Il8 inhibitors

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DESCRIPTION

Antibodies against the IL-8 receptor and their therapeutic uses.

Technical field

The present invention relates generally to cytokine inhibitors. More particularly, the invention relates to inhibitors of IL8 receptor 2 binding, including antibodies, that interact with the amino-terminal extracellular domain of IL8 receptor 2 and compete with IL8, and other related natural ligands, for the binding to the receptor.

Background of the invention

Cytokines are a group of hormone-like mediators produced by leukocytes. These agents serve as endogenous biological signals that act in conjunction with antigens to amplify host defense mechanisms, both localized and systemic, involving macrophages, lymphocytes, and other cell types. Representative cytokines include the various interleukins, interferons, GROa, GRO / i, and GROy, neutrophil activating peptide 2 (NAP-2), and ENA-78. Cytokines have been used to treat and prevent a wide variety of disorders based on the ability of these molecules to stimulate an immune response.

Interleukin-8 (IL8) is a cytokine originally derived from human macrophages (Suzuki, K. et al. (1989) J. Exp. Med. 169: 1895-1901; Schroder, JM et al. (1987) J. Immunol 139: 3474-3483; Schroder, JM et al. (1988) J. Immunol. 140: 3534-3540; Schroder, JM (1989) J. Exp. Med. 170: 847-861; Larson, CG et al. (1989) Science 243: 1464-1466). This factor has also been called polymorphonuclear chemotactic factor (PMN), monocyte-derived neutrophil activating peptide (MONAP), monocyte-derived neutrophil chemotactic factor (MDNCF) (“Monocyte-Derived Neutrophil Chemotactic Factor”), T lymphocyte chemotactic factor (TCF), lymphocyte derived neutrophil activating peptide (LYNAP) "Lymphocyte-derived Neutrophil-Activating Peptide") and neutrophil activating peptide 1 (NAP-1).

The IL8 molecule is produced in a wide variety of tissues and cells, including mononuclear phagocytes, endothelial cells, fibroblasts, epithelial cells, and alveolar macrophages, upon stimulation with agents such as lipopolysaccharide and phorbol myristate, phytohemagglutinin, Con A, or others. mitogenic preparations, and cytokines such as interleukin-1 (IL1) and tumor necrosis factor (TNF). The gene encoding human IL8 has been cloned. Matsushima, K. et al. J. Exp. Med. (1988) 167: 1883-1893; Mukaida, N. et al. J. Immunol. (1989) 143: 1366-1371. The gene encodes a precursor protein that is 99 amino acids long and is proteolytically cleaved into secreted IL8 polypeptides of varying lengths, such as those containing 69, 72, or 77 amino acid residues, with molecular masses of approximately 8,000 Daltons.

Human IL8 acts as a neutrophil chemoattractant molecule and induces granulocytosis after systemic injection, and a dermal reaction after local injection, in experimental animals. Bazzoni, F. et al. (1991) 173: 771-774; Van Damme, J. et al. (1988) J. Exp. Med. 167: 1364-1376; Ribeiro, RA et al. (1991) Immunology 73: 472-477. This molecule also activates the release of superoxide anions and induces the release of the primary constituents of neutrophil granules, which include myeloperoxidase, β-glucuronidase, and elastase. IL8 mediates these activities by binding to its receptor and triggering signal transduction, a cascade of reactions that ultimately result in a biological response.

The sequences of the two human IL8 receptors (here called "IL8R1" and "IL8R2") have been described. See, p. eg International Publication WO93 / 06229 (published April 1, 1993) and Holmes et al. Science (1991) 253: 1278-1280. These receptors have a similar affinity for IL8 and are members of the rhodopsin superfamily, which consists of seven helices, and extends across the membrane. These receptor molecules include seven transmembrane regions, linked by three intracellular loops and three extracellular loops, and possess an extracellular amino terminal tail and an intracellular carboxy terminal tail. Other naturally occurring cytokines are known to share a receptor with IL8. For example, GROa, GRO 'GROy, NAP-2 and ENA-78 all bind to one of the IL8 receptors on human neutrophils, as described in Baggiolini et al., FEBS Lett. (1992) 307: 97-101; Walz et al., J. Exp. Med. (1991) 174: 1355; Moser et al., J. Biol. Chem. (1991) 266: 10666; Geiser et al., J. Biol. Chem. (1993) 268: 15419-15424.

Gayle et al., J. Biol. Chem. (1993) 268: 7283-7289 and LaRosa et al., J. Biol. Chem. (1992) 267: 25402-25406 relate to an important determinant for the binding of natural agonists of IL8R2, GROa / MGSA and NAP-2, residues located in the receptor region that includes the extracellular amino terminal domain and a portion of the first transmembrane region.

Peptides based on the IL8 receptor sequence have been constructed in an attempt to determine the role of different regions of the IL8 receptor in IL8 binding. Gayle et al., J. Biol. Chem. (1993) 268: 7283-7289 describe peptides based on the amino terminal sequence of the IL8 receptor, and International Publication, WOA-92/04372 (published March 19, 1992) describes peptides and antibodies directed against them, based on the carboxy termination of the receptor.

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WO-A-92/18641 describes cDNAs encoding IL8 receptors, including the "low affinity" human IL8 receptor (IL8R2). Antagonists that interfere with the interaction between IL8 and the IL8 receptor are also proposed, including antibodies specific for the IL8 receptor.

Summary of the invention

The present invention provides substances that inhibit the binding of IL8 to IL8R2 through its interaction with the extracellular amino terminal domain of IL8R2. These inhibitors are useful modulators of IL8 receptor-mediated biological activity.

Accordingly, in one of the embodiments, the invention is directed to an inhibitor of IL8 receptor 2 binding, comprising an antibody, or one of its fragments, which is capable of interacting with the extracellular amino terminal domain of receptor 2 of IL8 and that it is able to compete with IL8 for receptor binding, wherein the antibody can be obtained by immunizing a mammal with an immunogen consisting essentially of a polypeptide selected from FEDFWKGEDLSNYSY, SSTLPPFLLDAAPC, and FLLD-AA-PCEPESLEI.

The invention further provides inhibitors of IL8 receptor 2 binding according to the invention, for use in a method of treating a human or animal organism, and pharmaceutical compositions for modulating an IL8 receptor mediated biological response, comprising an inhibitor of the IL8 receptor 2 binding of the invention, in combination with a pharmaceutically acceptable carrier.

The invention further provides the use of an inhibitor of the invention, in the manufacture of a medicament for use in a method of treatment, which comprises inhibiting the binding of IL8 to IL8R2.

The invention also provides the use of an inhibitor of the invention, in the manufacture of a medicament for use in a method of treatment, which comprises modulating an IL8 receptor 2-mediated biological response.

Brief description of the figures

Figure 1 shows the amino acid sequences of the extracellular amino terminal domain of human IL8R1 and human IL8R2 and represents the peptides used to generate the antisera.

Figure 2 shows the results of a FACS analysis of neutrophils using antisera against IL8R1 and IL8R2 amino terminal peptides, as described in the examples. Neutrophils were incubated with IgG prior to immunization, from sheep (Figures 2A and 2C), with anti-IL8R1 peptide IgG (Figure 2B) or with IL8R2 anti-peptide IgG (Figure 2D), each diluted in the ratio 1 : 150 in PBS + 1% BSA, then treated with fluorescein-labeled rabbit anti-sheep antibodies (1: 100 in PBS + 1% bSa) and subjected to FACS analysis, as described later.

Figure 3 depicts the results of receptor binding assays for IL8R1, as described in the examples. Sf9 cells were infected with a recombinant baculovirus responsible for the expression of IL8R1 and a binding assay of¹²⁵I-IL8 as described, after a 1 hour preincubation with IL8 or with antibodies:

1672 p, IgG fraction from a serum control prior to immunization, for the serum corresponding to peptide no. 1 of IL8R1; 1672 i, IgG fraction from an antiserum against peptide no. 1; 1672 i pur. af., affinity purified antibodies, directed against peptide no. 1 of IL8R1; 1673 p, IgG fraction from a serum control prior to immunization, for the serum corresponding to peptides nos. IL8R2 10-13;

1673 i, IgG fraction from an antiserum directed against peptides nos. 10-13 of IL8R2. The final concentrations of each of the effectors are indicated.

Figure 4 shows the inhibition of IL8 binding to IL8R1 by anti-peptide antibody no. 1 of IL8R1 in Sf9 cells. Sf9 cells were infected with a baculovirus responsible for IL8R1 expression, preincubated with antibodies, and assayed for binding of¹²⁵I-IL8 as described above. When indicated (Pur. Af. + Pept. 1), antibodies against peptide no. 1 of IL8R1, affinity purified, were pre-absorbed with a concentration of 50 mg / ml of peptide no. 1 before use. The data is corrected for nonspecific binding, measured in the presence of a 1 mg / ml concentration of unlabeled IL8.

Figure 5 depicts the results of receptor binding assays for IL8R2, as described above and described in the examples.

Figure 6 shows the inhibition of IL8 binding to IL8R2 by anti-peptide antibody no. 10 of IL8R2 in Sf9 cells. Sf9 cells were infected with baculovirus responsible for IL8R2 expression, preincubated with antibodies, and assayed for binding of¹²⁵I-IL8 as described above. The additions of the antibodies are as follows: IgG prior to immunization, fraction of IgG from a control of a serum prior to immunization; IgG anti-peptide R2, IgG fraction from an antiserum directed against peptides nos. IL8R2 10-13; Anti-pept. 10 pur. af. or Pur. af. - pept. 10, antibodies directed against peptide no. 10, affinity purified; Pur. Af. + pept. 10, antibody directed against peptide no. 10 pre-absorbed with

ES 2 302 774 T3 a concentration of 50 mg / ml of peptide no. 10, and affinity purified. The data is corrected for nonspecific binding, measured in the presence of a 1 mg / ml concentration of unlabeled IL8.

Detailed description of the invention

The practice of the present invention will utilize, unless otherwise indicated, conventional methods of virology, microbiology, molecular biology, and recombinant DNA techniques within the skill of the art. Those methods are explained in full detail in the literature. See, p. eg, Sambrook et al. Molecular Cloning: A Laboratory Manual (2^to Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I and II (D. Glover, editor); Oligonucleotide Synthesis (N. Gait, editor, 1984); Nucleic Acid Hybridization (B. Homes and S. Higgins, editors, 1985); Transcription and Translation (B. Names and S. Higgins, editors, 1984); Animal Cell Culture (R. Freshney, editor, 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

A. Definitions

In describing the present invention, the following terms and expressions will be employed and are intended to be defined as indicated below.

An "inhibitor of IL8 receptor 2 binding" is a substance (other than natural IL8 or other naturally occurring endogenous neutrophil IL8 receptor ligands such as GROa, β, y, NAP-2 and ENA-78) that interacts, either directly or indirectly, with IL8 receptor 2 and that competes with IL8 or other natural ligands, such as GROa, β, and, NAP-2 and ENA-78, for binding to receiver. The IL8 receptor 2 binding interaction includes both a covalent and non-covalent association between the inhibitor and IL8R2. The inhibitor of the invention binds to the IL8 receptor at a binding site for IL8 in the extracellular amino terminal domain and blocks the binding of IL8.

The ability of a molecule to bind IL8R2 and to compete with IL8 for binding to IL8R2 can be determined using standard receptor binding assays. For example, the ability of the inhibitor to bind to the receptor can be determined directly by labeling the inhibitor and demonstrating its binding to cells bearing the receptor, such as neutrophils. Inhibitor binding can also be demonstrated by methods such as chemical crosslinking, by recognition, by an anti-inhibitor antibody, of the presence of the inhibitor bound to cells bearing the receptor, as described in the examples, by means of NMR or X-ray crystallography of the inhibitor / receptor complexes, and / or by other appropriate methods for use with a given inhibitor, generally known to a skilled practitioner. The ability of the inhibitors of the present invention to compete with IL8 for binding to IL8R2 can be determined using standard competition assays such as the radioimmunoassays described by Gayle et al., J. Biol. Chem. (1993) 268: 7283- 7289; and LaRosa et al., J. Biol. Chem. (1992) 267: 25402-25406, and in the examples set forth herein. The molecule does not need to completely inhibit IL8 binding, but only needs to decrease the amount of binding that would normally occur in the absence of the inhibitor. Furthermore, an inhibitor of IL8 binding can be either an agonist, that is, a molecule capable of promoting at least one of the biological responses normally associated with IL8, or an antagonist, that is, a substance that opposes at least to one of the effects of IL8, thus decreasing the ability of IL8 to mediate biological responses normally associated with it.

The term "IL8 receptor" as used herein refers to any of a number of vertebrate IL8 receptors, or fragments thereof that include an IL8 binding domain. For example, both human IL8R1 and IL8R2 are encompassed by this expression. The inhibitors of the invention compete with IL8 for binding to IL8R2.

The term "IL8 receptor binding" or "IL8 binding" refers to the binding to any one of the IL8 receptors of any of IL8, GROa, GRO 'GROy, NAP-2 and ENA-78, their fragments and other naturally occurring ligands. Inhibitors of the invention bind IL8R2.

By "modulating a biological response mediated by an IL8 receptor" is meant an increase or decrease in the incidence of one or more cellular activities normally triggered by the binding of IL8 to its receptor. The nature of these activities can be biochemical or biophysical. For example, a substance would "modulate a biological response mediated by an IL8 receptor" if it does not stimulate the same signal transduction activity as IL8 when the inhibitor binds to an IL8 receptor. The rise or fall can be monitored using different assays, as described below, which also use molecules of an IL8 receptor as controls. The inhibitors of the invention compete with IL8 for its binding to IL8R2.

More particularly, a cascade of biochemical reactions is triggered when IL8 binds to its receptor. Accordingly, an IL8 inhibitor will "modulate an IL8 receptor mediated response" when it produces an increase or decrease in any one of these reactions. For example, receptors for IL8 are G protein-coupled proteins, which, when correct signal transduction activity occurs, trigger an increase in intracellular Ca levels.²⁺, IP₃ and DAG. Standard assays can be used to measure intracellular levels of these substances and thereby determine whether the IL8 receptor-mediated response has been modulated. Assays are known to measure Ca levels²⁺ cytosolic and are described, e.g. eg, in International Publication WO93 / 06229 (published April 1, 1993); Bazzoni, F. et al., (1991) J. Exp. Med. 173: 771-774 and

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Peveri, P. et al., (1988) J. Exp. Med. 167: 1547-1559. IP concentrations₃ and DAG levels can also be measured by known methods.

Other biological activities attributable to IL8, which can be measured in order to determine modulation, include, for example, chemotactic activity on neutrophils, measured using assays generally known in the art (see, eg, Schnoder, JM and col., (1987) J. Immunol. 139: 3474-3483; Fincham, NJ et al. (1988) J. Immunol. 140: 4294-4299; Larson, CG et al. (1989) Science 243: 1464-1466 ; Grob, PM et al. (1990) J. Biol. Chem. 265: 8311-8316; Strieter, RM et al. (1989) J. Biol. Chem. 264: 10621-10626); enzyme release assays, such as peroxidase release PMN activity assays, jd-glucuronidase release assays, O release₂ determined by cytochrome C assays and elastase release assays (see, e.g., Schroder,

JM et al., (1987) J. Immunol. 139: 3474-3483; Peveri, P et al. (1988) J. Exp. Med. 167: 1547-1559).

Two peptides will be "substantially the same" or "substantially identical" when at least about 50%, usually at least about 60%, more typically at least about 75%, and preferably at least about 90% - 95% of the amino acids , coincide along a defined length of the molecule. As used herein, "substantially the same" also refers to sequences that show identity to the specified polypeptide sequence.

By "natural IL8" is meant a polypeptide having an amino acid sequence that is identical to a sequence recovered from a source that produces IL8 in nature. Natural IL8 can be of various lengths, such as those containing 69, 72, and 77 amino acid residues, respectively.

The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, the definition includes peptides, oligopeptides, dimers, multimers, and the like. Both full-length proteins and their fragments are encompassed by the definition. These terms also include post-expression modifications of the polypeptide, eg, glycosylation, acetylation, phosphorylation, and the like.

The term "antibody" encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, modified antibodies, F (ab ') fragments₂, F (ab) fragments, F fragments_v, single domain antibodies, chimeric antibodies, humanized antibodies and their functional fragments, which retain their specificity for the IL8 binding sites of the extracellular amino terminal domain. For example, an antibody can include variable regions, or fragments of variable regions, that retain specificity for the extracellular amino terminal domain of an IL8 receptor molecule. The remaining part of the antibody can be derived from the species in which the antibody will be used. Thus, if the antibody is to be used in a human being, the antibody can be "humanized" in order to decrease its immunogenicity while retaining its activity. In connection with a description of chimeric antibodies, see, p. eg, Winter, G. and Milstein, C. (1991) Nature 349: 293-299; Jones, PT et al. (1986) Nature 321: 522-525; Riechmann, L. et al. (1988) 332: 323-327; and Carter, P. et al., (1992) Proc. Natl. Acad. Sci. USA 89: 4285-4289. Those chimeric antibodies can contain not only binding sites for the IL8 receptor, but also binding sites for other proteins. In this way, bifunctional reagents can be generated with a specificity directed to both external and internal antigens.

An "antigen" or an "immunogen" refers to a molecule that contains one or more epitopes that will stimulate the immune system of a host to generate a secretory, humoral, and / or cellular response specific to the antigen.

An "effective inhibitory amount" of an IL8 inhibitor refers to an amount of inhibitor sufficient to block the binding, in whole or in part, of IL8 to the IL8R2 receptor. The precise effective inhibitory amount will depend on the number and type of IL8R2 receptors present on the surface of the particular cell in question. That amount can be readily determined by one of ordinary skill in the art using routine experimentation and IL8 binding assays, such as standard neutrophil binding assays, as described in the examples.

The term "effective modulating amount" of an IL8 inhibitor refers to an amount of inhibitor sufficient to cause a change in an IL8 receptor 2-mediated biological activity, as described above. This amount will also depend on the number and type of IL8R2 receptors present on the surface of the particular cell in question and will vary depending on the biological activity to which the inhibitor is directed. Assays for determining changes in an IL8 receptor 2-mediated activity are described above.

B. General methods

Fundamental to the present invention is the discovery of IL8 inhibitors that are capable of suppressing the binding of different ligands, including IL8, GROa, GRO / 'Á GROy, NAP-2 and ENA-78, to IL8 receptors, modulating thus the biological responses triggered by this union. Consequently, these inhibitors are capable of blocking or modulating neutrophil chemotaxis and activation. The inhibitors are useful for the treatment and prevention of a wide variety of disorders in which neutrophils contribute to the disease pathology, including inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, as well as for the treatment and / or o prevention of tissue damage caused during diseases such as

ES 2 302 774 T3 septic shock and acute respiratory distress syndrome (ARDS). The inhibitors of the present invention will also find utility to stimulate a neutrophil-mediated inflammatory response in the treatment of cancerous processes, viral, bacterial, fungal and protozoal infections, as well as for the treatment of processes in which the immune system is compromised, such such as AIDS and other acquired and inherited immune disorders.

The inhibitors of the present invention act on the extracellular amino terminal domain of the IL8 receptor.

The extracellular amino terminal domain sequences of human IL8 receptors 1 and 2 (designated IL8R1 and IL8R2 respectively) are shown in Figures 1 and 2. Inhibitors for use in the present invention include antibodies raised against isolated peptide sequences, derived from contiguous sequences that extend throughout this region, as well as antibodies raised against their analogs (i.e., against sequences with substitutions, additions, or deletions of amino acids), which retain the ability to bind to the IL8R2 receptor and inhibit IL8 binding to it, as defined above. Surprisingly, it has been discovered that antibodies and antibody mixtures, raised against peptides derived from the extracellular amino terminal domain region of both IL8R1 and IL8R2, are capable of blocking the binding of Il8 to the receptor. Thus, the recognition site (epitope) for these antibodies appears to overlap with, or is close to, the recognition site for IL8. These antibodies can be polyclonal, monoclonal, chimeric, or their functional fragments, generated using standard techniques. In addition, useful antibody preparations can include mixtures of several different antibodies, as detailed below.

Several peptides have been synthesized based on the amino acid sequence of the extracellular amino terminal binding domain of IL8R1 and IL8R2. These peptides are shown in Tables 1 and 2, in the examples. These and other peptides can be synthesized by protein synthesis techniques, known to those of skill in the art. In general, these methods use solid phase or solution phase synthesis methods, well known in the art. See, p. eg, JM Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2^to Ed., Pierce Chemical Co., Rockford, IL (1984) and G. Barany and RB Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, in relation to solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, editors, The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, in relation to classical synthesis in solution.

Peptides can also be produced by recombinant methods, known in the art. For example, a DNA sequence encoding the peptide in question can be synthesized using standard methods. See, p. eg, Edge (1981) Nature 292: 756; Nambair et al. (1984) Science 223: 1299; Jay et al. (1984) J. Biol. Chem. 259: 6311. In general, the preferred codons will be selected for the host in which the sequence is desired to be expressed. Alternatively, the sequence can be derived from genomic DNA or cDNA. The DNA is cloned into a suitable expression vector, either prokaryotic or eukaryotic, using conventional methods, the host cells are transformed with the vector and cultured under conditions that allow the expression of the protein of interest.

Peptides can also be produced by enzymatic or chemical cleavage of the purified IL8R2 receptor or of a polypeptide containing the desired sequence. Those procedures are conventional and well known in the art.

In one embodiment, the inhibitors of the present invention can be polyclonal or monoclonal antibodies that can be generated in vitro or in vivo. Methods for generating those antibodies are known in the art.

Polyclonal antibodies directed against these peptides are generated by immunizing a suitable animal, such as a mouse, rat, rabbit, sheep, or goat, with the peptide of interest. In order to increase immunogenicity, the peptide can be bound to a carrier prior to immunization. Suitable transporters are typically large, slow metabolizing macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, (such as oil droplets or liposomes), and inactive viral particles. Such carriers are well known to those of ordinary skill in the art. Furthermore, the peptide can be conjugated with a bacterial toxoid, such as a toxoid from diphtheria, tetanus or cholera, etc., in order to increase its immunogenicity.

Rabbits, sheep and goats are preferred when large volumes of sera are desired. These animals constitute good design options also due to the availability of labeled anti-rabbit, anti-sheep and anti-goat antibodies. Immunization is generally carried out by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the sample or emulsion parenterally (generally subcutaneously or intramuscularly). The animal generally receives a booster dose 2-6 weeks later, with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. Antibodies can also be generated by in vitro immunization using methods known in the art. The polyclonal antisera are then obtained from the immunized animal.

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Monoclonal antibodies are generally prepared using the method of Kohler and Milstein, Nature (1975) 256: 495-96, or one of its modifications. Typically, a mouse or rat is immunized as described above. However, instead of drawing the animal's blood to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into isolated cells. If desired, spleen cells can be screened (after removing non-specifically adherent cells) by applying a suspension of the cells to a plate or well coated with the protein antigen. The B cells, which express the antigen-specific membrane-bound immunoglobulin, will bind to the plate and will not be washed away along with the rest of the suspension. The resulting B cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and cultured in a selective medium (eg, hypoxanthine, aminopterin, and thymidine medium, " HAT ”). The resulting hybridomas are plated by limiting dilution, and assayed for the production of antibodies that specifically bind to the immunizing antigen (and that do not bind unrelated antigens). The selected hybridomas that secrete the monoclonal antibody are then cultured either in vitro (eg, in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).

Functional fragments of inhibitory antibodies will also find utility in the present invention and can be produced by cleaving a constant region, not responsible for antigen binding, from the antibody molecule, using e.g. eg, pepsin, to produce F (ab ') fragments₂. These fragments will contain two antigen-binding sites, but will lack a portion of the constant region of each of the heavy chains. Similarly, if desired, Fab fragments can be produced, comprising a single antigen-binding site, e.g. eg, by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments, including only the heavy and light chain variable regions, can also be produced using standard techniques. These fragments are known as F_v.

Chimeric antibodies or humanized antibodies can also be produced for use in the present invention. These antibodies can be designed to minimize unwanted immunological reactions attributable to heterologous constant regions and species-specific, variable framework regions typically present in monoclonal and polyclonal antibodies. For example, if the antibodies are to be used as IL8 inhibitors in human subjects, chimeric antibodies can be created by replacing non-human constant regions, on one of the two chains, light or heavy, or both, with human constant regions, using methods generally known in the art. See, p. eg, Winter, G. and Milstein, C. (1991) Nature 349: 293-299; Jones, PT et al. (1986) Nature 321: 522525; Riechmann, L. et al. (1988) 332: 323-327; and Carter, P. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 4285-4289.

The above-mentioned preparations can be tested for their ability to bind to the IL8R2 receptor and to modulate an IL8 receptor-mediated activity, using known assays. Substances can be tested for their ability to compete with IL8 for receptor binding, using competition assays such as the radioimmunoassays described in Gayle et al., J. Biol. Chem. (1993) 268: 7283-7289; and in LaRosa et al., J. Biol. Chem. (1992) 267: 25402-25406 and in the examples included herein. That IL8-competing compounds interact with the receptor can be demonstrated by the methods described above. The ability of these molecules to interact with the extracellular amino terminal domain of the receptor can be demonstrated, also by the methods described above, using for example chimeric receptors that have been shown to bind IL8, which consist of an extracellular amino terminal domain. of the IL8R2 receptor and in another unrelated receptor.

Furthermore, the ability of inhibitors to modulate an IL8 receptor 2-mediated activity can be tested, as explained above, by measuring, e.g. eg, Ca levels²⁺ free cytosolic, from IP concentrations₃ intracellular and DAG levels (see, eg, International Publication WO93 / 06229 (published April 1, 1993); Bazzoni, F. et al. (1991) J. Exp. Med. 173: 771-774; Peveri, P. et al. (1988) J. Exp. Med. 167: 1547-1559). Other measures of IL8-mediated activity include chemotactic assays in neutrophils (see, eg, Schroder, JM et al. (1987) J. Immunol. 139: 3474-3483; Fincham, NJ et al. (1988) J Immunol 140: 4294-4299, Larson, CG et al. (1989) Science 243: 1464-1466; Grob, PM et al. (1990) J. Biol. Chem. 265: 8311-8316; Stricter, RM et al. (1989) J. Biol. Chem. 264: 10621-10626); enzyme release assays, such as peroxidase release PMN activity assays, jd-glucuronidase release assays, O2 release, as determined by cytochrome C assays, and elastase release assays (see, eg, Schroder, JM et al. (1987) J. Immunol. 139: 3474-3483; Peveri, P. et al. (1988) J. Exp. Med. 167: 1547-1559).

The inhibitory antibodies of the present invention are not only useful as modulators of an IL8 activity, but can also be used to identify homologous genes of IL8 receptors in other vertebrate species and to panning the IL8 receptor from sources that do so. produce. Finally, antibodies can also be used as targeting agents to deliver other IL8 agonists and antagonists to IL8 binding sites. To do this, antibodies can be conjugated to these agents or to fusion proteins, including at least the active binding region of the antibody, bound to at least a functionally active portion of an IL8 inhibitor, and can be constructed using DNA methods. recombinant well known in the art.

The inhibitors of the present invention can be provided in pharmaceutical compositions to inhibit the binding of IL8 to its receptor and to modulate a biological response mediated by an IL8 receptor. Compositions will generally include one or more "pharmaceutically acceptable excipients or carriers" such as water, saline, glycerol, ethanol, and the like. Additionally, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be present in those vehicles.

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The pharmaceutical compositions comprise an effective inhibitory or modulatory amount of an IL8 inhibitor, as defined above. The compositions are administered in a conventional manner parenterally, e.g. For example, by injection, either intravenously, subcutaneously, intramuscularly, or intraperitoneally. Additional formulations suitable for other modes of administration include formulations for oral and pulmonary administration, suppositories, and transdermal applications. Dosage treatment can follow a single dose regimen or a multiple dose regimen.

C. Experimental part

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Every effort has been made to ensure accuracy in relation to the figures used (eg quantities, temperatures, etc.), but of course some experimental error and bias should be allowed in them.

Example I

Preparation of peptides and antisera

Peptide synthesis, toxoid conjugation, immunization of mice and sheep, and determination of reactivity by ELISA against a pin-coupled peptide and against a plate-coated peptide were carried out in Chiron Mimotopes Pty. Ltd. (Clayton, Victoria 3168, Australia) using standard procedures. In particular, peptides exhibiting substantial homology to portions of the extracellular amino terminal region of human IL8 receptors, IL8R1 and IL8R2, as shown in Tables 1 and 2, were synthesized using standard techniques. Some of the peptides (as indicated in the Tables) were coupled through an internal cysteine ​​residue to a toxin to increase their immunogenicity. The N-terminal acetyl groups or the C-terminal j6-alanine-diketopiperazine groups are blocking groups that mimic the environment of the peptide sequence within the protein.

Antisera were generated in mice against peptides no. 1 and no. 3, and were generated in sheep against peptide no. 1 (Figure 1, Table 1). Antisera against the extracellular amino terminal domain of IL8R2 were generated in mice against peptides no. 6 and no. 7 and in sheep against a mixture of peptides no. 10, no. 11, no. 12, no. 13 (Figure 1, Table 2). Immunoserums, but not pre-immunization sera or antisera generated against peptides no. 6 and no. 7, showed a high titer against the immunizing peptide (s) as determined by ELISA against the peptide attached to pins and / or coated onto plates. The manufacturer cited technical difficulties as an explanation for the lack of immunoreactivity of the antisera against immunizing peptides no. 6 and no. 7. These particular antisera were no longer used and antisera raised against peptides no. 10 and no. 13.

The antisera were used, either untreated, or purified by chromatography on Protein G-Agarose (Pharmacia) or by affinity chromatography on Sepharose with peptide no. 1 (in the case of anti-IL8R1 immunoserums) or on Sepharose with peptide no. 10 (in the case of anti-IL8R2 immunoserums). Sepharose-conjugated peptides were prepared on Chiron Mimotopes.

TABLE 1

Peptides from the extracellular amino terminal domain of IL8R1

<td>Peptide</td><td></td>

<td> *1</td><td>amino-MSNITDPQMWDFDDLXC-dkp</td>

<td>Ib</td><td>amino-MSNITDPQMWDFDDLXG-amide</td>

<td>you</td><td>amino-MSNITDPQMWDFDDLXC-dkp-thiopropylsepharose</td>

<td> *3</td><td>acetyl-NFTGMPPADEDYSPXC-dkp</td>

<td>3b</td><td>acetyl-NFTGMPPADEDYSPXG-amide</td>

* = antiserum produced

X = β-alanine dkp = diketopiperazine (diketOOiperazine)

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TABLE 2

Peptides from the extracellular amino terminal domain of IL8R2

<td>Peptide</td><td></td>

<td> *6</td><td>amino-MESDSFEDFWKGEDLSNYSYXCX-dkp</td>

<td> *7</td><td>acetyl-SSTLPPFLLDAAPCEPESLELX-dkp</td>

<td> 8</td><td>amino-MESDSFEDFWKGEDLSNYSY-amide</td>

<td> 9</td><td>acetyl-SSTLPPFLLDAAPAEPESLEI-amide</td>

<td> *10</td><td>amino-MESDSFEDFWKGEDLCX-dkp</td>

<td>10b</td><td>amino-MESDSFEDFWKGEDL-amide</td>

<td>10c</td><td>amino-MESDSFEDFWKGEDL-dkp-thiopropylsepharose</td>

<td> *11</td><td>acetyl-FEDFWKGEDLSNYSYCX-dkp</td>

<td>11b</td><td>acetyl-FEDFWKGEDLSNYSY-amide</td>

<td> *12</td><td>acetyl-SSTLPPFLLDAAPCX-dkp</td>

<td>12b</td><td>acetyl-SSTLPPFLLDAAPA-amide</td>

<td> *13</td><td>acetyl-FLLDAAPCEPESLEIX-dkp</td>

<td>13b</td><td>acetyl-FLLDAAPAEPESLEI-amide</td>

★ = antiserum produced

X = β-alanine dkp = diketopiperazine

Example II

Neutrophil IL8 Receptor Recognition Assays

Antibodies were tested for their ability to recognize neutrophils as follows. Neutrophils were isolated from whole blood using Neutrophil Isolation Media (NIM) (Cardinal), essentially in the manner described by the manufacturer with the following modifications. 20 ml of NIM and 30 ml of blood were centrifuged in 50 ml tubes for 50 minutes. Contaminating red blood cells were removed by lysis in ice-cold water. The resulting neutrophils were resuspended in Hank's buffered saline (HBS) ("Hank's BufferedSaline") and stored on ice.

Neutrophils were incubated and subjected to FACS analysis as follows. Neutrophils were stained for FACS analysis by resuspending 1 x 10⁶ cells in 50 µl of IgG prior to immunization (Figures 2A and 2C), anti-IL8R1 peptide IgG (Figure 2B) or anti-IL8R2 peptide IgG (Figure 2D), from sheep, each diluted in proportion 1: 150 in PBS + 1% BSA. Cells were incubated 2 hours on ice, centrifuged at 1,000 RPM for 5 minutes, and then washed twice with PBS + 1% BSA. The washed cells were resuspended in 50 µl of anti-sheep IgG antibody (heavy chain + light chain), from rabbit, F (ab ') fragment₂, conjugated to FITC (1: 100 ratio in PBS + 1% BSA, Jackson ImmunoResearch Laboratories). Cells were incubated 1 hour on ice, centrifuged at 1,000 RPM for 5 minutes, washed in PBS + 1% BSA, and then washed in PBS. The cells to be analyzed were immediately resuspended in 500 ml of PBS. Propidium iodide was added at a concentration of 2.5 ng / ml as a viability stain. Neutrophils could be fixed in 1% paraformaldehyde and analyzed within up to 72 hours later. The labeled cells were analyzed on a FACSCAN (Becton Dickinson).

ES 2 302 774 T3

As can be seen in Figure 2, the antisera directed against peptide no. 1 of IL8R1 (Figure 2B) or against peptides nos. IL8R2 10-13 (Figure 2D), containing the extracellular amino terminal domain, but not the pre-immunization antisera (Figures 2A and 2C), recognized neutrophils. This indicates that antisera raised against peptide (s) from the extracellular amino terminal domain of IL8 receptors are capable of recognizing the natural receptor. The antiserum generated against peptide no. 3 IL8R1, despite its high titer against the immunizing peptide, did not recognize neutrophils (data not shown). This result gives no indication as to the role of the sequence of peptide no. 3 of IL8R1 in the recognition of ligands, but only means that peptide no. 3 was not present in sufficient amounts in a receptor-mimetic conformation to induce the generation of anti-receptor antibodies, as opposed to the exact anti-peptide antibodies. Antisera raised against peptide no. 6 or against peptide no. 7 IL8R2 showed a low titer against the immunizing peptides and were not used any further.

Example III

Receptor binding assays

Antibodies were tested for their ability to block the binding of IL8 to its receptor as explained below. IL8R1 and IL8R2, for use in receptor binding assays, were produced by recombinant techniques. DNA encoding IL8R1 and IL8R2 was isolated from human genomic DNA by PCR using oligonucleotide primers based on published IL8R1 sequences (Holmes et al. Science (1991) 253: 1278) and IL8R2 (Murphy and Tiffany, Science (1991) 253: 1280). The nucleotide sequence of the isolated genes was confirmed by dideoxy sequencing.

The sequence encoding IL8R1 (in the baculovirus transfer vector pVL1392, Invitrogen) or the sequence encoding IL8R2 (in the transfer vector pAcC13, described in Munemitsu et al. Mol. Cell. Biol. (1990) 10: 5977) was recombined into Autographa california baculovirus (AcNPV) by cotransfection of Sf9 cells with 2 pg of recombinant receptor transfer vector and 0.5 pg of linearized wild-type viral DNA, as described (Kitts et al. , Nuc. Acids. Res. (1991) 18: 5667). Recombinant baculoviruses were isolated by plaque purification (Smith et al., Mol. Cell. Biol. (1983) 3: 2156). Suspension cultures ~ 1.5 x 10⁶ Sf9 cells per ml were collected for analysis 40 hours - 48 hours after infection with the relevant baculovirus stock preparation at an MOI value of 2-10, in serum-free medium (Maiorella et al. Biotech. (1988 ) 6: 1406).

Insect Sf9 cells were infected with a recombinant baculovirus carrying IL8R1 or IL8R2 and seeded in Remova 96-well culture plates in ExCell 400 media. At 40 hours - 48 hours after infection, the culture medium was removed and cell monolayers were pretreated for 1 hour at room temperature with antibody, in Hepes-BSA binding buffer (25 mM Hepes, pH 7.5, 150 mM NaCl, CaCl₂ 5 mM, MgCl₂ 5 mM, 1 mg / ml bovine serum albumin). Was added¹²⁵I-IL8 until a final concentration of 0.2 nM was obtained and the incubation was continued at room temperature for 3 hours. Cells were washed once with Hepes-BSA binding buffer and the¹²⁵I-IL8 bound by measurement of gamma radiation counts. Nonspecific binding was measured in the presence of a concentration of 1 pg / ml of unlabeled IL8. Data are the mean of duplicate determinations.

The antiserum against peptide no. 1 of IL8R1, but not the preimmunization antiserum, blocked the binding of¹²⁵I-IL8 to IL8R1 receptors expressed in SF9 cells infected with the recombinant baculovirus (Figure 3). The antiserum against peptide no. 1 of IL8R1 did not inhibit the binding of IL8 to IL8R2 (Figure 5). Therefore this antiserum specifically neutralized the binding of IL8 to IL8R1. The epitope present on IL8R1 recognized by anti-peptide no. 1 of IL8R1 is close to, or overlaps with, the recognition site for IL8 present in IL8R1.

The ability of anti-peptide no. 1 of IL8R1 to neutralize the binding of IL8 to its receptor was abrogated by an excess of peptide no. 1 of IL8R1, confirming the specificity of the antiserum (Figure 4). Therefore, the epitope (s) responsible for the neutralization of IL8 binding are contained within the corresponding region of IL8R1, amino acid residues 1-15 of the extracellular amino terminal domain. These results suggest that the extracellular amino terminal domain of IL8R1 is involved in IL8 recognition.

The antiserum against the pooled material of peptide nos. IL8R2 10-13, but not the preimmunization antiserum, blocked the binding of¹²⁵I-IL8 to IL8R2 receptors expressed in Sf9 cells infected with the recombinant baculovirus. The antiserum against peptides nos. IL8R2 10-13 did not inhibit the binding of IL8 to IL8R1 (Figure 3). Thus, this antiserum specifically neutralized the binding of IL8 to IL8R2 and recognized the epitope (s) close to, or overlapping with, the IL8 recognition sequence of IL8R2.

The antiserum against peptides nos. 10-13 was fractionated by affinity chromatography in the presence of peptide no. 10. Antibodies against peptide no. 10 of IL8R2 inhibited the binding of IL8 to its IL8R2 receptors expressed on Sf9 cells (Figure 6). Peptide no. Free 10 blocked neutralization by this antibody and confirmed that peptide no. 10 includes an epitope (s) located at, or close to, the recognition site for IL8 present on IL8R2 (Figure 6). This epitope corresponds to a neutralizing epitope (s) contained in the region of amino acid residues 1-15 of IL8R2. Depletion of anti-peptide # 10 antibodies did not abolish the

ES 2 302 774 T3 ability of anti-IL8R2 peptide antiserum to inhibit binding of IL8 to IL8R2 (data not shown). This indicates that there is an additional epitope (s) contained in the remaining part of the extracellular amino terminal region of IL8R2, amino acid residues 16-41, which also contributes to the neutralizing activity of the anti-IL8R2 peptide antiserum. These results suggest that amino acids 1-41 of the IL8R2 extracellular amino terminal domain include sequences involved in IL8 recognition.

Thus, new IL8 inhibitors are described, as well as methods for the use thereof. Although the preferred embodiments of the present invention have been described in some detail, it is understood that obvious modifications can be made within the scope of the appended claims.