit of Mo1.

Description

METHOD OF PREVENTING INFLAMMATORY DAMAGE

Background of the Invention This invention relates to reducing or preventing organ damage caused by a neutrophil-mediated inflammatory response.

Under normal circumstances, neutrophils, or polymorphonuclear leukocytes (PMN's), circulate in the blood without infiltrating the endothelial cells lining the blood vessels or entering the tissue. Invasion of the body by infectious bacteria or foreign agents, however, results in a mobilization of the circulating PMN's to the afflicted site in order to limit the spread of infection. The initial trigger for this mobilization is provided by certain biochemical factors which may be produced either by the infectious agent or by the host itself. These factors serve to both stimulate and attract the neutrophils into sites of infection or trauma. In response to stimulation by such factors, the neutrophils undergo a number of biochemical and morphological changes to achieve an activated state. Once in the activated state neutrophils become tightly adherent to endothelial cells lining the capillaries and venules of the microvasculature through which they eventually pass (extravasate) , moving up the gradient of stimulatory factor(s) into the tissue by a process termed chemotaxis. During their migration toward the source of the stimulatory factors, the activated neutrophils begin to release cytotoxic molecules, such as oxygen radicals and hydrolytic enzymes, as a means of destroying the foreign cells or agents. Although this provides an important early protective response against infectious agents, it can also be highly damaging to bystander cells as well as the foreign target cells because these cytotoxic molecules are non-specific in their action.

T Neutrophil stimulating factors can also be produced in the absence of foreign cells or infectious agents. For example, ischemia can lead to the production of neutrophil stimulating factors, and PMN's have been shown to be important mediators of the microvascular injury which occurs during reperfusion after periods of low or no blood flow (reperfusion injury) . This has been demonstrated in models of occlusion/reperfusion in myocardial infarction as well as with models of hemorrhagic shock which are representative of whole body ischemia. Experiments in which neutrophils have been transiently removed or regulated prior to hemorrhagic shock have led to significant improvements in survival and reductions in the failure of sensitive organs such as the gut, liver, and brain secondary to the period of ischemia.

The association between PMN's and the endothelium is mediated by certain adhesion molecules certain of which rapidly appear on the surface of the neutrophils in response to activation. One class of molecules, which is of particular importance in neutrophil adhesion are the leukocyte integrins. The three leukocyte integrins which have been described have been termed LFA-1, Mol (or Mac-1) , and pl50,95. These molecules are exclusive to leukocytes and are differentially distributed according to cell type. For example, LFA-1 is the leukocyte integrin exclusively found on the majority of lymphocytes, while pl50,95 is the predominate integrin on macrophages, where no LFA-1 is found. All three integrins are found to be present on PMN's and LFA-1 and Mol have each been found to account for about half of the adhesive interaction between neutrophils and endothelium. The role of pl50,95 in this process has not been investigated. The primary structure of the leukocyte integrins has been recently determined. As with other integrins, the leukocyte integrins each consist of a non- covalently associated heterodi eric complex of an alpha and a beta subunit. The beta subunit is the product of a single gene and has been previously designated as CD18. The three alpha subunits are each distinct gene products and have been designated CDlla (alpha subunit of LFA-1, also αL) , CDllb (alpha subunit of Mol, also αM) and CDllc (alpha subunit of pl50,95, also αX) . A beta subunit can thus associate with any of the three alpha subunits to produce an intact and functional molecule.

Arfors, U.S. Patent No. 4,797,277 uses antibodies directed to the beta subunit (CD18) of the integrins to reduce tissue damage as evidenced by a reduction in microvascular permeability following hypovolemic shock. Wright et al. , EP Publication No. 0 346 078 also discloses the use of anti-CD18 antibodies for treating inflammation caused by sepsis or other infectious or non-infectious trauma. Mileski (Surgery, 1990, 2___8 ι206-212) uses anti-CD18 antibodies to reduce PMN-mediated organ injury after hemorrhagic shock in primates. Vedder et a! (Surgery, 1989, i_06:509-516) uses anti-CD18 antibodies to block organ damage and improve survival following hypovolemic shock in rabbits. It is not possible to know whether all or only one or two of the leukocyte integrins are important in the pathologic processes discussed in these references, as antibodies to CD18 affect all three leukocyte integrins equally.

Todd et al., U.S. Patent Nos. 4,935,234 and 4,840,793, hereby incorporated by reference, use an antibody specifically targeted to Mol (anti-CDllb) , designated MY904, to reduce tissue damage in a canine model of myocardial infarction. Ismail et al. (Blood, 1987, 6 :1167-1174) describe use of an anti-Mol antibody for prevention of pulmonary injury ex vivo in rat lungs perfused with activated human neutrophils. The Todd et al. work with canine myocardial infarction is not predictive of the effect of anti-CDllb antibodies in other organs, as much evidence suggests that the heart may be unigue. For example, cardiac myocytes produce ICAM-1 on their surface under certain conditions of stress. Neutrophils can adhere to myocytes by interacting with the expressed ICAM-1, and this interaction can be blocked by anti-CDllb antibodies but not anti-CDlla antibodies. This difference cannot be demonstrated when ICAM-1 is expressed on endothelial cells, where binding is mediated by both CDlla/CD18 and CDllb/CD18. This suggests that the ICAM-1 expressed on cardiac myocytes is unique and distinct from ICAM-1 expressed on endothelial cells. In addition, while it is generally expressed on endothelial cells and fibroblasts, ICAM-1 expression has not been demonstrated in organ - specific cells other than cardiac myocytes. The Ismail et al., supra, study does not allow prediction of protective effects of anti-CDllb antibodies or organs other than the heart in blood loss/transfusion circumstances because it is neither a homologous nor an in vivo model (isolated rat lungs exposed to human neutrophils) . Furthermore, the neutrophils were activated using a non-physiological means (phorbol esters) .

Rosen and Gordon (PCT Publication No. WO89/04174) report that an antibody directed to mouse CDllb is able to prevent neutrophil and monocyte accumulation in induced inflammatory loci. Additionally, these investigators demonstrate that this antibody is capable of blocking certain immunological functions such as delayed type hypersensitivity and the development of an autoimmune response by adoptive transfer. Summary of the Invention

The invention provides a method for inhibiting activated neutrophil-mediated damage to the gut, liver, kidneys, skin, brain, or lungs of a mammal resulting from blood loss followed by transfusion; the method involves administering to the mammal (preferably a human patient) , at the time of or following transfusion (preferably within one hour of transfusion) an organ damage-inhibiting amount of an antibody capable of

T binding to the CDllb subunit of the neutrophil integrin Mol, and being substantially incapable of binding to the CD18 subunit of Mol. Most preferably, administration is carried out by mixing the antibody with the transfused fluid so that the two are administered together, in mixed form.

By "transfusion" is meant intravenous infusion into a patient of whole blood, plasma, synthetic blood substitutes, or other fluid, following significant blood loss, in particular hemorrhagic blood loss.

Examples of suitable antibodies are MAb 17 and MY904; MY904 is publicly available (MY904 is described, e.g., in U.S. Patent No. 4,935,234, supra..

According to the invention, blocking of the CDllb subset of adhesion molecules alone is sufficient to prevent neutrophil-mediated organ damage caused by transfusion following significant blood loss. Anti-CDllb antibodies will not impair the functions of LFA-1 and pl50,95 integrins, the blockage of which could have potential adverse effects due to the involvement of these receptors in normal immunological function and wound healing, respectively. The invention, therefore, provides significant advantages over therapy using anti- CD18 antibodies. The inhibition of neutrophil-mediated damage may be observed as a protection from actue organ failure and/or as a protection from bacterial sepsis occurring later as a result of bacterial translocation across the damaged gut or lung mucosal barrier.

In another aspect, the invention features a method of inhibiting activated neutrophil-mediated damage to an internal organ of a mammal resulting from surgery on the mammal at a locus other than the organ; the method involves administering to the mammal prior to, during, or following the surgery an organ damage-inhibiting amount of an antibody capable of binding to the CDllb subunit of the neutrophil integrin Mol, and being substantially incapable of binding to the CD18 subunit of Mol. Administration of the antibody can be carried out by intravenous injection of a bolus just prior to surgery, or during surgery, by mixing the antibody with other IV fluids administered gradually to the surgical patient. In this aspect of the invention, systemic organ damage that can occur during surgery, particularly in compromised patients, is reduced by the systemically administered antibody. The method is particularly useful in prevention of Adult Respiratory Distress Syndrome (ARDS) , which is a common and serious complication of surgical procedures on compromised patients, particularly cigarette smokers, cancer patients whose immune systems are suppressed because of chemotherapy or radiation, and patients infected with microbial pathogens.

In another aspect, the invention features inhibition of activated neutrophil-mediated inflammation in a mammal by administering to the mammal two synergistically acting anti-inflammatory agents, the first being an anti-CDllb antibody as described above, and the second being an agent which interferes with the process of neutrophil activation; the two agents are administered together, or close enough in time so that they act synergistically. Examples of neutrophil activation-blocking agents are methylprednisone, ibuprofen, and pentoxifylline. This method is particularly useful in prevention of inflammation resulting from blood loss followed by transfusion, and in preventing systemic inflammation resulting from surgery.

In another aspect, the invention features the method of treating sepsis in a mammal by administering to the mammal two synergistically acting sepsis-inhibiting agents, the first of which is an anti-CDllb antibody as discussed above, and the second of which is an agent which blocks the action of a pathogen-mediated activator of inflammation; the first and second agents are administered together, or close enough in time so that they act synergistically. Preferably, the second, inflammation activator-blocking agent is an antibody, e.g. , one specific for bacterial lipopolysaccharide, or - 1 - one which binds specifically to a mammalian inflammation activator, e.g., tumor necrosis factor (described in EP Appln. No. 0351789) , whose release from mammalian cells is triggered by a pathogen. other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Detailed Description of the Invention The method of the present invention involves administration of antibodies directed against the CDllb subunit of the Mol glycoprotein to prevent or inhibit generalized or systemic neutrophil-mediated inflammatory damage in organs other than the heart resulting from blood loss followed by transfusion. The invention makes possible the early treatment or prevention of inflammatory episodes to prevent such episodes from progressing into serious conditions resulting in organ damage or immune paralysis. In more detail, we believe that the protective effect provided by the anti-CDllb antibody can be described as follows.

Hemorrhagic shock or hypovolemic shock, occurring from transient blood loss followed by transfusion, leads to damage to gut, liver, lung, and brain. Organ failure related to this damage is frequently irreversible and can result in death. The previously demonstrated protective effect of adhesion- blocking anti-CD18 antibodies and neutrophil depletion in hemorrhagic shock confirms that neutrophils play a role in the pathogenesis of this disorder. The mechanisms by which neutrophils become activated in hemorrhagic shock have been shown to be multiple and include complement activation and the production of inflammatory cytokines such as TNF-alpha and IL-6. Antibodies to CD18 which block or alter leukocyte adhesion affect all three leukocyte integrins equally. Thus, in previous studies using anti-CD18 antibodies, it is not possible to determine whether neutrophil adhesion relevant to tissue damage is dependent on all three integrins, only one, or a combination of two. Certain of the cognate ligands (contra-receptors) for LFA-1 and Mol on endothelial cells have been identified. For example,

ICAM-1, which is expressed on endothelial and certain other cells, is a ligand for both LFA-1 and Mol. ICAM-1 is believed to be important in mediating neutrophil adhesion and extravasation and is increased during periods of inflammation. A second ligand, ICAM-2, is only bound by LFA-1 and not by Mol. The role of ICAM-2 in the inflammatory process is not well understood. Other ligands for LFA-1 and Mol may also exist but have not yet been identified. At present, no ligand for pl50,95 has been unambiguously identified. The complexity of the leukocyte integrins' interaction with their various cognate ligands means that it cannot be known whether blockade of any single integrin or ligand would be sufficient to inhibit organ damage. Further complicating the assignment of primacy of neutrophil adhesion to any one leukocyte integrin is the demonstration that LFA-1 and Mol are equal contributors to neutrophil adhesion to endothelial cells in vitro. In other words, antibodies specific to either integrin (e.g. either anti-CDllb) are individually capable of blocking only about 50% of the adhesive interaction. Using a combination of the antibodies leads to complete blockage of adhesion, which can also be achieved by treatment with an anti-CDlδ antibody. Anti-CDllb Antibody

The invention can employ any antibody capable of specifically binding to the CDllb subunit of the Mol neutrophil cell surface receptor. Such specifically- binding antibodies can block one or more relevant, potentially organ-damaging neutrophil functions including (1) the interaction between the neutrophil and vascular endothelium which initiates the influx of the neutrophils into the extravascular tissue, (2) the production of deleterious reactive oxygen intermediates by the neutrophil NADPH oxidase, and (3) the homotypic aggregation of the neutrophils which can impede microvascular flow. The particular antibody utilized in the examples described below is a monoclonal antibody identified as MAb 17.

MAb 17 was made by scientists at the Dana- Farber Cancer Institute, Boston, MA, by immunizing female Balb/C mice with cells from the peripheral blood of an adult patient with monocytic leukemia using the standard procedures described by Kohler and Milstein (Nature, 1975, 256:495-497) . Specifically, mice were injected intraperitoneally with 20 x 10⁶ cells, and were boosted at weekly intervals for 4 weeks with 20 x 10⁶ cells and for an additional 2 weeks with 15 x 10⁶ cells. Four days following the final boost, the spleen was removed and a single cell suspension was made of the splenocytes. The cells were mixed with mouse myeloma NS-1 cells and monoclonal antibody-producing hybridoma clones were characterized by their ability to block neutrophil aggregation and other Mol ependent adhesive interactions in both human and rat cells. MAb 17 was identified as an antibody of the IgM isotype that reacts with neutrophils, monocytes, macrophages and certain NK cells.

Other monoclonal antibodies useful in the method of the invention can be made by immunizing mice with human neutrophils or monocytes, fusing the murine spleen cells with appropriate fusion partners, and screening the antibodies produced by the resultant hybridoma lines for the ability to bind, and the failure to bind the CD18 subunit thereof. Alternatively, other cells known to express the leukocyte CDllb/CD18 complex can be used as immunogens. Where monocytes are used for hybridoma formation, a dual screening process can be carried out. In the first step, antibodies are screened for their ability to bind human granulocytes and monocytes, but not normal T-lymphocytes,

B-lymphocytes, or red blood cells. Those selected in the first screening assay are then evaluated for their ability to demonstrate a 6 to 8 fold increase in reactivity with human neutrophils activated with f- MetLeuPhe. Selected antibodies are finally screened for the ability to inhibit both neutrophil adhesion to plastic surfaces and homotypic neutrophil aggregation induced by neutrophil activating factors.

Monoclonal antibody MY904 can also be used. MY904 is on deposit with the American Type Culture Collection (ATCC) and is assigned ATCC Accession Number HB 9510. Antibodies exhibiting the binding specificity of MY904 can be made according to the procedure described for making MY904 in Todd et al. U.S. Patent No. 4,935,234, supra.

The invention can employ not only intact antibody molecules, e.g. , intact monoclonal or polyclonal antibodies, but also an immunologically-active antibody fragment, for example, the Fab fragment or the (Fab)₂ fragment; an antibody heavy chain; an antibody light chain; a genetically engineered single-chain F_v molecule; or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions of the antibody are of human origin. In general, it may be advantageous to modify the antibody to be more compatible with human use. For example, MAb 17, a murine IgM which is specific for the CDllb subunit of the Mol receptor on human neutrophils may be converted into a (Fab)₂ fragment which retains the same binding properties as the intact antibody but which does not have the disadvantages associated with the immunologic effector functions carried by the Fc portion of the antibody. Therapy Sufficient antibody is provided to prevent substantial organ damage which would otherwise result from substantial blood loss followed by transfusion, or systemic organ damage resulting from surgery at a locus other than the organ. Typically, this may be achieved with doses of from about 1 to about 10 mg/kg. The preferred dosage is in the range 1-2 mg/kg.

The invention is primarily concerned with the treatment of humans and administration is preferably systemic, i.e., the antibody is administered in such a way that it can reach the circulation without being substantially inactivated. Most preferably, the antibody is added to the transfusion fluid prior to or during transfusion, or with other fluids, administered prior to or during surgery. When used in combination with another anti- inflammatory agent or an agent which blocks the action of a pathogen-mediated activator of inflammation, anti-CDllb antibody is provided in the doses described above. When the second agent is a neutrophil activation blocking agent, such as methylprednisone, ibuprofen or pentoxyfylline, dosages can be those typically used for use of these agents in known therapies. For ibuprofen, for example, doses of from about 1 to about 25 mg/kg; preferably about 10 to about 20 mg/kg can be used.

When the second agent is an antibody which blocks the action of a pathogen-mediated activator of inflammation, doses of from about 1 to about 25 mg/kg; preferably in the range of from about 10 to about 20 mg/kg can be used. The specific dose will be dependent on the circulating half-life of the particular antibody and will generally be determined by providing an amount sufficient to maintain about 2-20 ug/ l in the blood. The methods of the invention may be used to limit inflammatory responses in any mammal, for example, humans, domestic pets, or livestock. Where a non-human mammal is treated, the antibody employed is preferably specific for that species.

What is claimed is:

Claims

1. Use of an antibody capable of binding to the CDllb subunit of the netrophil integrin Mol, and being substantially incapable of binding to the CD18 subunit of Mol, for the manufacture of a pharmaceutical composition for the inhibition of activated neurtophil- mediated damage to the gut, liver, brain, skin, kidneys or lungs of a mammal resulting from blood loss followed by transfusion.

2. The method of claim 1 wherein said pharmaceutical composition further comprises fluid for transfusion into a mammal.

3. Use of an antibody capable of binding to the CDllb subunit of the netrophil integrin Mol, and being substantially incapable of binding to the CD18 subunit of Mol, for the manufacture of a pharmaceutical composition for inhibiting activated neutrophil-mediated damage to an internal organ of a mammal resulting from surgery on said mammal at a locus other than said organ.

4. A pharmaceutical composition for inhibiting activated neutrophil-mediated inflammation in a mammal, said composition comprising two anti- inflammatory agents, the first agent being an antibody capable of binding to the CDllb subunit of the neutrophil integrin Mol, and being substantially incapable of binding to the CD18 subunit of Mol, and the second agent being an agent which interferes with the process of neutrophil activation.

5. A pharmaceutical composition for treating sepsis in a mammal, said composition comprising two sepsis-inhibiting agents, the first agent being an antibody capable of binding to the CDllb subunit of the neutrophil integrin Mol, and being substantially incapable of binding to the CD18 subunit of Mol, and the second agent being an agent which blocks the action of a pathogen-mediated activator of inflammation.

6. The composition of claim 5 wherein said second agent is an antibody.

7. The composition of claim 6 wherein said antibody binds specifically to bacterial lipopolysaccharide.

8. The composition of claim 6 wherein said antibody binds specifically to tumor necrosis factor.