WO1997000893A1 - Interleukin-8 receptor binding compounds - Google Patents

Abstract

The present invention presents sequences of polypeptides capable of modulating IL8 receptor binding and IL8 receptor-mediated biological response. Also, polynucleotides encoding the instant polypeptides and methods of producing the polypeptides are also described.

Description

Interleukin-8 Receptor Binding Compounds Inventors

Mary Ellen Wernette-Hammond, Ventakrishna Shyamala, Michael Siani, Jeff Blaney, and Patricia Tekamp-Olson.

Description

Technical Field

The invention relates generally to compounds capable of binding to IL8R1. More specifically, the compounds comprise a hydrophobic pocket capable of interacting with IL8 receptor 1 or receptor 2 (BL8R1 or IL8R2). Additionally, the invention relates to methods of screening for candidates that comprise a hydrophobic pocket and capable of binding to either EL8R1 or IL8R2. The IL8 receptor binding compounds of the present invention can be used to screen for candidates capable of binding to the IL8 hydrophobic pocket to inhibit IL8 receptor binding. Background of the Invention

Cells utilize diffusible mediators, called cytokines, to signal one another. A superfamily of cytokines are the chemokines, which includes EL8. A review article of the chemokine superfamily was written by Miller et al., Crit Rev Immun 12(1,2): 17-46 (1992) and by Baggiolini et al. Adv Immunol 55: 97-179 (1994), herein incorporated by reference.

Native human IL8 acts as a chemoattractant for neutrophils, and induces granulocytosis upon systemic injection and skin reaction upon local injection in

experimental animals. See Bazzoni et al., (1991) 173: 771-774; Van Damme et al., J Exp Med 167: 1364-1376; Ribero et al., Immunology 73: 472-477 (1991). The molecule also activates the release of superoxide anions and elicits release of primary granule constituents of neutrophils, including myeloperoxidase, β-glucuronidase, and elastase. Native human IL8 mediates these biological activities by binding to its receptor and triggering

transduction, a cascade of reactions ultimately resulting in a biological response.

Presently, two IL8 binding receptors have been identified and are termed "IL8R1' and "IL8R2." The amino acid sequence of these polypeptides are described in Murphy et al, Science 253: 1280 (1991) and Holmes et cd., Science 253: 1278 (1991), herein incorporated by reference. Other proteins can compete with IL8 to bind to IL8R2, such as GROα, GROβ, and GROγ. NAP-2 AND ENA-78 have been implicated with EL8R2 binding by cross-desensitization experiments with native IL8 by measuring Ca²⁺.

These other proteins which can compete for EL8 binding are members of the chemokine family. The chemokines are a group of structurally and functionally related cytokines. Recent studies indicated that these proteins function in the recruitment and activation of leukocytes and other cells at sites of inflammation and, therefore, appear to be important inflammatory mediators. Structurally, these molecules exhibit common secondary protein structure and display four conserved cysteine residues. The common secondary structures of a chemokine include: (1) an amino terminal loop; (2) a three- stranded antiparallel β sheet in the form of a Greek key; and (3) an C-terminal α helix. Because a systematic nomenclature for these proteins has not yet been generally agreed upon, the proteins can be divided into two families according to the spacing of the first two cysteine residues of the mature proteins. The families are referred to as the CXC and CC family. The first two cysteine residues of the CXC family members are separated by an amino acid residue. For the CC family, the cysteines are not separated. To date, seventeen chemokines have been described. Six are members of the CXC family and include, platelet factor 4 ("PF4"); β-thromboglobulin ("βTG"); NAP-1/TL8; gro α, β, and γ; IP-10; mig; and ENA-78. The CXC family is also known as the α family. The remaining chemokines are part of the CC family: macrophage inflammatory proteins ("MlP-1α" and " MIP-1β"); monocyte chemoattractant ρrotein-1/JE ("MCP-1 "/"JE") RANTES; HC-14; C10; and I- 309. This family has also been designated as the β family.

Researchers have identified regions of native human IL8 that are implicated in both DL8R1 and IL8R2 binding. However, at this time, no chemokine is known to compete with native IL8 for IL8R1 binding. Disclosure of the Invention

It is one of the objects of the present invention is to provide peptides comprising at least 5 hydrophobic residues, wherein the peptide can exhibit a conformation permitting the docking of a phenyl ring.

Another object of the invention is to provide polypeptides exhibiting a chemokine protein structure capable of IL8R1 or IL8R2 binding.

It is another object of the invention to provide a method of inhibiting IL8R1 signal transduction by contacting an effective concentration of the instant IL8R1 compounds comprising a hydrophobic pocket to compete with IL8 for IL8R1 binding.

Yet another object of the invention is a method of inhibiting receptor binding of native IL8 comprising:

(a) providing the peptides or polypeptides of the instant invention; and

(b) contacting the receptor with an effective inhibiting amount of the polypeptide.

Also, another object of the invention is a method of modulating an IL8 receptor-mediated biological response comprising:

(a) providing the peptides or polypeptides of the instant invention; and

(b) contacting a cell that produces an IL8 receptor with an effective modulating amount of the polypeptide.

Also, it is an object of the invention to screen for IL8 antagonists by determining which compounds bind to the IL8 receptor binding compounds comprising a hydrophobic pocket.

Modes of Carrying Out The Invention

A. Definitions

A "native IL8 polypeptide" refers to a polypeptide which is identical to a sequence recovered from a source which naturally produces IL8, such as human, bovine, porcine or other mammalian sources. Native EL8 may be vary in length from species to species. An example of native IL8 is the native human IL8 which has the amino acid sequence shown in SEQ ID NO: 1.

"Hydrophobic amino acids" comprise R-groups that have a tendency to shun an aqueous environment. These radical groups are generally non-polar, and polar molecules, like water, will preferentially form hydrogen bonds with other polar molecules rather than the non-polar R groups. Hydrophobic amino acids include alanine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, and valine.

Native IL8 comprises a "hydrophobic pocket," a group of hydrophobic residues. Atypically, this group of residues is not positioned in the interior of the protein shielded from an aqueous environment, but exposed on the surface of the protein. The size and shape of the pocket can accommodate a phenyl ring. The phenyl ring can dock in the pocket just as a coin slips in a coin slot. This pocket is not implicated in IL8 dimerization. More specifically, the hydrophobic pocket of native human IL8 comprises of the following amino acids: tyr13, phe17, ile22, val41, leu43, leu51, and leu49. The entrance to the pocket of native human IL8 is surrounded by tyr13, lys15, phe21, and arg47.

Peptides and polypeptides of the instant invention comprises a hydrophobic region surface exposed in contact with an aqueous environment. The hydrophobic residues of the hydrophobic region do not need to be contiguous the primary sequence. The peptides and polypeptides of the instant invention will typically comprise 5 hydrophobic residues; more typically 4 to 7 residues; even more typically, 4 to 11 hydrophobic residues. The hydrophobic region of the instant peptides and polypeptides mimic the IL8 hydrophobic pocket by exhibiting a similar ability to permit a phenyl ring to dock in the hydrophobic region. This ability can be measured using TL8 receptor binding, signal transductions, and biological assays, described supra. The instant peptides and

polypeptides exhibit altered IL8 receptor binding characteristics as compared to native chemokines, native human IL8 and GROα, as examples.

NMR and X-ray crystallography experiments revealed that the three dimensional structure of the chemokines is similar, herein referred to as the "chemokine protein structure." The structure of the native human IL8 has been solved and is a model for the chemokine protein structure. The structure includes an amino-terminal loop, a three-stranded antiparallel β sheet (Greek key), and a carboxy-terminal α helix.

By "modulating an IL8 receptor-mediated biological response" is meant either increasing or decreasing the incidence of one or more cellular activities normally triggered by the binding of IL8 to its receptor. The nature of these activities may be biochemical or biophysical. For example, a substance would "modulate an IL8 receptor- mediated biological response" if it does not stimulate the same signal transduction activity as IL8 when the polypeptides of the instant invention bind to an IL8 receptor.

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 biological response" when it causes an increase or decrease in any one of these reactions. Other biological activities attributable to IL8 which can be measured in order to determine modulation include, for example, neutrophil chemotactic activity, measured using assays described in Schroder et al., J Immunol. 139: 3474-3483 (1987). Also, IL8 has been implicated in rapid mobilization of hematopoietic stem cells (Laterveer, et al. Blood 85: 8 2269-2275 (1995)) and implicated signal transduction in T-lymphocytes (Bacon et al., J Immunology 154: 3654-3666 (1995)).

An "effective inhibiting amount" of the polypeptides of the instant invention refers to an amount sufficient to block the binding, in whole or in part, of native IL8 to an IL8 receptor. Typically, an effective amount inhibits at least 20% of the native IL8 receptor binding. More typically, the polypeptides inhibit at least 40%, even more typically the polypeptides inhibit at least 60% of the native IL8 receptor binding; most preferably at least 70%.

The term "effective modulating amount" of a polypeptide of the instant invention refers to an amount sufficient to cause a change in an IL8 receptor-mediated biological activity, as described above. Typically, an effective amount causes a change at least 20% compared to the response to native IL8 receptor-mediated biological response. More typically, the polypeptides cause a change of least 40%, even more typically at least 60% of the native IL8 receptor binding; most preferably at least 100%. B. General Method

The peptides of the instant invention comprise 5 hydrophobic residues separated by four spacers. The following formula depicts the peptides of the instant invention from amino to carboxyl terminus:

- S₁ - H₁ - S₂ - H₂ - S₃ - H₃ - S₄ - H₄ - S₅ - H₅- S₆ - wherein

H₁ can be any hydrophobic amino acid; preferably, H₁ is leucine, methionine, phenylalanine, threonine, serine, histidine, alanine, tyrosine or isoleucine; most preferably, H₁ is tyrosine or leucine;

H₂ can be aspartic acid as well as any hydrophobic amino acid; preferably,

H₂ is phenylalanine, isoleucine, valine, aspartic acid, or leucine; most preferably, H₂ is phenylalanine;

H₃ can be any hydrophobic amino acid; preferably, H₃ is isoleucine, leucine, or valine; most preferably, H₃ isoleucine;

H₄ can be any hydrophobic amino acid; preferably H₄ is leucine or methionine; most preferably H₄ leucine;

H₅ can be arginine as well as any hydrophobic amino acid; preferably, H₅ is leucine, alanine, isoleucine, or arginine; most preferably H₅ is leucine.

Further, the spacers comprise:

S₁ can be any length from 0 to 20 amino acid residues; preferably, S₁ comprises glutamine-leucine-arginine- at or near the amino terminus of S₁; most preferably, S₁ is 9-18 amino acid residues in length and comprises glutamine-leucine-arginine- at the amino terminus of S₁;

S₂ is preferably from 1 to 5 amino acid residues in length; more preferably, 2 to 3 amino acid residues in length; even more preferably, S₂ is serine-lysine-proline;

S₃ is preferably from 1 to 6 amino acid residues in length; more preferably, 2 to 4 residues in length; even more preferably, S₃ is histidine-proline-lysine-phenylalanine;

S₄ preferably is from 0 to 24 amino acid residues in length; more preferably 0 to 2 amino acid residues in length;

if S₄ is greater than 0 then S₄ comprises a hydrophobic amino acid residue; more preferably, the hydrophobic residue is valine, alanine, or isoleucine; even more preferably, valine, wherein the hydrophobic residue is at or near the carboxyl terminus of S₄,

S₅ preferably is preferably from 3 to 8 amino acid residues in length;

more preferably 5 to 6 amino acid residues; even more preferably S₅ is serine-aspartic acid- glycine-arginine-glutamic acid;

S₆ is preferably from 0 to 30 amino acid residues in length; more preferably 0 to 4 amino acid residues, wherein if S₆ is greater than 0, S₆ comprises a hydrophobic residue; preferably the hydrophobic residue is leucine. Preferably, leucine is positioned near the amino-terminus of S₆, more preferably, leucine is at the first or second position of S₆; even more preferably, at the second position.

Polypeptides of the instant invention exhibiting a chemokine protein structure comprise of one of the following three domains.

Domain 1 (D₁) comprises amino acids 1-18 of native human IL8; preferably amino acids 4-18 of native human IL8; more preferably amino acids 4-17 of native human IL8; even more preferably, Glu-Leu-Arg-Cys-Xaa-Cys-Ile-Lys-Thr-Xaa₁-Xaa-Lys-Xaa- Xaa₂, wherein Xaa₁ and Xaa₂ are hydrophobic residues.

Domain 2 (D₂) comprises amino acids 18-32 of native human IL8, preferably, His-Pro-Lys-Phe-Ile-Lys-Glu-Leu-Arg-Val-Ue-Glu-Ser-Gly-Pro; even more preferably, His-Pro-Lys-Xaa-Ile-Xaa-Xaa-Xaa-Xaa-Val-Xaa-Xaa-Xaa-Gly-Pro.

Domain 3 (D₃) comprises amino acids 41-53 of native human IL8;

preferably, Val-Lys-Leu-Ser-Asp-Gly-Arg-Glu-Leu-Cys-Leu- Asp-Pro; even more preferably, Xaa₃-Xaa-Xaa₄-Xaa-Xaa-Gly-Xaa-Xaa-Leu-Cys-Leu-Xaa-Pro, wherein Xaa₃ and Xaa₄ are hydrophobic residues, most preferably Xaa₃ is valine and Xaa₄ is leucine; most preferably D₃ is Gly-Xaa-Xaa-Leu-Cys-Leu-Xaa-Pro.

Preferably, if the polypeptide of the instant invention comprises D₁ and D₂,

D₁ is amino terminal to D₂. Further, D₃ is carboxy terminal to D₂, if the polypeptide of the instant invention comprises D₂ and D₃. In addition, D₃ is carboxy of D₁, if the polypeptide of the instant invention comprises D₁ and D₃.

The polypeptides of the present invention exhibiting a chemokine protein structure comprise four conserved cysteine residues which are positioned to align with the cysteine residues of other chemokine superfamily members. An example of an alignment of chemokines is shown in Miller et al., Crit Rev Immun 12(1,2): 17-46 (1992). The cysteines form disulfide bounds that aid the formation of a chemokine protein structure. A polypeptide of the present invention exhibiting a chemokine protein comprises an amino terminal portion, which includes a loop; a three-stranded β sheet in the form of a Greek key; and a C-terminal α-helix.

The amino terminal portion contains a tail which retains no particular structure and a loop. Preferably, amino acid sequence of Domain 1 is incorporated in the amino terminal portion. The entire portion including the tail and loop is from about 25 to 14 amino acid residues; more preferably, from about 22 to about 18 amino acid residues. The loop comprises from about 15 to about 6 amino acid residues; more preferably, about 12 to about 8 residues.

The three stranded β sheet of the polypeptides of the instant invention is preferably of similar size to those found in chemokines. For example, the strands of the β sheet are about 12 to 3 amino acid residues in length; more preferably, from about 10 to 3 residues; even more preferably, 7 to 3 amino acid residues; even more preferably, 6 to 8 amino acid residues. Domain 2 and 3 are preferably incorporated into the β sheet.

The length of the C-terminal α helix of the polypeptides of the present invention is not critical. Usually, the length of the α helix is from about 9 to 25 residues; more usually, from about 12 to 22 residues; even more usually 15 to about 19 residues. Typically, the α helix is amphipathic helix that may be positively or negatively charged. Most chemokine helices are positively charged. The charge of the helix can be chosen depending if similar or dissimilar biological activity is desired.

Constructing a chimeric chemokine is one means of constructing a polypeptide of present invention. The domains can be spaced using fragments from other chemokines that are homologous to those regions in native human IL8 which space the domains. For example, the C-terminal helix of GROγ can be substituted into the native human IL8 polypeptide. In such an embodiment, the polypeptide can exhibit non-native IL8 biological activity due to the presence of GROγ α helix.

In addition, mutants, fragments, and fusions of the amino acid sequences of chemokines can be assembled together to construct a polypeptide of the present invention.

For example, a polypeptide of the present invention may comprise the amino terminal portion and one strand of the β sheet of native human IL8, two strands of the β sheet of NAP-2 and the α helix of the GROγ. The amino acid sequences to be utilized as spacers to construct a chemokine protein structure need not be identical to the sequences found in native chemokines to exhibit the desired secondary structure. For example, the amino acid sequences may be mutants, fragments, or fusions.

Specific chimeric chemokines include G18I32G, I46G53I, and I18G46I53G. The G in the specific chimeras refer to GROγ sequence and the I refers to native human IL8 sequence. G18I32G comprises of the amino terminus of GROγ to the homologous position 17 of native IL8 immediately adjacent position 18-32 of native human IL8 and C- terminal fragment of Groγ, wherein the amino terminal amino acid of the Groγ fragment is homologous to position 33 of native human IL8. All the other chimeric chemokines can be deciphered in the same way. The numbers refer either the native human IL8 position or the homologous position on Groγ. Constructing the Desired Polypeptides

The polypeptides can be produced by synthesizing the desired amino acid sequence and refolding the polypeptide. See, for example, Clark-Lewis et al., J Biol Chem 269(23): 16075-1081 (1994). Alternatively, the coding sequences of the desired amino acid sequence can be synthesized. See Urdea et al, Proc. Natl. Acad. Sci. USA 80: 7461 (1983).

Production of IL8R1 Binding Compounds by Host Cells

At the minimum, an expression vector will contain a promoter which is operable in the host cell and operably linked to the desired coding sequence. Expression vectors may also include signal sequences, terminators, selectable markers, origins of replication, and sequences homologous to host cell sequences. These additional elements are optional but can be included to optimize expression.

A promoter is a DNA sequence upstream or 5' to the desired coding sequence to be expressed. The promoter will initiate and regulate expression of the coding sequence in the desired host cell. To initiate expression, promoter sequences bind RNA polymerase and initiate the downstream (3') transcription of a coding sequence {e.g. structural gene) into mRNA A promoter may also have DNA sequences that regulate the rate of expression by enhancing or specifically inducing or repressing transcription. These sequences can overlap the sequences that initiate expression. Most host cell systems include regulatory sequences within the promoter sequences. For example, when a repressor protein binds to the lac operon, an E. coli regulatory promoter sequence, transcription of the downstream gene is inhibited. Another example is the yeast alcohol dehydrogenase promoter, which has an upstream activator sequence (UAS) that modulates expression in the absence of a readily available source of glucose. Additionally, some viral enhancers not only amplify but also regulate expression in mammalian cells. These enhancers can be incorporated into mammalian promoter sequences, and the promoter will become active only in the presence of an inducer, such as a hormone or enzyme substrate (Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237).

Functional non-natural promoters may also be used, for example, synthetic promoters based on a consensus sequence of different promoters. Also, effective promoters can contain a regulatory region linked with a heterologous expression initiation region. Examples of hybrid promoters are the E. coli lac operator linked to the E. coli tac transcription activation region; the yeast alcohol dehydrogenase (ADH) regulatory sequence linked to the yeast g.yceraldehyde-3-phosphate-dehydrogenase (GAPDH) transcription activation region (U.S. Patent Nos. 4,876,197 and 4,880,734, incorporated herein by reference); and the cytomegalovirus (CMV) enhancer linked to the SV40 (simian virus) promoter.

The desired coding sequence may also be linked in reading frame to a signal sequence. The signal sequence fragment typically encodes a peptide comprised of hydrophobic amino acids which directs the desired polypeptide to the cell membrane.

Preferably, there are processing sites encoded between the leader fragment and the gene or fragment thereof that can be cleaved either in vivo or in vitro. DNA encoding suitable signal sequences can be derived from genes for secreted endogenous host cell proteins, such as the yeast invertase gene (EP 12 873; JP 62,096,086), the A-factor gene (U.S. Patent No. 4,588,684), interferon signal sequence (EP 60 057). A preferred class of secretion leaders, for yeast expression, are those that employ a fragment of the yeast alpha-factor gene, which contains both a "pre" signal sequence, and a "pro" region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (typically about 25 to about 50 amino acid residues) (U.S. Patent Nos. 4,546,083 and 4,870,008, incorporated herein by reference; EP 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast signal sequence, but a pro-region from a second yeast alpha-factor. (See e.g., PCT WO

89/02463.)

Typically, terminators are regulatory sequences, such as polyadenylation and transcription termination sequences, located 3' or downstream of the stop codon of the coding sequences. Usually, the terminator of native host cell proteins are operable when attached 3' of the desired coding sequences. Examples are the Saccharomyces cerevisiae alpha-factor terminator and the baculovirus terminator. Further, viral terminators are also operable in certain host cells; for instance, the SV40 terminator is functional in CHO cells.

For convenience, selectable markers, an origin of replication, and homologous host cells sequences may optionally be included in an expression vector. A selectable marker can be used to screen for host cells that potentially contain the expression vector. Such markers may render the host cell immune to drugs such as ampicillin, chloramphenicol, erythromycin, neomycin, and tetracycline. Also, markers may be biosynthetic genes, such as those in the histidine, tryptophan, and leucine pathways. Thus, when leucine is absent from the media, for example, only the cells with a biosynthetic gene in the leucine pathway will survive.

An origin of replication may be needed for the expression vector to replicate in the host cell. Certain origins of replication enable an expression vector to be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the 2μ and autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells. Expression vectors may be integrated into the host cell genome or remain autonomous within the cell. Polynucleotide sequences homologous to sequences within the host cell genome may be needed to integrate the expression cassette. The homologous sequences do not always need to be linked to the expression vector to be effective. For example, expression vectors can integrate into the CHO genome via an unattached dihydrofolate reductase gene. In yeast, it is more advantageous if the homologous sequences flank the expression cassette. Particularly useful homologous yeast genome sequences are those disclosed in PCT WO90/01800, and the HIS4 gene sequences, described in Genbank, accession no. J01331.

The choice of promoter, terminator, and other optional elements of an expression vector will also depend on the host cell chosen. The invention is not dependent on the host cell selected. Convenience and the level of protein expression will dictate the optimal host cell. A variety of hosts for expression are known in the art and available from the American Type Culture Collection (ATCC). Bacterial hosts suitable for expressing the desired polypeptide include, without limitation: Campy lobacter, Bacillus, Escherichia, Lactobacillus, Pseudomonas, Staphylococcus, and Streptococcus. Yeast hosts from the following genera may be utilized: Candida, Hansenula, Kluyveromyces, Pichia,

Saccharomyces, Schizosaccharomyces, and Yarrowia. Immortalized mammalian host cells include but are not limited to CHO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and other cell lines. A number of insect cell hosts are also available for expression of heterologous proteins: Aedes aegypti, Bombyx mori, Drosophila melanogaster, and Spodopterafrugiperda (PCT WO 89/046699; Carbonell et al, (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al, (1983 ) Mol. Cell. Biol. 3:2156: and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).

Transformation

After vector construction, the desired polypeptide expression vector is inserted into the host cell. Many transformation techniques exist for inserting expression vectors into bacterial, yeast, insect, and mammalian cells. The transformation procedure to introduce the expression vector depends upon the host to be transformed. Methods of introducing exogenous DNA into bacterial hosts are well- known in the art, and typically protocol includes either treating the bacteria with CaCl₂ or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation or viral infection. Transformation procedures usually vary with the bacterial species to be transformed. See e.g., (Masson et al. (1989) FEMS

Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP Publ. Nos. 036 259 and 063 953; PCT WO 84/04541, Bacillus), (Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter), (Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al (1988) Nucleic Acids Res.

16 : 6127; Kushner (1978) "An improved method for transformation of Escherichia coli with ColEl -derived plasmids in Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H.W. Boyer and S. Nicosia); Mandel et al.

(1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophvs. Acta 949:318; Escherichia), (Chassy et al. (1987) FEMS Microbiol. Lett. 44: 173 Lactobacillus); (Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas); (Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus), (Barany et al (1980) J. Bacteriol. 144:698; Harlander (1987) "Transformation of Streptococcus lactis by electroporation," in Streptococcal Genetics (ed. J. Ferretti and R Curtiss III); Perry et al. (1981) Infec. Immun. 32: 1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Eyr. Cong. Biotechnology 1:412, Streptococcus).

Transformation methods for yeast hosts are well-known in the art, and typically include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Electroporation is another means for transforming yeast hosts. See for example, Methods in Enzymology. Volume 194, 1991, "Guide to Yeast Genetics and Molecular Biology. " Transformation procedures usually vary with the yeast species to be transformed. See e.g., (Kurtz et al. (1986) Mol. Cell. Biol. 6: 142; Kunze et al. (1985) J. Basic Microbiol. 25:141: Candida); (Gleeson et al. (1986) J. Gen. Microbiol. 132:3459: Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula); (Das et al. (1984) J. Bacteriol. 158: 1165; De Louvencourt et al. (1983) J. Bacteriol. 154: 1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces); (Cregg et al. (1985) Mol. Cell. Biol.

5:3376; Kunze et al (1985) J. Basic Microbiol. 25:141: U.S. Patent Nos. 4,837,148 and 4,929,555; Pichia); (Hinnen et al (1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et al. (1983) J. Bacteriol. 153: 163 Saccharomyces); (Beach and Nurse (1981) Nature 300:706: Schizosaccharomyces); (Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia).

Methods for introducing heterologous polynucleotides into mammalian cells are known in the art and include viral infection, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

The method for construction of an expression vector for transformation of insect cells for expression of recombinant herein is slightly different than that generally applicable to the construction of a bacterial expression vector, a yeast expression vector, or a mammalian expression vector. In an embodiment of the present invention, a baculovirus vector is constructed in accordance with techniques that are known in the art, for example, as described in Kitts et al, BioTechniques 14: 810-817 (1993), Smith et al, Mol. Cell. Biol. 3: 2156 (1983), and Luckow and Summer, Virol. 17: 31 (1989). In one embodiment of the present invention, a baculovirus expression vector is constructed substantially in accordance to Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Moreover, materials and methods for baculovirus/insect cell expression systems are commercially available in kit form, for example, the MaxBac® kit from

Invitrogen (San Diego, CA).

Also, methods for introducing heterologous DNA into an insect host cell are known in the art. For example, an insect cell can be infected with a virus containing the desired coding sequence. When the virus is replicating in the infected cell, the desired polypeptide will be expressed if operably linked to a suitable promoter. A variety of suitable insect cells and viruses are known and include following without limitation.

Insect cells from any order of the Class Insecta can be grown in the media of this invention. The orders Diptera and Lepidoptera are preferred. Example of insect species are listed in Weiss et al, "Cell Culture Methods for Large-Scale Propagation of Baculoviruses," in Granados et al. (eds.), The Biology of Baculoviruses: Vol. II Practical

Application for Insect Control, pp. 63-87 at p. 64 (1987). Insect cell lines derived from the following insects are exemplary: Carpocapsa pomeonella (preferably, cell line CP-128); Trichoplusia ni (preferably, cell line TN-368); Autograph calif ornica; Spodoptera frugiperda (preferably, cell line Sf9); Lymantria dispar; Mamestra brassicae; Aedes albopictus; Orgyia pseudotsugata; Neodiprio sertifer; Aedes aegypti; Antheraea eucalypti; Gnorimoschema operceullela; Galleria mellonella; Spodoptera littolaris; Blatella germanic; Drosophila melanogaster; Heliothis zea; Spodoptera exigua; Rachiplusia ou; Plodia interpunctella; Amsaeta moorei; Agrotis c-nigrum, Adoxophyes orana; Agrotis segetum; Bombyx mori; Hyponomeuta malinellu;, Colias eurytheme; Anticarsia germmetalia; Apanteles melanoscelu; Arctia caja; and Porthetria dispar. Preferred insect cell lines are from Spodopterafrugiperda, and especially preferred is cell line Sf9. The Sf9 cell line used in the examples herein was obtained from Max D. Summers (Texas A & M University, College Station, Texas, 77843, U.S.A.) Other S. frugiperda cell lines, such as IPL-Sf-21AE III, are described in Vaughn et al, In Vitro 13: 213-217 (1977).

The insect cell lines of this invention are suitable for the reproduction of numerous insect-pathogenic viruses such as parvoviruses, pox viruses, baculoviruses and rhabdcoviruses, of which nucleopolyhedrosis viruses (NPV) and granulosis viruses (GV) from the group of baculoviruses are preferred. Further preferred are NPV viruses such as those from Autographa spp., Spodoptera spp., Trichoplusia spp., Rachiplusia spp., Gallerai spp., and Lymantria spp. More preferred are baculovirus strain Autographa calif ornica NPV (AcNPV), Rachiplusia ou NPV, Galleria mellonella NPV, and any plaque purified strains of AcNPV, such as E2, R9, S1, M3, characterized and described by Smith et al, J Virol 30: 828-838 (1979); Smith et al, J Virol 33: 311-319 (1980); and Smith et al, Virol 89: 517-527 (1978).

Typically, insect cells Spodopterafrugiperda type 9 (SF9) are infected with baculovirus strain Autographa calif ornica NPV (AcNPV) containing the desired coding sequence. Such a baculovirus is produced by homologous recombination between a transfer vector containing the coding sequence and baculovirus sequences and a genomic baculovirus DNA. Preferably, the genomic baculovirus DNA is linearized and contains a dysfunctional essential gene. The transfer vector, preferably, contains the nucleotide sequences needed to restore the dysfunctional gene and a baculovirus polyhedrin promoter and terminator operably linked to the desired coding sequence. (See Kitts et al,

BioTechniques 14(5): 810-817 (1993).

The transfer vector and linearized baculovirus genome are transfected into SF9 insect cells, and the resulting viruses probably containing the desired coding sequence. Without a functional essential gene the baculovirus genome cannot produce a viable virus. Thus, the viable viruses from the transfection most likely contain the desired coding sequence and the needed essential gene sequences from the transfer vector. Further, lack of occlusion bodies in the infected cells are another verification that the desired coding sequence was incorporated into the baculovirus genome.

The essential gene and the polyhedrin gene flank each other in the baculovirus genome. The coding sequence in the transfer vector is flanked at its 5' with the essential gene sequences and the polyhedrin promoter and at its 3' with the polyhedrin terminator. Thus, when the desired recombination event occurs the desired coding sequence displaces the baculovirus polyhedrin gene. Such baculoviruses without a polyhedrin gene will not produce occlusion bodies in the infected cells. Of course, another means for determining if coding sequence was incorporated into the baculovirus genome is to sequence the recombinant baculovirus genomic DNA. Alternatively, expression of the desired polypeptide by cells infected with the recombinant baculovirus is another verification means.

Assays for Determining the IL8R1 Binding

Receptor binding assays herein may utilize cells that naturally produce the IL8R1 or R2 receptor, such as human neutrophils. Alternatively, a polynucleotide encoding a native IL8R1 or IL8R2 can be introduced into a cell to produce a IL8R1 or IL8R2. For the assay, either whole cells or membranes can be used to determine receptor binding. Typically, the assay for receptor binding is performed by determining if the present polypeptide can compete with radioactive, native IL8 or IL8 receptor binding compounds for binding to IL8 receptors. The less radioactivity measured the less native IL8 bound to the receptor. See Herbert et al., J Biol Chem 266(28): 18989-18994 (1991) for examples of receptor binding assays. The IL8 receptor binding can also be measured utilizing signal transduction assays. The IL8 receptor binding compounds which inhibit IL8 activity can compete with native IL8 to modulate signal transduction. Typical signal transduction assays measure Ca2⁺, IP₃, and DAG levels as described herein.

Most cellular Ca2⁺ ions are sequestered in the mitochondria, endoplasmic reticulum, and other cytoplasmic vesicles, but binding of IL8 to IL8R1 will trigger the increase of free Ca²⁺ ions in the cytoplasm. With fluorescent dyes, such as fura-2, the concentration of free Ca²⁺ can be monitored. The ester of fura-2 is added to the media of the host cells expressing IL8 receptor polypeptides. The ester of fura-2 is lipophilic and diffuses across the membrane. Once inside the cell, the fura-2 ester is hydrolyzed by cytosolic esterases to its non-lipophilic form, and then the dye cannot diffuse back out of the cell. The non-lipophilic form of fura-2 will fluoresce when it binds to the free Ca²⁺ ions, which are released after binding of a ligand to IL8 receptor. The fluorescence can be measured without lysing the cells at an excitation spectrum of 340 nm or 380 nm and at fluorescence spectrum of 500 nm.

The rise of free cytosolic Ca²⁺ concentrations is preceded by the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Hydrolysis of this phospholipid by the plasma- membrane enzyme phospholipase C yields 1,2-diacylglycerol (DAG), which remains in the membrane, and the water-soluble inositol 1,4,5-triphosphate (EP3). Binding of IL8 or IL8 agonists will increase the concentration of DAG and IP3. Thus, signal transduction activity can be measured by monitoring the concentration of these hydrolysis products.

To measure the IP₃ concentrations, radioactively labelled ³H-inositol is added to the media of host cells expressing IL8 receptor polypeptides. The ³H-inositol taken up by the cells, and after stimulation of the cells with IL8, the resulting inositol triphosphate is separated from the mono and di-phosphate forms and measured.

Alternatively, Amersham provides an inositol 1,4,5-triphosphate assay system. With this system Amersham provides tritylated inositol 1,4,5-triphosphate and a receptor capable of distinguishing the radioactive inositol from other inositol phosphates. With these reagents an effective and accurate competition assay can be performed to determine the inositol triphosphate levels. Screening for Candidates Comprising a Hydrophobic Pocket Capable of IL8R1 Binding TL8R1 binding compounds comprising a hydrophobic pocket can also be used to screen peptide libraries to find other IL8R1 binding compounds. Other binding compounds can be constructed based on the dimensions of the hydrophobic pocket of native human EL8. NMR and X-ray crystallography experiments have been reported the conformation and dimension of native human IL8. See, for example, Clore et al., J Mol Bio 217: 611-620 (1991). A library of compounds can be screened in a competition assay for IL8R1 binding utilizing already constructed IL8R1 binding compounds comprising a hydrophobic pocket.

A "library" of peptides may be synthesized following the methods disclosed in U.S. Pat. No. 5,010,175, and in PCT WO91/17823, both incorporated herein by reference in full. Briefly, one prepares a mixture of peptides, which is then screened to determine the peptides exhibiting the desired signal transduction and receptor binding activity. In the '175 method, a suitable peptide synthesis support (e.g., a resin) is coupled to a mixture of appropriately protected, activated amino acids. The concentration of each amino acid in the reaction mixture is balanced or adjusted in inverse proportion to its coupling reaction rate so that the product is an equimolar mixture of amino acids coupled to the starting resin. The bound amino acids are then deprotected, and reacted with another balanced amino acid mixture to form an equimolar mixture of all possible dipep- tides. This process is repeated until a mixture of peptides of the desired length (e.g., hexa- mers) is formed. Note that one need not include all amino acids in each step: one may include only one or two amino acids in some steps (e.g., where it is known that a particular amino acid is essential in a given position), thus reducing the complexity of the mixture. After the synthesis of the peptide library is completed, the mixture of peptides is screened for binding to the selected IL8 receptor polypeptide. The peptides are then tested for their ability to compete with native IL8 for IL8 receptor binding. Peptides exhibiting the desired binding are then isolated and sequenced.

The method described in '17823 is similar. However, instead of reacting the synthesis resin with a mixture of activated amino acids, the resin is divided into twenty equal portions (or into a number of portions corresponding to the number of different amino acids to be added in that step), and each amino acid is coupled individually to its portion of resin. The resin portions are then combined, mixed, and again divided into a number of equal portions for reaction with the second amino acid. In this manner, each reaction may be easily driven to completion. Additionally, one may maintain separate "subpools" by treating portions in parallel, rather than combining all resins at each step. This simplifies the process of determining which peptides are responsible for any observed receptor binding or signal transduction activity.

In such cases, the subpools containing, e.g., 1-2,000 candidates each are exposed to IL8 receptor. Each subpool that produces a positive result is then resynthesized as a group of smaller subpools (sub-subpools) containing, e.g., 20-100 candidates, and reassayed. Positive sub-subpools may be resynthesized as individual compounds, and assayed finally to determine the peptides, which exhibit a high binding constant. Then, these peptides can be tested for their ability to compete with native IL8 for IL8 receptor binding. The methods described in '17823 and U.S. Patent No. 5,194,392 (herein incorporated by reference) enable the preparation of such pools and subpools by automated techniques in parallel, such that all synthesis and resynthesis may be performed in a matter of days.

C. Examples

The examples presented below are proved as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.

(i) Materials for the following examples:

Recombinant human IL8, GROα (GRO/MGS A), and ¹²⁵I-IL8 were prepared as described in Hammond, M.E.W. et al., J. Immunol. 155: 1428-1433 (1995).

Recombinant GROβ (MIP2α) and GROγ (MIP-2β) were produced in yeast and purified by chromatography on heparin-Sepharose and Sephacryl S-100 HR.

(ii) Construction of Chimeric Chemokines-

DNA encoding chimeric IL8/GROγ were generated by recombinant Polymerase chain reaction ("PCR") and/or by ligation with synthetic oligonucleotides encoding the desired amino acid. The PCR was performed as described in Higuci et al., PCR Protocols (Innis et al., eds.), pages 177-183, Academic Press, Inc., San Diego, California, USA.

A synthetic IL8 gene, il8syn, and a MIP2β cDNA clone, MIP540 were used as intitial templates for IL8 GROγ sequences. H8syn was constructed as described in Miller et al., Protein Expression. Purif. 6: 357-362 (1995). The IL8 sequence is also described in Aschauer et al. , PCT WO89/04836 and Gregory et al., Proc. Natl. Acad. Sci. USA 85 : 9199-9203 (1988).

MIP540 was isolated according to Tekamp-Olson et al., J. Exp. Med. 172: 911-919 (1990); PCT WO92/00327; and PCT WO92/00326.

PCR primers and and synthetic oligonucleotides linkers are as follows below: Table I.

For G18I, sequences between the Asp718 and M1ul restriction sites were generated with oligonucleotides AccX, AM1uI, Xacc, and 1luX.

For I18G, sequences between Asp718 and Bg1ll were from oligonucleotides AccZ ZBg1ll, Zacc, and Bg1IIZ.

Chimeric DNA were used as PCR templates. G46I53G was used for template of

I18G46I53G. Constructs wre ligated, as Asp718/Sall fragments into a transfer vector containing the GAP promoter fused to the α-factor leader. The promoter/leader sequence is described in Brake et al., Methods Enzymol. 185: 408-421 (1990) and EP 324274. Protein Expression and Purification

Expression cassettes for yeast secretion were transferred as BamHI restriction fragments into vector pAB24 and introduced into Sacchormyces cerevisae strain MB2-1 by electroporation. Vector pAB24 is described Brake et al., supra.

Chimeric and mutant chemokines were purified from 50-200 mL of yeast culture broth by batch adsorption on S-Sepharose FF (Pharmacia Biotech Inc., Uppsala, Sweden) after adjustment to pH5.5 with 50 mM sodium acetate and eluted in 20 mM HEPES, pH 8.3 1 M NaCl to a final concentration of 0.2-2 mg.mL.

SDS-polyacrylamide gel electrophoresis on 18% Tris/glycine gels indicated 80-95% purity. Protein conentrations were estimated by Coomassie-stained polyacrylamide gels and by BCA protein assays. Amino acid composition and amino-terminal sequencing were perfomred on selected proteins and agreed with predicted protein sequences.

Binding Assays- Competitive binding assays for chimeric proteins were performed on CHO- IL8R1and CHO-IL8R2 essentially as described in Hammond et al, supra. Assays were performed in triplicate and data were analyzed by GraFit version 2.0, Erithacus Software, Ltd., Staines United Kingdom, written by Leatherbarrow.

Chemotaxis Assays

Assays were perfomred in triplicate on freshly isolated human neutrophils as described in Hammond et al, supra, chemotaxix to f-Met-Leu-Phe (100 nM) was measured as a postive control for each experiment.

Homology Modeling

A homology-based model of GROγ was built based on the NMR- solution-derived structro of IL8, described in Clore et al., Biochem 29: 1689-1696 (1990). The LOOK software program was used to align the GROγ and IL8 sequences. The LOOK program is available from Molecular Applications Group, Mountain View, California, USA. The methodology of the software program is also described in Needleman et al., J. Mol. Biol. 48: 443-453 (1970).

A three dimensional model of GROγ was constructed by the Levitt' s automatic segment matching and further refined by restrained energy minimization and molecular dynamics. These techniques are described in Levitt et al, J. Mol. Biol. 226: 507-533 (1992) and Levitt et al, J. Mol. Biol. 170: 723-764 (1983). Deposit Information

The following materials wre deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852:

S. cerevisae MB2-1 (ρYMIP540) deposited 20 June 1990, ATCC no. 74002.

These materials were deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-Organisms for Purposes of Patent

Procedure. These deposits are provided as a convenience to those of skill in the art, and do not represent an admission that a deposit is required under 35 USC Section 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the written description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

Claims

WHAT IS CLAIMED:

1. A polypeptide comprising an amino acid sequence of the following:

from amino to carboxyl terminus:

- S₁ - H₁ - S₂ - H₂ - S₃ - H₃ - S₄ - H₄ - S₅ - H₅- S₆ - wherein

H₁ is a hydrophobic amino acid;

H₂ is aspartic acid or a hydrophobic amino acid;

H₃ is a hydrophobic amino acid;

H₄ is a hydrophobic amino acid;

H₅ is arginine or a hydrophobic amino acid;

S₁ is any length from 0 to 20 amino acid residues;

5₂ is 1 to 5 amino acid residues in length;

5₃ is 1 to 6 amino acid residues in length;

S₄ is from 0 to 24 amino acid residues in length;

S₅ is preferably from 3 to 8 amino acid residues in length; and S₆ is preferably from 0 to 30 amino acid residues in length.

2. The polypeptide of Claim 1, wherein H₁ is selected from the group consisting of leucine, methionine, phenylalanine, threonine, serine, histidine, alanine, tyrosine and isoleucine.

3. The polypeptide of Claim 2, wherein H₁ is tyrosine or leucine.

4. The polypeptide of Claim 1, wherein H₂ is selected from the group consisting of phenylalanine, isoleucine, valine, aspartic acid, and leucine.

5. The polypeptide of Claim 4, wherein H₂ is phenylalanine.

6. The polypeptide of Claim 1, wherein wherein H₃ is selected from the group consisting of isoleucine, leucine, and valine.

7. The polypeptide of Claim 6, wherein H₃ isoleucine.

8. The polypeptide of Claim 1, wherein wherein H₄ is selected from the group consisting of leucine and methionine.

9. The polypeptide of Claim 8, wherein H₄ leucine;

10. The polypeptide of Claim 1, wherein wherein H₅ is selected from the group consisting of leucine, alanine, isoleucine, and arginine.

11. The polypeptide of Claim 10, wherein H₅ is leucine.

12. The polypeptide of Claim 1, wherein wherein S₁ comprises glutamine-leucine-arginine- at or near the amino terminus of S₁.

13. The polypeptide of Claim 12, wherein S₁ is 9-18 amino acid residues in length and comprises glutamine-leucine-arginine- at the amino terminus of S₁.

14. The polypeptide of Claim 1, wherein wherein S₂ is 2 to 3 amino acid residues in length.

15. The polypeptide of Claim 14, wherein, S₂ is serine-lysine- proline.

16. The polypeptide of Claim 1, wherein wherein S₃ is 2 to 4 residues in length.

17. The polypeptide of Claim 16, wherein S₃ is histidine-proline- lysine-phenylalanine.

18. The polypeptide of Claim 1, wherein S₄ is 0 to 2 amino acid residues in length.

19. The polypeptide of Claim 18, wherein S₄ is greater than 0 and comprises a hydrophobic amino acid residue.

20. The polypeptide of Claim 1, wherein wherein S₅ is 5 to 6 amino acid residues in length.

21. The polypeptide of Claim 20, wherein S₅ is serine-aspartic acid-glycine-arginine-glutamic acid.

22. The polypeptide of Claim 1, wherein wherein S₆ is 0 to 4 amino acid residues in length.

23. The polypeptide of Claim 22, wherein S₆ is greater than 0 and comprises a hydrophobic residue.

24. A polypeptide comprising an amino acid sequence of

-Tyr-Ser-Lys-Xaa₁-Phe-Xaa₂-Xaa₃-Lys-Phe-Ile-Leu-Ser-Asp-Xaa₄-Arg-Xaa₅-Leu-Xaa₆-, wherein the following can be selected independently from

Xaa₁ is Pro or Ser;

Xaa₂ is His or Ser;

Xaa₃ is Pro or Ser;

Xaa₄ is Gly or Ser;

Xaa₅ is Glu or Ser; and

Xaa₆ is Cys or Ser.

25. A polypeptide comprising an amino acid sequence selected from the group consisting of

26. A polypeptide comprising a sequence of amino acids, amino to carboxy,

(a) amino acids 1 to 18 of SEQ ID NO: 2 immediately adjacent to

(b) amino acids 18 to 32 of SEQ ID NO: 1 immediately adjacent to

(c) amino acids 34 to 73 SEQ ID NO:2.

27. A polypeptide comprising a sequence of amino acids, amino to carboxy,

(a) amino acids 1 to 46 of SEQ ID NO: 1 immediately adjacent to

(b) amino acids 48 to 54 of SEQ ID NO:2 immediately adjacent to (C) amino acids 54 to 73 of SEQ ID NO: 1.

28. A polypeptide comprising a sequence of amino acids, amino to carboxy,

(a) amino acids 1 to 18 of SEQ ID NO:1 immediately adjacent to

(b) amino acids 20 to 46 of SEQ ID NO:2 immediately adjacent to

(c) amino acids 46 to 53 of SEQ ID NO:1 immediately adjacent to

(d) amino acids 55 to 73 of SEQ ID NO:2.

29. A method of screening for a candidate comprising a hydrophobic pocket capable of binding IL8R1, wherein said method comprises:

(a) providing a compound comprising a hydrophobic pocket capable of competing with interleukin-8 (IL8) for IL8 receptor 1 (IL8R1) binding, wherein the compound is not IL8;

(b) providing a IL8R1 polypeptide;

(b) exposing said IL8R1 polypeptide to said compound and said candidate; and

(c) measuring the IL8R1 binding by said compound.

30. A method of screening for a candidate capable of binding to an IL8 hydrophobic pocket comprising:

(a) providing compound comprising a hydrophobic pocket capable of competing with interleukin-8 (IL8) for IL8 receptor 1 (TL8R1) binding, wherein the compound is not IL8;

(b) exposing said compound to said candidate; and

(c) measuring the binding of the candidate to said compound.

31. A method of inhibiting IL8 receptor binding comprising: (a) providing an effective amount of a compound comprising a hydrophobic pocket and capable of competing with interleukin-8 (IL8) for IL8 receptor 1 (IL8R1) binding, wherein said compound is not IL8; and

(b) exposing a cell expressing IL8R1 to the effective amount said compound.

32. A polynucleotide comprising a sequence encoding the polypeptide of Claim 1.

33. The polynucleotide of Claim 32, further comprising a promoter that is operably linked to the polypeptide coding region.

34. A host cell comprising a polynucleotide that comprises

(a) a sequence encoding the polypeptide of Claim 1; and

(b) a promoter that is operably linked to the polypeptide coding region.

35. A method of producing a polypeptide capable of modulating IL8 receptor-mediated biological responses; comprising

(a) providing a host cell comprising a polynucleotide that comprises (i) a sequence coding for the polypeptide of Claim 1 and (ii) a promoter that is operably linked to said coding sequence; and

(b) culturing said cell under conditions that permit production of said polypeptide.