WO1992006697A1 - Anti-inflammatory composition - Google Patents

Anti-inflammatory composition Download PDF

Info

Publication number
WO1992006697A1
WO1992006697A1 PCT/US1991/007841 US9107841W WO9206697A1 WO 1992006697 A1 WO1992006697 A1 WO 1992006697A1 US 9107841 W US9107841 W US 9107841W WO 9206697 A1 WO9206697 A1 WO 9206697A1
Authority
WO
Grant status
Application
Patent type
Prior art keywords
antibody
neutrophil
activating factor
method
neutrophils
Prior art date
Application number
PCT/US1991/007841
Other languages
French (fr)
Inventor
Daniel P. Witt
Original Assignee
Repligen Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2845Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Abstract

An anti-inflammatory composition comprising a neutrophil activating factor and an antibody which is capable of binding to a neutrophil cell surface receptor of the integrin family.

Description

ANTI-INFLAMMATORY COMPOSITION Background of the Invention This invention relates to reducing inflammation. Inflammation, both acute and chronic, can often result in destruction of host tissue. By "inflammation" is meant a pathologic process consisting of a complex set of cytologic and histologic reactions occurring in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent. Neutrophils are cellular components of the blood which, when activated, produce substances that are cytotoxic and amplify the inflammatory response. Neutrophils possess cell surface glycoproteins which promote intercellular adhesion with, e.g., endothelial cells.

Neutrophils are capable of migrating through vessel walls into tissue, e.g., into a site of inflammation, and adhering to endothelial cells lining the vasculature.

One neutrophil cell surface glycoprotein Mol (or Mac-l) is a heterodimeric protein containing two subunits, CDllb and CD18, and is a member of the integrin class of receptor molecules (Kishimoto et al.. Cell, 1987, 48:681). Todd et al. (Structure and Function of Molecules Involved in Leukocyte Adhesion, 1989, Springer, ed. , Springer-Verlag, New York) report that administration of an adhesion blocking monoclonal antibody in a canine model of myocardial infarction followed by reperfusion resulted in a 30-40% reduction in infarct size. The monoclonal antibody was directed to a molecule, CDllb, which is present on the neutrophil cell surface (Simpson et al., Circulation, 1990, 81:226). Arfors, U.S. Patent No. 4,797,277, and Todd et antibodies to reduce tissue injury. Bajaj et al. (Circulation, 1989, 70:645) uses a chemical, Fluosol-DA, which suppresses neutrophil chemotaxis and lysozyme release functions and reduces injury associated with inflammation in an animal model system.

Gimbrone et al. (Science, 1989, 246:1601) reports that activated endothelial cells produce a factor, called endothelial interleukin 8 (IL-8) , which inhibits neutrophil adhesion and protects endothelial cells from neutrophil- mediated damage. The amino acid sequence of IL-8 has been determined (Lindley et al. PNAS 1988, 85:9199). IL-8 promotes chemotaxis and degranulation of neutrophils (Djeu et al., J. Immunol., 1990, 144:2205).

Kishimoto et al. (Science, 1989, 245:1238) demonstrates that a second class of adhesion proteins, exemplified by the MEL-14, antigen is present on the neutrophil surface and is involved in adhesion of neutrophils to endothelial cells. This class of molecules has been variously termed Lec-CAMs and selectins. Neutrophil activation causes rapid shedding of these molecules in vitro, and may prevent extravasation into and damage of normal tissues by the activated neutrophils.

Hechtman et al. (FASEB J. , 1990, 4: A890) report that IL-8 can function as a leukocyte adhesion inhibitor and thus reduce neutrophil accumulation at intradermal sites of inflammation, concluding that systemically administered human IL-8 may have anti-inflammatory effects. However, Colditz et al. (Am. J. Pathol., 1989, 134:755) demonstrated the opposite results, i.e., that IL-8, also known as neutrophil activating factor (NAF) , elicits a rapid inflammatory response in rabbits with massive neutrophil emigration. Summary of the Invention The invention provides methods and compositions which effectively treat inflammation by blocking CDllb/CD18 molecules. The invention employs two agents which, individually, would be expected to function imperfectly or cause toxic side effects, but which, when combined, operate synergistically to simultaneously expose and block the cryptic CDllb/CD18 fraction to inhibit most of the potentially inflammatory Mol molecules, while avoiding the severe toxicity problems presented by the use of a single agent alone.

Accordingly, the invention provides an anti- inflammatory composition composed of a neutrophil activating factor and an antibody which is capable of binding to a neutrophil cell surface receptor of the integrin family.

Integrins are heterodimeric surface receptors comprising an α-subunit and a /3-subunit, associated in a non-covalent interaction. On leukocytes there are three types of α- subunits present; CDlla, CDllb, and CDllc in association with a single 5-subunit; CD18. Particularly predominant on neutrophils is the CDllb/CD18 complex, which has also been termed Mac-1 or Mo-1.

In preferred embodiments, the neutrophil activating factor is IL-8/LAI (defined below) or a fragment or derivative thereof possessing the ability to degranulate neutrophils and cause the selective release of adhesion molecules of the selectin class from the neutrophil membrane. The antibody used in the invention preferably is capable of binding to the Mol cell surface antigen, in particular the CD18 or CDllb subunit thereof; a particular example is the MY904 monoclonal antibody.

The neutrophil activating factor and antibody can be administered simultaneously, or near enough in time so that the antibody inhibits systemic toxicity potentially caused by the neutrophil activating factor.

The invention provides effective inflammation inhibition without the toxicity otherwise presented by neutrophil activating factors such as IL8/LAI. The invention, because it uses two components acting synergistically, provides increased efficacy over that achievable with a single component, as well as lowered toxicity. 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 The drawing will first be described. Drawing

The Figure recites the amino acid sequences of LAI and IL-8, respectively. Antibodv/Neutrophil Activating Factor Interaction

The invention provides treatment of inflammatory disorders using a combination of a neutrophil activating factor and an antibody which binds to integrin receptors on the surfaces of neutrophils. Reduction of damage caused by an inflammatory response is accomplished according to the invention by a process involving 1) eliciting, with the neutrophil activating factor, the release of selectins from neutrophils to discourage selectin-mediated adhesion of neutrophils to endothelial cells, and 2) blocking integrin- mediated adhesion events with an integrin-specific antibody. In compositions of the invention, synergy is obtained between the two components in that, in addition to functioning as summarized above, the integrin-specific antibody provides protection against potential adverse effects of the neutrophil activating factor while the neutrophil activating factor improves the efficacy of the antibody.

In more detail, I believe the synergistic effect provided by the neutrophil activating factor in combination with the therapeutic antibody can be described as follows. A majority of the CDllb/CD18 in a resting neutrophil is present in a cryptic form in secretory granules within the cytoplasm. Until the cell is activated during inflammation, the CDllb/CD18 sequestered in the granule membrane is neither capable of interacting with a cognate receptor on an endothelial cell nor available for reaction with an adhesion-blocking antibody such as anti-Mol. Therapeutic administration of antibody, e.g. prior to reperfusion in the canine infarct model, permits binding and inhibition of only that sub-fraction of the receptor molecules which was already exposed on the cell surface of the unactivated cells. The majority of circulating neutrophils will typically not be in an activated state; however, upon initiation of reperfusion of ischemic tissue, activation of neutrophils in the afflicted vasculature will occur due to localized pathophysiologic processes such as complement activation and cytokine production. Local activation results in degranulation by affected neutrophils and a rapid increase in the level of CDllb/CD18 on the cell surface. For inhibition of the adhesive properties of the CDllb/CD18 integrin to be effected, antibody binding must occur prior to interaction of exposed CDllb/CD18 with its cognate endothelial receptor. Even if the entire, system-wide dose of therapeutically administered antibody is sufficient to saturate potentially all of the CDllb/CD18, both exposed and cryptic, in circulating neutrophils, the concentration of antibody present in the icroenvironment of the inflammatory site where local activation and adhesion of neutrophils is taking place may be inadequate to neutralize the rapidly increasing amount of cryptic CDllb/CD18 becoming exposed on the cell surfaces. In such a microenvironment, the ratio of free adhesion-blocking antibody to endothelial integrin receptors will progressively decrease as the antibody binds to exposed CDllb/CD18. Limited local amounts of antibody and rapid surface appearance of large amounts of active CDllb/CD18 integrin, combined with potential kinetic limitations of the CDllb/CD18 interaction with antibody versus the interaction with cognate endothelial receptors, will result in an incomplete inhibition of the neutrophil- endothelium interaction. Neither CDllb- nor CD18- directed antibodies are highly effective at reversing a neutrophil- endothelial cell interaction once it has occurred. The activation of neutrophils by IL-8/LAI leads to a complex of interrelated processes and changes in the cell. For example, at a low concentration of IL-8/LAI, such as might be found under both physiological and pathophysiological conditions of acute or chronic inflammation, neutrophils may become bound to endothelium primarily by selectin-mediated interactions. Under the influence of a gradient of IL-8/LAI which is emanating from the source tissue, neutrophils would then be attracted towards the region of higher concentration of IL-8/LAI and begin to migrate, shedding selectin molecules in the process. As increasing concentrations are encountered, the cells begin to degranulate selectively, releasing hydrolytic enzymes and exposing additional CDllb/CD18 receptor on the cell surface. Production of toxic reduced oxygen intermediates may also commence at this point, as the cell prepares to breach the basement membrane which underlies the endothelial cell layer. This is regarded to be the natural sequence of events leading to extravasation of neutrophils into the tissue which is the source of the chemotactic/activating agent.

The basis for the protective effect of the antibody against the adverse consequences of the systemically- administered neutrophil activating factor I believe can be described as follows. Intravenous administration of IL-8/LAI or any other neutrophil-activating factor will result in undesired neutrophil accumulation in certain sensitive organs such as the lung. One reason why activated neutrophils accumulate in the lung is because they form ulticellular aggregates upon stimulation by a variety of neutrophil activating factors. This process has been termed homotypic aggregation. The activated neutrophils and neutrophilic aggregates have a significantly increased adhesiveness to endothelium as a result of the increased surface expression of newly exposed CDllb/CD18. When these aggregated and hyperadhesive neutrophils encounter the extensive fine capillary beds of the lung, they tend to become trapped in the lung microvasculature. These activated cells which accumulate in the microvasculature release elastase and other hydrolytic enzymes, as well as toxic reduced oxygen species leading to acute pulmonary edema and other aspects of inflammation. Production of these degradative agents in the confined vascular microenvironment can ultimately lead to extensive damage to the endothelium, basement membrane, and interstitial tissue. The resulting pulmonary disorder can be life threatening, particularly in the elderly and in infants.

Under non-physiological conditions, such as can be achieved in vitro and which can be encountered in pharmacological use, increasing the concentration of IL- 8/LAI beyond about 0.1 μg/ml without establishing a concentration gradient leads to the following potentially adverse effects: further degranulation exposing additional Mol molecules and increasing the cell's adhesiveness to selected microvasculature, activation of superoxide production, and homotypic aggregation of the neutrophils. Concentrations of IL-8/LAI which have been evaluated intravenously as pharmaceutical agents (Hechtman et al., FASEB J. (1990) 4., A890) produce these changes systemically, and thus will not be safe, by themselves, for general clinical use. The phenomenon of activated neutrophil retention in the lung and other sensitive tissues would seem to preclude the systemic use of IL-8/LAI, or any other agent with neutrophil-activating activity, as an anti-inflammatory agent. Nonetheless, if activation-induced accumulation of neutrophils in the lung and other tissues could be avoided, advantage could then be taken of the process of selectin shedding to reduce the initial high-affinity adhesive interaction.

According to the invention, the adhesion-inhibiting properties of neutrophil activating factors such as

IL-8/LAI can be exploited without promoting excess tissue- specific accumulation of activated neutrophils, by combining such a factor with a CDllb/CD18-directed monoclonal antibody. The inhibition of CDllb/CD18 by a therapeutic antibody can in turn be optimized, by combining such an antibody with a neutrophil-activating factor. Thus the combination of these two agents produces a desirable synergistic effect. The Neutrophil Activating Factor Any physiologically acceptable neutrophil activating factor can be used in the combination therapy of the invention. By "neutrophil activating factor" is meant an agent which elicits the release of selectins from the surface of a neutrophil and which stimulates neutrophil degranulation. In addition to IL-8 and LAI, other previously identified or as yet unidentified neutrophil activating factors useful in the methods of this invention may be identified using the in vitro assay described in detail below. Examples of neutrophil activating factors known or expected to elicit selectin release and stimulate neutrophil degranulation, and which are therefore likely to be useful in the invention, include melanoma growth stimulating activity (MGSA) ; formylated peptides, for example, Fmet-leu-phe; and complement fragments, for example, C5a and C5adβsA .

LAI and IL-8, two neutrophil activating factors exemplified herein, are, respectively 77 and 72 amino acid peptides which are identical in primary structure except that LAI contains an additional 5 amino acids at the amino terminus (Figure) . These two peptides can be used interchangeably for the purposes of this invention and are referred to herein collectively as IL-8/LAI. In addition to intact, native IL-8/LAI molecule, the invention can employ neutrophil-activating fragments or derivatives thereof. By "neutrophil-activating fragment or derivative of IL-8/LAI" is meant any portion of IL-8/LAI which elicits selectin release and neutrophil degranulation. Such fragments or derivatives may be isolated from any animal tissues or cells which naturally produce such a fragment or derivative or which can be induced to do so; or can be produced by standard chemical synthesis; or can be isolated by standard techniques of recombinant DNA technology. Such fragments or derivatives can be tested for neutrophil-activating activity by measuring the effect of the fragment or derivative on the adherence of neutrophils to endothelial cell surfaces in vitro (as described below) . Integrin Receptor Binding Antibody

The invention can employ any antibody capable of specifically binding to a neutrophil cell surface receptor of the integrin family. By "neutrophil cell surface receptor" is meant a protein on the surface of a cell (for example, an endothelial cell) which facilitates a specific interaction between a neutrophil and a receptor-bearing cell. Antibodies utilized in the examples described below are monoclonal antibodies identified as MY904 (American Type Culture Collection, Access Number HB 9510) and IB4 (Wright et al, Proc. Natl. Acad. Sci., U.S.A. (1983) J3(), 5699). Monoclonal antibody MY904 has been shown to potently inhibit the homotypic aggregation of neutrophils induced by various neutrophil-activating agents such as formylated peptides, as well as non-physiologic agents such as phorbol esters (general activators of protein kinase C) . Other functional properties of MY904 and IB4 which are relevant to their use as described herein are their ability to inhibit 1) the extravasation of neutrophils, 2) the production of hydrogen peroxide by neutrophils, and 3) the adhesion of neutrophils to unactivated endothelial cells. MY904, unlike other anti- Mol monoclonal antibodies, inhibits adhesion-dependent functions, but not binding of the iC3b component of complement.

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 requisite Mol binding properties. 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.

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, hereby incorporated by reference.

Also effective in the methods of the invention is antibody IB4, a murine monoclonal antibody of the subclass IgG2a that is specific for the CD18 subunit of the Mol receptor (Wright et al, Proc. Natl. Acad. Sci., U.S.A. (1983) 80, 5699).

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 Fv 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, MY904, a murine IgGl 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. Antibodies useful in the invention may be identified as those antibodies which inhibit IL-8-enhanced binding of neutrophils to unactivated endothelial cells in vitro (using methods described below) . Any antibody shown to be effective in this assay (in particular, any effective Mol- directed antibody) may be used in the methods of the invention. Therapy

The dose of neutrophil activating factor in the therapeutic composition disclosed herein depends on the type and quality of the factor and the body type of the individual to be treated. Optimally, a sufficient quantity of IL-8/LAI is provided to produce maximal release of specific granule contents and exposure of all of the CDllb/CD18 pool on the ceil surface. This dose can be up to 25 μg/kg body weight in the disclosed formulation without producing adverse effects. The preferred range is 5-10 μg/kg body weight.

Optimally, a sufficient quantity of antibody is provided to saturate all of the CDllb/CD18 present on circulating neutrophils. This may typically be achieved with doses of 0.1 mg/kg or greater. The preferred dosage is in the range of 0.1-2.0 mg/kg.

The invention is primarily concerned with the treatment of humans, and the administration is preferably systemic, i.e., the composition is given in such a way that its active components can reach the circulation without being substantially inactivated. The agents may be combined in a single infusion or may be administered sequentially. If administered sequentially, it is preferable to infuse the antibody first, followed within 10 to 60 minutes by the neutrophil activating factor. This provides ample time for the aggregation-inhibiting antibody to disseminate throughout the vascular space. In certain cases, it may be necessary to give repeated administrations, depending on the metabolism of the components of the pharmaceutical formulation or the severity of the inflammatory episode.

The compositions and 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.

The following examples illustrate the invention, and should not be construed as limiting. Examples

The experiments described below can be carried out to demonstrate that the combination of a neutrophil activating factor and an antibody directed against a neutrophil receptor of the integrin class are effective in decreasing neutrophil adhesion to endothelial cells in vitro and neutrophil infiltration in vivo. In vitro experiments described below are to demonstrate that the combination of a neutrophil activating factor, IL-8, and an antibody (MY904) directed against the CDllb subunit of the neutrophil CDllb/CD18 integrin is more effective in decreasing adhesion of neutrophils to endothelial cells than is IL-8 alone. In addition, MY904 can decrease in vitro neutrophil homotypic aggregation induced by IL-8. The proposed in vivo experiments presented below are to demonstrate that the combination of IL-8 and MY904 is more effective than IL-8 alone in reducing neutrophil accumulation induced by a proinflammatory agent. IL-8 and MY904 Decrease Neutrophil Adhesion in vitro

In the following experiments neutrophil-endothelial cell adhesion is tested by suspending the neutrophils above a monolayer of endothelial cells for a fixed period and then either removing the non-adherent neutrophils by gentle washing or detaching weakly adherent neutrophils by centrifugation. The adherent neutrophils are quantified by light microscopy. Human neutrophils are collected from normal volunteers and purified according to conventional techniques. Human endothelial cells are derived from the umbilical vein and cultured as described by Tonneson et al. (J. Clin. Invest. 74., 1581, 1984) . IL-8 is produced by recombinant means in E. coli and purified in active form as described by Lindley et al. (Proc. Nat'l. Acad. Sci. USA 85. 9199, 1990). The activity of the IL-8 preparation is confirmed by its dose dependent ability to attract human neutrophils and to release neutrophil granule enzymes. MY904 monoclonal antibody is purified from hybridoma cells (ATCC HB 9510) using standard techniques (Current Protocols in Molecular Biology, Ausubel et al., eds., Wiley Interscience. New York) .

In an experiment to evaluate the effect of IL-8 on neutrophil adherence, neutrophils are exposed to either 0.5μg/ml IL-8 or buffered saline for 10 minutes prior to suspension on an endothelial monolayer. After a 20 min co- incubation, during which the neutrophils settle onto the endothelial monolayers, the non-adherent neutrophils are removed by gently washing the monolayers with fresh serum- free culture medium; this procedure allows weakly adherent cells to remain associated with the endothelial monolayer. Under these conditions, IL-8 treatment causes a significant increase in the percentage of neutrophils binding to the endothelial monolayer (60% treated; 30-35% untreated). In a second experiment, neutrophils are activated with IL-8, but in one group the IL-8 treated neutrophils are also exposed to 10 μg/ml of the anti-CDllb antibody, MY904, following the IL-8 activation step. The treated cells are incubated with unactivated endothelial cells as described above, and the non-adherent neutrophils are removed by gentle washing. Exposure of the activated neutrophils to MY904 prior to co-incubation inhibits not only the IL-8 induced increase in the percentage of cells bound but also the basal percentage of cells bound to the unactivated endothelial cells. This is to demonstrate that the anti- CDllb antibody is capable of effectively blocking at least the weak adhesive interaction between activated neutrophils and unactivated endothelial cells.

A third set of experiments is to examine the effect of IL-8 activation and MY904 antibody on the adherence of neutrophils to activated endothelial cells. Neutrophils treated for 10 min with either IL-8 (0.5 μg/ml in a buffered saline solution) or buffered saline are suspended above a monolayer of cultured endothelial cells which has been previously activated with 10 U/ml interleukin Iβ for at least four hours (Gimbrone et al.). After a 20 min co- incubation the cell monolayers are inverted and centrifuged at 250g for 5 minutes. This procedure, in contrast to gentle washing, removes weakly adherent cells but permits strongly adherent cells to remain bound. Evaluation of neutrophil adherence by light microscopy will reveal that exposure of neutrophils to IL-8 leads to a 75% decrease in the number of strongly adherent cells, demonstrating that IL-8 can decrease the strong component of the adherence of neutrophils to activated endothelial cells. In contrast, when the number of weakly adherent neutrophils to activated endothelium is assessed by gentle washing, neutrophils will largely remain associated with the endothelial monolayer regardless of IL-8 treatment. Inclusion of 10 μg/ml MY904 in the incubation buffer in addition to IL-8 will further eliminate this weak component of the binding, demonstrating that the antibody and activating factor can act cooperatively to inhibit both strong and weak components of neutrophil adhesion. MY904 Decreases Homotypic Aggregation of Neutrophils

Human neutrophils, collected under non-activating conditions, are found to rapidly self-associate into multicellular aggregates containing at least 2-3 cells/cluster (homotypic aggregation) when exposed to concentrations of IL-8 of greater than 1 μg/ml. Extended incubation leads to partial reversal of this process over a time course of minutes. This self association can be assessed quantitatively by increases in light scattering. MY904 antibody can be added to the incubation buffer at 5 μg/ml, to decrease homotypic aggregation. Treatment with IL-8 and MY904 Decreases Neutrophil Infiltration in vivo Neutrophils are collected from mongrel dogs, purified by standard techniques, and labelled in vitro with indium-Ill by incubation of IO7 neutrophils/ml with 10 μCi of 113 n, according to the method of Tonnesen et al., J. Clin. Invest 83/ 637 (1989) . The labelled cells are washed in buffered saline and put back into the same animals from which they had been removed as an autologous infusion. Interleukin-8 is then injected intravenously, 10 μg/kg, into the test animals and an injection of 0.1 nM/site of the proinflammatory agent LTB4 (leukotriene B4) is made intradermally into a shaved area of the back. After 6 hours, the inflammatory lesions are excised from the treated and control animals, and neutrophil infiltration is assessed by radioactive counting and expressed as cpm/gram tissue. Treatment by intravenous IL-8 will significantly decrease neutrophilic infiltration. Gamma camera imaging of the animals at 6 hours will be carried out to reveal accumulation of neutrophils in the lungs, and histopathological evaluation of the lungs will be carried out to confirm that a significant accumulation of aggregated neutrophils in the pulmonary vasculature occurred and that pulmonary edema and other characteristic features of acute inflammation was evident.

A second in vivo experiment can be done, similar to that described immediately above, except that 1 mg/kg of monoclonal antibody MY904 is administered by intravenous infusion in combination with IL-8. As before, a reduction in neutrophil infiltration into the inflammatory locus will be achieved. In this study, however, a significantly greater reduction will be observed than in the preceding study, an enhancement of the effect of the intravenous IL-8 by MY904. Gamma camera imaging of the animals will also be carried out to verify that the pulmonary accumulation of neutrophils is significantly reduced relative to that seen in control animals which did not receive the protective antibody. Histopathology of the lungs of the animals treated with a combination of MY904 and IL-8 will reveal reduced pulmonary edema and acute inflammation. What is claimed is: COMPUTER SUBMISSION OF DNA AND AMINO ACID SEOϋENCES

(1) GENERAL INFORMATION:

(i) APPLICANT: Witt, Daniel P.

(ii) TITLE OF INVENTION: Anti-Inflammatory Composition

5(iii) NUMBER OF SEQUENCES:

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: One Financial Center

(C) CITY: Boston

10 (D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP CODE: 02111-2658

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb storage 15 (B) COMPUTER: IBM PS/2 Model 50Z or 55SX

(C) OPERATING SYSTEM: IBM P.C. DOS (Version 3.30)

(D) SOFTWARE: WordPerfect (Version 5.0)

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: 20 (B) FILING DATE: 23-Oct-1990

(C) CLASSIFICATION: - 19 (vii) PRIOR APPLICATION DATA:

Prior applications total, including application described below:

i (A) APPLICATION NUMBER: (B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Clark, Paul T.

(B) REGISTRATION NUMBER: 30,162

10 (C) REFERENCE/DOCKET #: 00231-039001

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (617) 542-8906

(C) TELEX: 200154

TOTAL NUMBER OF SEQUENCES TO BE LISTED:

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78

(B) TYPE: nucleic acid

20 (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(E) NAME: SEQ. LISTING (ϋ) SEQUENCE DESCRIPTION FOR SEQUENCE ID NUMBER: 1

INSERT SEQUENCE #1 HERE!

AVLPRSAKEL RCQCIKTYSK PFHPKFIKEL RVIESGPHCA NTEIIVKLSD GRELCLDPKE 60 NWVQRWEKF LKRAENS 78

Claims

CLAIMS:
1. An anti-inflammatory composition comprising a neutrophil activating factor and an antibody which is capable of binding to a neutrophil cell surface receptor of the integrin family.
2. The composition of claim 1 wherein the neutrophil activating factor is IL-8/LAI or a fragment or derivative thereof possessing the ability to degranulate neutrophils and cause the selective release of adhesion molecules of the selectin class from the neutrophil membrane.
3. The composition of claim 1 wherein the antibody is capable of binding to the Mol cell surface antigen.
4. The composition of claim 3 wherein the antibody is selected from the group consisting of monoclonal antibodies MY904, 60.3, and IB4.
5. The composition of claim 3 wherein the antibody binds to the CD18 subunit of the Mol antigen.
6. The composition of claim 3 wherein the antibody binds to the CDllb subunit of the Mol antigen.
7. The composition of claim 6 wherein the antibody has the cell surface molecule binding characteristics of the monoclonal antibody MY904.
8. A method of inhibiting an inflammatory response in a mammal, said method comprising administering to said mammal an inflammation inhibiting amount of a neutrophil activating factor and a pharmaceutically effective amount of an antibody capable of binding to a neutrophil cell surface receptor of the integrin family, said neutrophil activating factor and said antibody being administered simultaneously or near enough in time so that said antibody inhibits systemic toxicity potentially caused by said neutrophil activating factor.
9. The method of claim 8 wherein the neutrophil activating factor and the antibody are administered simultaneously.
10. The method of claim 9 wherein the neutrophil activating factor and the antibody are administered sequentially.
11. The method of claim 10 wherein the antibody is administered prior to administration of the neutrophil activating factor.
12. The method of claim 10 wherein the neutrophil activating factor is IL-8.
13. The method of claim 1 wherein said antibody does not inhibit binding of the iC3b component of complement.
14. The method of claim 10 wherein the antibody is capable of binding to the Mol cell surface antigen.
15. The method of claim 10 wherein the antibody is monoclonal antibody MY904.
16. A method of treating a human or animal host with the intent of reducing tissue damage occuring at an inflammatory site, said method comprising: administering a neutrophil activating factor which will elicit the release of selectins from the surface of neutrophils, and an antibody which will bind to neutrophil receptors of the integrin family thereby inhibiting adhesion dependent intercellular reactions of neutrophils.
17. The method of claim 16 wherein the neutrophil activating factor and the antibody are administered simultaneously.
18. The method of claim 17 wherein the neutrophil activating factor and the antibody are administered sequentially. - 23 -
19. The method of claim 18 wherein the antibody is administered prior to administration of the neutrophil activating factor.
PCT/US1991/007841 1990-10-23 1991-10-22 Anti-inflammatory composition WO1992006697A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US60216190 true 1990-10-23 1990-10-23
US602,161 1990-10-23

Publications (1)

Publication Number Publication Date
WO1992006697A1 true true WO1992006697A1 (en) 1992-04-30

Family

ID=24410227

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/007841 WO1992006697A1 (en) 1990-10-23 1991-10-22 Anti-inflammatory composition

Country Status (1)

Country Link
WO (1) WO1992006697A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997031099A1 (en) * 1996-02-22 1997-08-28 Icos Corporation Human beta 2 integrin alpha subunit
US5677426A (en) * 1994-03-03 1997-10-14 Genentech, Inc. Anti-IL-8 antibody fragments
US5854070A (en) * 1992-07-16 1998-12-29 Icos Corporation Murine and humanizer 23F2G antibodies and cell lines expressing said antibodies
US5874080A (en) * 1994-03-03 1999-02-23 Genentech, Inc. Anti-IL-8 monoclonal antibodies for treatment of asthma
US6737059B1 (en) 1992-07-16 2004-05-18 Board Of Regents Of The University Washington Methods of inhibiting inflammatory processes and alleviating symptoms associated with multiple sclerosis
WO2007048186A1 (en) * 2005-10-25 2007-05-03 Baker Medical Research Institute Leukocyte-binding polypeptides and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4840793A (en) * 1987-06-11 1989-06-20 Dana-Farber Cancer Institute Method of reducing tissue damage at an inflammatory site using a monoclonal antibody
US4935234A (en) * 1987-06-11 1990-06-19 Dana-Farber Cancer Institute Method of reducing tissue damage at an inflammatory site using a monoclonal antibody

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4840793A (en) * 1987-06-11 1989-06-20 Dana-Farber Cancer Institute Method of reducing tissue damage at an inflammatory site using a monoclonal antibody
US4935234A (en) * 1987-06-11 1990-06-19 Dana-Farber Cancer Institute Method of reducing tissue damage at an inflammatory site using a monoclonal antibody

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CLINICAL EXPERIMENTAL IMMUNOLOGY, Vol. 81 (3), issued September 1990, LEUWENBERG et al., "Adhesion of polymorphonuclear cells to human endothelial cells. Adhesion molecule dependant and Fc receptor mediated adhegion moleculeindependant mechanisms", see medline abstract No. 90374553. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6737059B1 (en) 1992-07-16 2004-05-18 Board Of Regents Of The University Washington Methods of inhibiting inflammatory processes and alleviating symptoms associated with multiple sclerosis
US5854070A (en) * 1992-07-16 1998-12-29 Icos Corporation Murine and humanizer 23F2G antibodies and cell lines expressing said antibodies
US5874080A (en) * 1994-03-03 1999-02-23 Genentech, Inc. Anti-IL-8 monoclonal antibodies for treatment of asthma
US5686070A (en) * 1994-03-03 1997-11-11 Genentech, Inc. Methods for treating bacterial pneumonia
US5702946A (en) * 1994-03-03 1997-12-30 Genentech, Inc. Anti-IL-8 monoclonal antibodies for treatment of inflammatory disorders
US5707622A (en) * 1994-03-03 1998-01-13 Genentech, Inc. Methods for treating ulcerative colitis
US5677426A (en) * 1994-03-03 1997-10-14 Genentech, Inc. Anti-IL-8 antibody fragments
WO1997031099A1 (en) * 1996-02-22 1997-08-28 Icos Corporation Human beta 2 integrin alpha subunit
WO2007048186A1 (en) * 2005-10-25 2007-05-03 Baker Medical Research Institute Leukocyte-binding polypeptides and uses thereof

Similar Documents

Publication Publication Date Title
Van Vliet et al. Focal myocarditis associated with pheochromocytoma
Sezer et al. RANK ligand and osteoprotegerin in myeloma bone disease
Von Asmuth et al. Tumour Necrosis Factor Alpha (TNF‐α) and Interleukin 6 in a Zymosan‐Induced Shock Model
Ding et al. Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration
US5198424A (en) Functionally active selectin-derived peptides
Keane et al. Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis
Condliffe et al. Priming differentially regulates neutrophil adhesion molecule expression/function
Furze et al. Neutrophil mobilization and clearance in the bone marrow
Andersson et al. The acute inflammatory response to lipopolysaccharide in CNS parenchyma differs from that in other body tissues
US6190660B1 (en) Anti-endoglin monoclonal antibodies and their use in antiangiogenic therapy
Bonder et al. Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis
Goldenberg Etanercept, a novel drug for the treatment of patients with severe, active rheumatoid arthritis
Chen et al. Suppression of tumor formation in lymph nodes by L-selectin–mediated natural killer cell recruitment
Smith et al. Chemotactic factors regulate lectin adhesion molecule 1 (LECAM-1)-dependent neutrophil adhesion to cytokine-stimulated endothelial cells in vitro.
Hom et al. In vivo administration with IL-1 accelerates the development of collagen-induced arthritis in mice.
US6492325B1 (en) Use of α1β1 integrin receptor inhibitors and TGF-β1 inhibitors in the treatment of kidney disease
US6555340B1 (en) Nucleic acid encoding bovine extracellular rage binding protein (en-rage)
Lambrigts et al. Discordant organ xenotransplantation in primates: world experience and current status
Dinarello et al. Anticytokine strategies in the treatment of the systemic inflammatory response syndrome
Isaacs et al. CAMPATH-1H in rheumatoid arthritis—an intravenous dose-ranging study
Movat Tumor necrosis factor and interleukin-1: role in acute inflammation and microvascular injury
Choi et al. Antisense inhibition of macrophage inflammatory protein 1-α blocks bone destruction in a model of myeloma bone disease
US5597567A (en) Blocking cell adhesion molecules and treating animals with ocular inflammation
Bhatia Inflammatory response on the pancreatic acinar cell injury
US5538724A (en) Method of control leukocyte extravasation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: CA