EP0766737A2 - Polypeptides with interleukin 8 receptor 1 (il8r1) binding domains - Google Patents

Abstract

This invention relates to a method of conferring IL8R1 specific binding domains on a polypeptide and to polypeptides that contain altered or unaltered IL8R1 specific binding domains. A method of using such polypeptides as agonists or antagonists is also provided, as well as a method producing such polypeptides.

Description

Polvpeptides with Tntf lmilrin ^~ Rftn .tor 1 (IL8R1 . Binding Domains

Description

Technical Field

The invention relates generally to IL8R1 binding domains. More specifically, the invention relates to (1) polypeptides, other than native IL8, comprising one or more IL8R1 specific binding domains; and (2) polypeptides comprising one or more altered IL8R1 specific binding domains. This invention also relates to polynucleotides eacoding the polypeptides of the present invention, a method of using die polypeptides and a method of producing the polypeptides of the present invention utilizing these polynucleotides. The polypeptides of the present invention can thus act as either antagonists or agonists of IL8 for IL8R1 or IL8R2 binding.

Background of the Invention

Cells utilize diffusible mediators, called cytolrines, to signal one another. A tupαfuuϋy of cyto ines are the chemo-rines, which includes IL8. A review aiticle about the chemokine siφexfamily was written by Miller et al- Grit Rev Irnrnun 12(1.2): 17-46 (1992) and Baggiolini et al.. Adv Immunol 55: 97-179 (1994), herein incorporated by reference.

SUBSTITUTE SHEET (RU 2B) The chemolάnes arc a group of structurally and functionally related cytokines. Recent studies indicate 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 are small secreted proteins that exhibit common secondary protein structure and display four conserved cysteine residues. The common secondary structure of a chemokine, exhibit the following features: (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, which lies over the β-sheet Because a systematic nomenclature for these proteins has not yet been generally agreed, the proteins are 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 or CC family. In the CXC family, the first two cysteine residues are separated by an amino acid residue; the first two cysteine residues in the CC family are not To date, seventeen chemolάnes have been described. Six are members of the CXC family and include, platelet factor 4 (PF4); β- thromboglobulin; NAP-1/IL8; gro α, β, and γ, ff-10; πύg; ENA-78. The CXC family is also known as the α family. The remaining chemolάnes are part of the CC family: macrσphage inflammatory proteins (MlP-l and MEP-lβ); monocyte chemoattractant protein-1 JE (MCP-l/JE); RANTES; HC-14; CIO, and 1-309. This family has also been designated as the β family.

Of interest, 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. I EXP Med 167: 1364-1376; Ribero et al., Immnnologv 3: 472-477 (1991). The molecule also activates the release of superoxide anions and elicits release of the primary granule constituα ts of neutrophils, including my operoxidase, β-glucuronidase and elastase. Native human IL8 mediates these biological activities by binding to its receptor and triggering signal transduction, a cascade of reactions ultimately resulting in a biological response. Presently, two IL8 binding receptors have been identified and are termed "EU-Rl" and "IL8R2." The amino acid sequence of these polypeptides are described in Mnrohv et aim. Science 253: 1280 (1991) and Holmes et al.. Science 253: 1278 (1991), herein incorporated by reference. Other chemokines can compete with IL8 to bind to the IL8R2, such as GROα, GROβ, GROγ. NAP-2 and ENA-78 have been implicated with IL8R2 binding by cross-desensitization experiments with native IL8 by measuring Ca^2*. Others have identified regions of native human IL8 that are implicated in both IL8R1 and IL8R2 binding. However, at this time, no chemokine is known to compete with native IL8 for the IL8R1 specific binding.

Disclosure of the Invention

It is one of the objects of the present invention to confer HL8R1 specific binding to a polypeptide other than IL8 and, in particular a polypeptide possessing a chemokine protein structure like IL-8, by introducing one or more IL8R1 specific binding domains. The polypeptides other than IL8 that posses a chemokine protein structure includes, for example, PF4, β-thromboglobulin, GROα, GROβ, GROγ, IP- 10, mig, ENA-78, MlP-lα, MlP-lβ, MCP-l/JE, RANTES, HC-14, CIO, and 1-309. The binding domains are introduced into the chemokine protein structure such that the spacing of the binding domains permit IL8R1 binding.

It is an object of the present invention to provide a modified IL8 molecule so that its binding affinity to IL8R1 is either enhanced or reduced.

Another object of the present invention is to provide an altered IL8R1 binding domain to render a polypeptide possessing a chemokine protein structure capable of modulating IL8R1 specific binding affinity. An example of a chemokine other than IL8 that is provided with a functional characteristic of EL8, i_e., banding to IL8R1, may be a GllOγ protein, ao that the resdt__αg cfciιnen The altered domain may be made in the native IL8 or be introduced into another polypeptide, for example, that possesses a C-iemolάne protein structure. Yet another object of the invention includes providing polynucleotides that encode the instant desired polypeptides, vectors, and host cells that are capable of producing such polypeptides from the polynucleotides. Further, methods of producing the instant polypeptides are also provided.

Further, it is an object of the invention to provide a method of inhibiting or increasing the biological activity of native IL8 by contacting a target cell with the polypeptide of the present invention.

Modes of Carrying Out The Invention

The inventors herein have identified the amino acid sequences of two IL8R1 specific binding domains within the native IL8 polypeptide. In accordance with the objects of the present invention, therefore, polypeptides comprising one or more IL8R1 specific binding domains are provided, as well as polynucleotides, vectors and host cell containing such. Also provided is a method of producing the polypeptides and a method of using them.

More specifically, native H 8 is known, presently, to bind to two receptors, IL8R1 and IL8R2 on the surface of certain cell types, such as neutrophils. The amino acid sequence of these binding domains specifically affect the ability of native IL8 to bind to H 8R1. The binding domains identified herein can be linked with other amino acid sequences to construct polypeptides, other than native IL8, that are capable of binding to IL8R1. Preferably, such other amino acid sequences are effective to preclude rapid degradation of the polypeptide.

Preferably, these binding domains are linked with amino acid sequences derived from polypeptides of the superfctmily of proteins called die chemokines. Thus, tiie IL8R1 binding domains can be linked with fragments derived from other chemokines to construct polypeptides that exhibit die common secondary structures of chemokines. Polypeptides exhibiting these secondary structures will permit the binding domain(s) to assume a similar conformation as found in native IL8. Examples of such chemokines include PF4, β-thromboglobulin, GROα, GROβ, GROγ, IP-10, mig, ENA-78, MJP-lα, MlP-lβ, MCP-l/JE, RANTES, HC-14, CIO, and 1-309.

The amino acid sequence of native IL8 can be altered within its binding domains to increase or decrease its IL8R1 binding affinity, for example, by substitution or deletion of amino acid residues.

The present polypeptides can be divided into two classes:

(1) polypeptides, other than native IL8, comprising at least one IL8R1 specific binding domain; and

(2) polypeptides comprising an altered IL8R1 specific binding domain.

The polypeptides of the present invention may or may not exhibit a chemokine protein structure. The instant polypeptides having similar or enhanced IL8R1 binding affinity as compared to native IL8 and can compete with native IL8 for IL8R1. Also, polypeptides widi decreased binding affinity to IL8R1 as compared to native IL8 can be effective competitors of native IL8 for die other receptor, IL8R2.

A. Definitions

The terms as defined herein form part of the disclosure of the present invention.

The NMR and X-ray crystallography experiments revealed that the three dimensional structure of the chemokines is remarkably similar, herein referred to as the "chemokine protein structure." The structure of the native human IL8 has been solved and is a model for die chemokine protein structure. The structure includes an ammo-terminal loop, a three-stranded antiparallel β sheet (Greek key), and a carboxy-terminal α helix. The α heKx extends over die top of the β sheet Further, native human IL8 forms a bocnodπner with a 2-fold axis of symmetry, a six-stranded β sheet widi a pair of α helices lying atop die β sheet The placement of die cystdnes and the size of the β sheet are also factors in the three dimensional structure.

SUBSTITUTE SHEET (RULE 2B, The present inventors have determined herein the polypeptide regions of native IL8 that affect specific IL8R1 binding. The domains identified by the inventors herein are those that specifically affect IL8R1 binding and not IL8R2 binding. These regions are referred to as TL8R1 specific binding domains." These domains are found in die an_άu_ Heπninal loop and in strand 3 of die β sheet of native IL8. Though these domains may not interact directly with IL8R1, the IL8R1 binding affinity of a IL8 polypeptide can be drastically reduced when these domains are replaced by homologous domains from other chemokines, such as GROγ, an IL8R2 agonist Using the binding domains of native human IL8 as an example, the amino acid sequence of an IL8R1 binding domain contains a sequence: Ser-Ala-Lys-Glu-Leu-Arg-Cys-Gln-Cys-De-Lys-Thr- Tyr-Ser-Lys-Pro-Phe-His, (amino add residues of 1 to 18 of SEQ ID NO:l); more preferably, die amino acid sequence contains die sequence: Glu-Leu-Arg-Cys-Gln-Cys-De- Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His (residues 4 to 18 of SEQ ID NO:l); even more preferably, die amino add sequence contains the sequence: Lys-Thr-Tyr-Ser-Lys (residues 11 to 15 of SEQ ID NO:l). The amino add sequence of an IL8R1 specific binding domain can also contain die sequence: Lys-Xaa-Tyr-Xaa-Lys, (SEQ ID NO:3). These amino acid sequences are examples of "amino terminal" binding domains because the sequences are based on die sequence of d e amino terminal portion of native IL8.

Another example of the amino add sequence of an HL8R1 binding domain contains the sequence: Gly-Arg-Glu-Leu-Cys-Leu-Asp-Pro (residues 46 to 53 of SEQ ID NO-1); more preferably, die amino add sequence contains die sequence Arg-Glu-Leu-Cys- Leo-Aφ-Pro (residues 47 to 53 of SEQ ID NO.1). The amino add sequence of an IL8R1 specific binding domain can contain die sequence Arg-Glu-Leu-Xaa-Xaa-Xaa-Pro, (SEQ ID NO-4). These sequences are examples of "β sheet" binding domains because the sequences are based on die sequence of he β sheet of native IL8.

■ The binding domains of σtiier native IL8, such as native bovine IL8, porcine IL8, etc- are within the contemplation of the present invention, and can be determined by sequence alignment for example, according to the conserved cysteine residues to native IL8.

Preferably, die present polypeptides contain two IL8R1 specific binding domains "spaced within the polypeptide to permit IL8R1 binding." In this regard, the binding domains are spaced appropriately within die primary sequence of die polypeptide. Consequently, when the polypeptide assumes its three dimensional conformation, die binding domains are positioned to effidently interact widi the other portions Of the polypeptide and/or die receptor to permit IL8R1 binding. Preferably, the present polypeptide possesses a chemokine protein structure to mimic die three dimensional configuration of these domains found in the native HL8.

The amino acid sequence of the IL8R1 specific binding domains can be "altered," for example, by amino acid substitutions, deletions or insertions, to rither increase or decrease EL8R1 specific binding affinity. Alternatively, one or more sequences of amino adds can be inserted, deleted, or substituted to truncate or exdse the binding domain from the polypeptide, such as native IL8. IL8R1 binding domains can be excised from native human IL8 and replaced with amino add sequence from a corresponding region of other homologous chemokines, such as GROγ. The amino add residues that are of particular interest for IL8R1 specific binding have been identified herein as residues 11 (Lys), 13 (Tyr), 15 (Lys), 47 (Arg), 48 (Glu), 49 (Leu), and 53 (Pro) of die native human IL8 (SEQ ID NO:l). These amino acid residues are maintained in die present polypeptides to confer IL8R1 specific binding affinity or are altered or deleted to reduce or enhance IL8R1 specific binding affinity.

Binding of a polypeptide to a receptor is often time a matter of degree. Consequently, as used by those skilled in die art, die receptor binding is usually assessed by die "binding affinity" of die polypeptide. One means of determining binding affinity is to measure the ability of the polypeptide to compete widi native IL8 for IL8R1. The ICg, concentration is die concentration that inhibits 509b of the maximal receptor binding of d e native IL8; the smaller die IC-_JD. die greater the binding affinity. Therefore, a polypeptide is considered to bind to IL8R1 if, for example, its ICs, is above background or a negative control.

The instant polypeptides can be used to "modulate an IL8 receptor-mediated biological response." Such biological responses include, for example, those cellular activities which are triggered by die binding of IL8 to its receptor. Modulation occurs when the instant polypeptides compete widi die native HL8 for HL8R1 and result in either an increase or decrease of at least one of these cellular activities. The nature of these activities may be biochemical or biophysical. For example, a polypeptide modulates an IL8 receptor-mediated response if it does not stimulate die same signal transduction as IL8 when the polypeptide binds to an IL8 receptor. The increase or decrease can be monitored using various assays, described further below, which also utilize IL8 receptor molecules as controls.

More particularly, a cascade of biochemical reactions is triggered when IL8 binds to its receptor. As d e term is applied herein, die instant polypeptides will modulate an IL8 receptor-mediated response when it causes an increase or decrease in any one of these reactions. For example, IL8 receptors are G-coupled proteins which, when proper signal transduction activity occurs, triggers an increase of intracellular Ca * and an activation of phospholipase C. Signal transduction can be measured by observing die levels of inositol triphosphate (IP,) and diacylglycerol (DAG), which are increased due to phospholipase C activation and cyclic AMP (cAMP). Conventional assays can be used to measure die intracellular levels of Ca*, IP,, and DAG to determine whether the IL8 receptor-mediated response has been modulated. Assays for measuring levels of free cytosolic Ca are known.

"Native IL8" refers to a polypeptide having an amino add sequence which is identical to a sequence recovered from a source which naturally produces IL8, such as human, bovine, porcine or other mammalian sources. Native IL8 may be of vary in length from species to species. An example of native IL8 is the human IL8 which has die amino add sequence as shown in SEQ ID NO:l. The term "IL8 receptor," as used herein refers to any of the several vertebrate IL8 receptors, or fragments thereof which are capable of binding to IL8. For example, human IL8R1 and IL8R2 are encompassed by this term.

The term "chemokine" refers to a superfamily of naturally occurring proteins, which are diffusible mediators that cells use to signal one another. A review article by Miller et al., loc. cit., describes the chemokine superfamily. The chemokines are structurally and functionally related. Recent studies indicate 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 are small secreted proteins that display four conserved cysteine residues. To date, about seventeen different chemokines have been described. They include platelet factor 4 (PF4); β-diromboglobulin; NAP-1/IL8; gro α, β, and γ; IP-10; mig; ENA-78; macrophage inflammatory proteins (MEP-lα and MlP-lβ); monocyte chemoattractant protein- 1/JE (MCP-l/JE); RANTES; HC-14; CIO, and 1-309. Other chemokines can be identified by their amino add homology to die known chemokines and by their similarity in secondary protein structures and biological activities to the known chemokines.

A "modulating amount" of the present polypeptide refers to the amount needed to enhance or reduce die IL8 receptor mediated biological response of a cell producing IL8 receptor. Such biological responses can be monitored by the assays described below.

An "inhibiting amount" of the present polypeptide refers to die amount needed to inhibit IL8 binding to the IL8 receptors. Though IL8 binding may not be completely extinguished, less IL8 will be bound to its receptors in the presence of an inhibiting amount of die present polypeptide than in die absence.

Examples of "functional characteristics" of an altered or unaltered binding domain or polypeptide include receptor binding affinity, ability to trigger a biological response, signal transduction, etc. Thus, a IL8 GROγ chimera exhibits the same functional characteristic as IL8 if die chimera has die same receptor binding affinity, for example. A composition containing A is "substantially free of B when at least 85% by wright of the total A+B in the composition is A. Preferably, A comprises at least about 90% by weight of the total of A+B in the composition, more preferably at least about 95% or even 99% by weight

A "promoter" is a DNA sequence that initiates and regulates the transcription of a coding sequence when the promoter is operably linked to the coding sequence. A promoter is "heterologous" to die coding sequence when the promoter is not operably linked to die coding sequence in nature. In contrast a "native" or "homologous" promoter is operably linked to the coding sequence in nature.

An "origin of replication" is a DNA sequence tiiat initiates and regulates replication of polynucleotides such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the oelL Examples of such origins are die 2μ and autonomously replicating sequences, which are effective in yeast; and die viral T-antigen, effective in COS-7 cells. Other origins of replication are known in the art and can be utilized in the appropriate host

An "expression vector" is a polynucleotide that comprises polynucleotides that regulate the expression of a coding sequence and includes, for example, a promoter, a terminator and an origin of replication.

Host cells capable of producing die present polypeptides are cultured "under conditions inducing expression." Such conditions allow transcription and translation of die po-yπocleσtide encoding die polypeptide. These conditions include cultivation temperature, oxygen concentration, media composition, pH, etc. For example, if the trp promoter is stilized in die expression vector, the media will lack tryptophan to trigger the promoter and induce expression. The exact conditions will vary from host cell to host cell and from expression vector to expression vector. B. General Method

Determination of the Amino Add Sequence of EL8R1 Specific Binding Domains

The present inventors have determined die polypeptide regions of native IL8 that affect specific IL8R1 binding. The domains identified herein are those that specifically affect IL8R1 binding and not IL8R2 binding. Thus, an embodiment of die present invention, die ability to bind IL8R1 is conferred on any polypeptide by introducing at least one IL8R1 binding domain, tiius producing an antagonist of IL8 binding to IL8R1. In a preferred embodiment die polypeptide of die present invention contains two binding domains. One domain is selected from die group of amino terminal binding domains and the other domain is selected from the group of β sheet domains as described in greater detail below.

The following amino add sequences are examples of amino terminal binding domains. The group was diusly named because the sequences are based on die amino acid sequence of die amino terminal portion of native IL8. Using die binding domains of native human IL8 as an example, die amino add sequence of an IL8R1 binding domain is Ser-Ak-Ly_ Glu-__-eu-Arg-C_ys><ϊ-n-Cys-B^^ (amino acid residues of 1 to 18 of SEQ ID NO.1); more preferably, die amino add sequence is Glu- Leu-Arg-Cys-Gln-Cys-ne-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His (residues 4 to 18 of SEQ ID NO:l); even more preferably, die amino acid sequence is Lys-Thr-Tyr-Ser-Lys (residues 11 to 15 of SEQ ID N0.1). The amino add sequence of an IL8R1 specific binding domain can also be Lys-Xaa-Tyr-Xaa-Lys (SEQ ID NO:3), where Xaa represents any amino acid residue.

Another group of IL8R1 binding domains is the group of β sheet binding domains. The amino acid sequences of these domains are based on die sequence of the third strand of die β sheet of native IL8. An example of die amino add sequence of such an IL8R1 specific binding domain is Gly-Aig-Glu-Leu-Cys-Leu-Asp-Pro (residues 46 to 53 of SEQ ID NO:l); more preferably, die amino acid sequence is Arg-Glu-Leu-Cys-Leu- Asp-Pro (residues 47 to 53 of SEQ ID NO: 1). The amino acid sequence of an IL8R1 specific binding domain can also be Arg-Glu-Leu-Xaa-Xaa-Xaa-Pro (SEQ ID NO:4).

The binding domains of other native IL8, such as native bovine IL8, can be determined by sequence alignment of the native human IL8 to other native IL8.

The presence of any one of the binding domains above may be necessary by not be optimal to influence receptor binding, particularly when placed in the context of a non-IL8 polypeptide. In a preferred embodiment of the present invention, therefore, a polypeptide comprising IL8R1 binding domains possess a chemokine protein structure so that the IL8R1 specific binding domains may assume a configuration similar to the one in native IL8. Once the primary sequence of the binding domain is determined to be used in the present polypeptides, in one embodiment of the invention, the domains are spaced within the polypeptide to permit IL8R1 binding.

A polypeptide exhibiting a chemokine protein structure and comprising one or more IL8R1 specific binding domains can be exemplified by the following formula:

A - B - C; where B represents an IL8R1 specific binding domain. Optionally, B can represent a sequence containing more than one IL8R1 specific binding domain, such as represented by the formula -b- - X - j - where b_ and b₂ each represent an IL8R1 specific binding domain and X represents one or more amino acid residues. Preferably, the amino acid sequence of bj is selected from a group of amino terminal binding domains, and the amino acid sequence of b₂ is selected from the group of β sheet binding domains as disclosed above. Also, preferably, together A - B - C exhibit the secondary structural features of a chemokine.

The polypeptides of the present invention exhibiting a chemokine protein structure comprise four conserved cysteine residues when properly aligned with other chemokine superfamily members. The chemokines can be aligned utilizing typical sequence alignment programs. An example of an alignment of the chemokines is shown in Miller et al., Crit Rev Immun 12(1.2): 17-46 (1992). The conserved cysteines form disulfide bonds that aid the formation a chemokine protein structure. A polypeptide of the present invention exhibiting a chemokine protein structure, preferably, therefore, 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 that lies over die β sheet

The three stranded β sheet of the polypeptides of die present invention is preferably of - rrdlar size to those found in chemokines. For example, the strands of the β sheet are about 12 to 3 amino add residues in length; more preferably, from about 10 to 3 amino add residues; most preferably, 7 to 3 amino acid residues. The amino acid sequence of the β sheet IL8R1 specific domains are preferably incorporated into this secondary structure; more preferably, the sequence of the domain is placed in die third strand of die β sheet

The C-terminal α helix of the polypeptides of die present invention having a chemokine protein structure lies over the β sheet The length of die α helix is not critical and may or may not overhang the edge of the β sheet Usually, the length of the α-helix is from about 9 to 25 residues; more usually, from about 12 to 22; even more usually 15 to about 19 residues. Typically, the α helix is an 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.

The amino terminal portion contains an tail which retains no particular structure and an loop. Preferably, amino add sequence of die amino terminal IL8R1 binding domains are incorporated in die loop portion. The entire portion including tail and loop is from about 25 to 14 amino add residues; more preferably, from about 22 to about 18 amino acid re&duoes. The loop comprises from about 15 to about 6 amino add residues; more preferably about 12 to about 8 ammo acid residues. In addition, preferably, die tail of the amino terminal portion comprises the amino acid sequence Glu-Lea-Arg sequence. This sequence is non-specific sequence for IL8 receptor binding. ln addition, die present polypeptides contemplated herein may contain sequences that are not specific for IL8R1 binding but are sequences that are non-specific. These sequences, like Glu-Leu-Arg can afreet binding of eitiier IL8 receptors.

More specifically, constructing a chimeric chemokine is one means of constructing a polypeptide of d e present invention having binding domains appropriately to permit IL8R1 binding. For example, the IL8R1 specific binding domains can spaced to permit IL8R1 binding by substituting the domains for the homologous regions in the GROγ polypeptide. Alternatively, the C-terminal α helix of GROγ can be substituted into die native human IL8 polypeptide. Therefore, the binding domains retain their native configuration. In such an embodiment die polypeptide can exhibit non-native IL8 biological activity due to the presence of die GROγ α helix. Thus, in one embodiment of die present invention, die binding domain of IL8R1 can be placed in a polypeptide having a chemokine protein structure so as to displaced the corresponding native chemokine sequences. Thus, conferring T 8R1 binding activity to that polypeptide while maintaining the chemokine protein structure.

In an alternative embodiment it may be desirable to insert an IL8R1 binding domain in a polypeptide without removal of any sequences. The techniques for insertion, deletion and substitution of amino add residues by altering polynucleotide sequences encoding die polypeptide to be altered are conventional in die art

In addition, fragments of the amino add sequences of the chemokines can be assembled together to construct a polypeptide of die present invention. For example, the present polypeptide may possess die amino add sequence of he amino terminal of native human IL8, the first two strands of the β sheet suuαme of NAP-2, die tiiird strand of die β sheet of IL8, and die α helix of GROβ. The amino add sequences to be utilized to construct die polypeptide of the present invention do not have to be identical to the sequences found in the chemokines to exhibit die deared secondary structure features. For example, die amino add sequences may be mutants or fusions of die sequences found in the chemokines. Mutants of the chemokines can be constructed by making conservative amino add substitutions of such. The following are examples of conservative substitutions: Gly <-> Ala; Val <→ Be <→ Leu; Asp <→ Glu; Lys <→ Arg; Asn <→ Gin; and Phe <→ Tip <-+ Tyr. Also, insertions and (deletions can be made to the amino acid sequences of the chemokines provided that die chemokine protein structure is maintained.

The choice of amino acid sequence for die present polypeptides can also be chosen for their ability to confer functional characteristics. For example, the α helix sequence of GROγ may be chosen for the present polypeptide to confer a biological activity of GROγ. Further, it is contemplated the sequences of the present polypeptide can be altered to reduce or enhance the biological activities. For example, the amino acid sequence a GR0γ/TL8 chimera exhibiting IL8R1 specific binding can be altered to reduce its ability to trigger HL8R1 signal transduction.

The binding domains can be altered to increase or decrease the binding affinity of the present polypeptide to HL8R1. Such polypeptides widi altered binding affinities can be used as agonists or antagonists of IL8 as desired. Mutants of the binding domains can be constructed, for example, by making amino acid substitutions that maintain or enhance or reduce die binding affinity of the polypeptide to IL8R1. Other altered landing domains can be made by deleting or inserting residues to die amino add sequence of die unaltered binding domains so as to alter the binding affinity of the polypeptide. Additional amino add residues can be incorporated at the N- or C-terminus. In particular, some or all of die amino add residues of die binding domain can be exdsed to decrease die binding affinity of die present polypeptide.

The amino acid residues that are of particular interest for IL8R1 specific binding are highlighted in the binding domains below: Ser-A__»-Ly*-Glu-Leυ-A_^-Cys-Gln-C_y^

(amino acid residues of 1 to 18 of SEQ ID NO_1); <Hu-___eu-Aιg-Cys-G-n-Cys-.^

(residues 4 to 18 of SEQ ID NO:l); Lys-Thr-Tyr-Sex-Lys, (residues 11 to 15 of SEQ ID NO: 1 );

Lys-Xaa-Tyr-Xaa-Lys, (SEQ ID NO:3); (residues 46 to 53 of SEQ ID NO:l);

Art-Glu-Leβ-Cys-Leu- Asp-Pro, (residues 47 to 53 of SEQ ID NO:l); and

Ars-Giv- ejU-Xaa-Xaa-Xaa-Pro, (SEQ ID NO:4). The highlighted amino acid residues correspond to residues 11, 13, 15, 47, 48, 49, and 53 of SEQ ID NO:l. Preferably, these amino acid residues in die binding domains are altered by substitution, deletion, or insertion of another amino add residue to enhance or decrease the binding affinity of the domain.

Constructing Polynucleotides Encoding the Polypeptides. Expression Vectors, and Host Cells

Once die amino add sequence of die present polypeptides is determined, then polynucleotides encoding the polypeptides can be constructed. The polynucleotide sequences can be isolated from known libraries. The appropriate sequences can be ligated together to produce a coding sequence. Known linkers or restrictions sites can be used to construct the various fragments. These sequences can be altered using polymerase chain reaction (PCR) or site specific mutagenesis. Alternative, the polynucleotide sequence can be synthesized with a commercially available synthesizer.

The polynucleotide encoding die present polypeptides can be used to construct an expression vector to produce die polypeptide. At the minimum, an expression vectcn- will contain a promoter which is operable in die host odl and operably finked to the polynucleotide encoding die present polypeptides. Expression vectors may also include signal sequences, terminators, selectable markers, origins of replication, and sequences homologous to host odl sequences for purposes of integration into the host genome. These additional elements are optional but can be included to optimize expression.

A promoter is a DNA sequence upstream or 5' to the polynucleotide encoding die present polypeptide. The promoter will initiate and regulate expression of the coding sequence in die desired host cell. To initiate expression, promoter sequences bind RNA polymerase and initiate die 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 represser protein binds to die lac operon, an E. coli regulatory promoter sequence, transcription of die 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 die promoter will become active only in die presence of an inducer, such as a hormone or enzyme substrate (Sassone-Corsi and Borelli (1986) Trends Genet 2:215: Maniatis et al. (1987) Sdence 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 widi a heterologous expression initiation region. Examples of hybrid promoters are the E. coli lac operator linked to die E. coli tac transcription activation region; the yeast alcohol dehydrogenase (ADH) regulatory sequence finked to the yeast glyceralddιyde-3-phosphate-dehydrogenase (GAPDH) transcription activation region (U.S. Patent Nos. 4,876,197 and 4,880,734, incorporated herein by reference); and die cytomegalovirus (CMV) enhancer linked to the SV40 (simian virus) promoter.

The polynucleotides encoding the present polypeptides may also be linked in leading frame to a signal sequence. The signal sequence fragment typically encodes a peptide comprised of hydrophobic amino acids which directs the present polypeptide to die 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), die 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 die full-length pre-pro alpha factor leader (about 83 amino add residues) as well as truncated alpha-factor leaders (typically about 25 to about 50 amino add 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 polynucleotide encoding the present polypeptide. Usually, the terminator of native host cell proteins are operable when attached 3' of die polynucleotide encoding die present polypeptide. Examples are die 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, sdectaUe markers, an origin of replication, and homologous host cells sequences may optimally be included in an expression vector. A selectable marker can be used to screen for host cells that potentially contain die expression vector. Such markers may render die host cell immune to drugs such as ampidϋin, chloramphenicol, e-ythromycin, neomycin, and tetracycline. Also, markers may be biosyntiiet-C 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 die leucine pathway will survive.

An origin of replication may be needed for die depression vector herein to lepficate 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 die ceH. Examples of origins that can be used herein are the 2p and autonomously replicating scquπre., which are effective in yeast; and die viral T-antigen, effective in COS-7 cells.

Expression vectors herein may be integrated into die host odl genome or remain autonomous within the cell. Polynucleotide sequences homologous to sequences within the host cell genome may be needed in the expression vector to integrate die expression cassette. Alternative, die homologous sequences are not linked to the expression vector. For example, expression vectors can integrate into die CHO genome via an unattached dϋiydrofolate reductase gene. In yeast it is more advantageous if the homologous sequences flank die expression cassette. Particularly useful homologous yeast genome sequences are those disclosed in PCT WO90 01800, and die HIS4 gene sequences, described in Genbank, accession no. J01331.

The choice of promoter, terminator, and other optional dements of an expression vector wiD also dqpend on the host cell chosen. The invention is not dependent on the host cell selected. Convenience and die level of protein expression will dictate die optimal host cell. A variety of hosts for expression herein are known in the art and available from the American Type Culture Collection (ATCC). Bacterial hosts suitable for fj.|-_raaing die present polypeptides include, without limitation: Canφylobacter, Bacillus, Etckerickia, Loctobacill s, Pseudomonas, Staphyiococcus, and Streptococcus. Yeast hosts from the following genera may be utilized: Candid-, Hansenula Kluyveromyces, Pichia, Saeck umyus, Schiiosaccharcmtyce-, and Yarrσwia. Immortalized mammalian host cells that can be used herein include but are not limited to CHO cdls, IfcLa cells, baby hamster kidney (BHK) cdls, monkey kidney cells (COS), human hepatocdlul-g 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 Spodoptera frugiperda as described in 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) fa vitro Cell. Dev. Biol. 25:225.

Transformation

After vector construction, the expression vector comprising a polynucleotide encoding the present polypeptide is inserted into the host cell. Many transformation techniques exist in the art for inserting expression vectors into bacterial, yeast insec and mammalian cells. The transformation procedure to introduce die expression vector depends upon the host to be transformed.

Methods of introducing exogenous DNA into bacterial hosts, for example, are well-known in the art, and typically protocol includes dther treating ti e bacteria widi CaClj or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cdls by decn-oporation or viral infection. Transformation procedures usually vary with die bacterial spedes to be transformed as described in e.g., (Masson et al. (1989) FEMS Microbiol. Lett 60:273: Palva et al. (1982) Proc. Nad. Acad. Sd. USA 79:5582: EP Publ. Nos. 036 259 and 063 953; PCT WO 84 04541, Bacillus), (Miller et al. (1988) Proc. Nad. Acad. Sd. 85:856: Wang et al. (1990) 3. Bacteriol. 172:949. Canψylobacte ), (Cohen et al. (1973) Proc. Nad. Acad. Sd. 69:2110: Dower et al. (1988) Nucleic Adds Res. 16:6127: Kushner (1978) "An improved method for transformation of Escherichia coli widi CoIEl-derived plasπήds in Genetic Engineering: Proceedings of the Intemational Symposium on Genctfc Engineering (eds. H.W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159: Taketo ⁽1988⁾ Biochim. Bionfavs. Acta 949:318: Escherichia), (Chassy et al. (1987) FEMS Microbioi Lett. 44:173 Lactobaάlhis); (Fiedler et al. (1988) (Augustin et al. (1990) FEMS Microbiol. Lett &.203, Staphyiococcus), (Barany et al. (1980) J. Bacteriol. 144:698: Hariander (1987) "Transformation of Streptococcus lactis by electroporation," in Streptococcal Genetics (ed. J. Ferretti and R. Curtiss D3); Peπy et al. (1981) Infec. Immun. 32:1295; Powell et al. (1988) APPI. Environ. Microbiol. 54:655: Somkuti et al. (1987) Proc. 4th Eyr. Con^g. Biotechnology 1:412. Streptococcus).

Transformation methods for yeast hosts are also well-known in die art, and typically include tither the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Electroporation is another means for transforming yeast hosts. These methods are described in, for example, Methods in Enzvmologv. Volume 194, 1991, "Guide to Yeast Genetics and Molecular Biology." Transformation procedures usually vary widi de yeast spedes to be transformed, e.g., Kurtz et al. (1986) Mol. Cell. Biol. 6; 142 and Kunze et al. (1985) J. Basic Microbiol. 25:141: for Candida; Gleeson et al. (1986) J. Gen. Microbiol. 132:3459 and Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302 for Hansenula, Das et al. (1984) J. Bacteriol. 158:1165. and De Louvencourt et al. (1983) J. Bacteriol 154:1165. Van den Bag et al. (1990) Bio Technolo^gy 8:135. for Kluyveromycey, Cregg et al. (1985) Mol. Cell. Biol. 5:3376: and Kunze et al. (1985) Basic Microbiol. 25:141. and U.S. Patent Nos. 4,837,148 and 4,929,555; for Pichia; Hinnen et al. (1978) Proc. Nad. Acad. Sd. USA 75:1929. and Ito et al. (1983) J. Bacteriol. 153:163 for Saccharomyces; Beach and Nurse (1981) Nature 300:706 for Schizo- saccharomyces; Davidow et al. (1985) Cmr. Genet 10:39. and Gaillaπiin et al. (1985) Curr. Genet 10:49 for Yarrσwia.

Methods for introducing heterologous polynucleotides into mammalian cells are known in die art and include viral infection, dextran-mediated transfection, cakium phosphate precipitation, polyfarene mediated transfection, protoplast fusion, electroporation, encapsulation of die polynucleotides) in liposomes, and direct m roinjection of the DNA mto nudeL.

The method for construction of an expression vector for transformation of insect cdls for expression of recombinant herein is slightiy different than that generally applicable to die construction of a bacterial expression vector, a yeast expression vector, or a mammalian expression vector. In an embodiment of die present invention, a baculovirus vector is constructed in accordance witit techniques tiiat arc known in the art, for example, as described in Kitts et al., BioTechniαues 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 die 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 mvitrogen (San Diego, CA).

Also, methods for introducing heterologous DNA into an insect host cell are known in die art For example, an insect cell can be infected widi a virus containing an polynucleotide encoding die present polypeptides. When die virus is replicating in the infected cell, die present polypeptides 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 die Class Insecta can be grown in the media of this invention. The orders Diptera and Lepidoptera are preferred. Example of insect spedes are Hsted in Wdss et al., "Cdl 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 that can be used herein are exemplary: Carpocapsa pomeonella (preferably, cell line CP-128); Trichoplusia i (preferably, cell line TN-368); Autograph mmmφonάc.- Spodoptera frugiperda (preferably, cdl fine Sf9); Lymantria dispar, Mamestra brassicar, Aedes albopictus; Orgyia pseudotsugata. Neodφrio serti eπ Aedes aegypti; Autheraea eucalypti; Gnorimoschema operce llela; Galleria meUonella; Spodoptera Bttolaris; BlateUa germankr. Drosophila melanogaster. HeUothis tea; Spodoptera eήgua; Rachiplusia on; Plodia iraerpunctella Amsaeta moorev, Agrotis c-nigrum, Adoxophyes σranar, Agrotis segetu , Bo byx mori; Hyponomeuta maUnellu;, Colias e rythemr, Attticarsia germmetalia Apa teles melanoscelu; Arena caja; and Porthetria dispar. Prefβred insect cell lines arc from Spodoptera frugiperda, and especially preferred is cell line Sf9. The Sf9 cell line can be used in herein and obtained from Max D. Summers (Texas A & M University, College Station, Texas, 77843, U.SΛ.) Odier S.frugiperda cell fines, such as IPL-Sf-21AE m, 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 ormea NPV (AcNPV), Rachiplusia ou NPV, Galleria mellonella NPV, and any plaque purified strains of AcNPV, such as E2, R9, SI, 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., Vσol £9: 517-527 (1978).

Typically, insect cells Spodoptera frugiperda type 9 (SF9) are infected with baculovirus strain Autographa californica NPV (AcNPV) containing a polynucleotide encoding die present polypeptides. Such a baculovirus is produced by homologous recombination between a transfer vector containing die 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 die nucleotide sequences needed to restore the dysfunctional gene and a baculovirus polybedrin promoter and terminator operably linked to die polynucleotide encoding die present polypeptides, as described in See Kitts et al.. BioTechnioues 14(5): 810-817 (1993).

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

The essential gene and die polyhedrin gene flank each odier in die baculovirus genome. The coding sequence in the transfer vector is flanked at its 5^* with the essential gene sequences and die polyhedrin promoter and at its 3' with die polyhedrin terminator. Thus, when the desired recombination event occurs the polynucleotide encoding d e present polypeptide 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 die recombinant baculovirus genomic DNA. Alternatively, expression of the present polypeptide by cells infected wid die recombinant baculovirus is another verification means.

Isolation of die Polypeptides

Based on die physical characteristics of the present polypeptides, well known methods can be sdected to purify die polypeptide of die present invention. Such physical characteristics include hydrophobidty, isoelectric point size, solubility, antigenicity, etc. Specifically, naturally occurring IL8 are found as dimer of identical submits.

Separation techniques can be chosen for convenience and optimization. A single method may suffice, or a combination of techniques may be needed to purify the present polypeptides to the deared purity. The separation technique sdected is not critical to the invention. Many techniques are available. For example, the following are separation techniques differentiating size: dialysis, ultrafiltration, gd filtration, and SDS polyaαylamide gd dectrophoresάs. Ion-exchange chromatography separates different electrically charged components. Antibodies to the present polypeptides can also be used in affinity chromatography to separate the desired polypeptides from antigenically dissimilar proteins. Reverse-phase high performance liquid chromatography is a separation method based on differences in hydrophobicity.

Assays

L Receptor Binding Assays

Receptor binding assays herein may utilize cells that naturally produce the IL8R1 or IL8R2 receptors, such as human neutrophils. Alternatively, a polynucleotide encoding either the IL8R1 or IL8R2 can be introduced into a cell to produce the desired receptor. For die assay, dther whole cells or membranes can be used to determine receptor binding. Typically, die assay for receptor binding is performed by determining if die present polypeptide can compete with radioactive, native IL8 for binding to IL8R1. The less die radioactivity measured die less die native EL8 was binding to the receptor.

ii. Biological Activity Assays

Signal Transduction Assays

One means of measuring die biological activity of die present polypeptide is by a signal transduction assay. Typical signal transduction assays measure Ca**, IP,, and DAG levels as described in more detail below.

Most cdlular Ca¹* ions are sequestered in the mitochondria, endoplasmic rcticulum, and odier cytoplasmic vesicles, but binding of present polypeptide to die IL8R1 will trigger an increase of free Ca** ions in die cytoplasm. Widi fluorescent dyes, such as fura-2, die concentration of free Ca** can be monitored. The ester of fura-2 is added to die culture media of die host cdls expressmg IL8R1 or IL8R2 receptor polypeptides. The ester c& fura-2 is ϋpophilic and diffuses across the membrane. Once inside die cdl, die fura-2 ester is hydrolyzed by cytosolic esterases to its non-lipophilic form, and then die 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 arc released after binding of a ligand to the 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. Sakurai et al., EP 480 381 and Adachi et α FEBS Lett 311(2): 179-183 (1992) describe some examples of assays measuring free intracellular Ca** concentrations.

The rise of free cytosolic Ca** concentrations is preceded by die hydrolysis of phosphatidylinositol 4,5-bisphosphate. Hydrolysis of this phospholipid by the plasma- membrane enzyme phospholipase C yidds 1,2-diacylglycerol (DAG), which remains in the membrane, and die water-soluble inositol 1,4,5-triphosphate (IPj). Binding of IL8 or IL8 agonists will increase the concentration of DAG and _P₃. Thus, signal transduction activity can be measured by monitoring die concentration of these hydrolysis products.

To measure die _P₃ concentrations, radioactively labelled Η-inositol is added to die media of host cells expressing IL8R1 or IL8R2. The ^sH-inositol taken up by the cells and after stimulation of die cells with die present polypeptide, die resulting inositol triphosphate is separated from die mono and di-phosphate forms and measured. Sakurai et al., EP 480 381 describes one example of measuring inositol triphosphate levels. 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 die radioactive inositol from other inositol phosphates. With tiiese reagents an effective and accurate competition assay can be performed to determine die inositol triphosphate levds.

Mydoperoxidase Assay

A mydoperoxidase (MPO) assay is an example of another method for measuring die biological activity of a IL8R1 mediated biological activity. Biologically active MIP-2 polypeptides can stimulate neutrophil degranulation. During degranulation, MPO is released and can be measured according to die procedures described in Suzuki et al.. Anal Biochem 132: 345-352 (1983). Chemotaxis Assays

Neutrophils chemotaxis is another IL8R1 mediated biological activity that can be measured herein. The assays can be performed on fluorescendy labeled neutrophils, essentially as described in DeForge et al.. J Immunol 148: 2133-2141 (1992).

C Examples

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

Example 1: IL8 Mutants - Altering the B Sheet Binding Domain Polypeptides:

The amino add sequence of the polypeptides depicted below is as found in SEQ ID NO:l except as follows:

Host Cell: Yeast Saccharomyces cerevisae or Bacteria: Escherichia coli

Polypeptide Isolation:

A protocol for purification of the present polypeptide is set forth bdow.

Create a cation exchange resin shiny of 1:1 Fast Flow S Sepharose® resin and 50 mM sodium acetate pH 5.4. Load 30-50 mLs of yeast supernatant pH adjusted to 5 S. in 50 mL plastic screw cap tube and add 400 pL of resin slurry. Rock tube overnight at 4°C. Centrifuge tube at 3,000 rpm at 4^βC for twenty minutes. Pour off supernatant, unbound material.

To wash the resin, transfer die resin pellet to an 1.5 mL Eppendorf tube. Wash out 50 mL tube with about 1 mL of 50 mM sodium acetate pH 5.4. Centrifuge Eppendorf tube at 2,000 rpm for ten minutes. Remove the supernatant Add about 1 mL of 50 mM sodium acetate to tube. Vortex tube to resuspend pellet Repeat wash steps.

To ute die present polypeptides, add 100 pL of 50 mM HEPES pH 8.3, 1.0 M NaCl. Rock tube at 4°C for twenty minutes. Centrifuge the tube at 2,00 rpm for ten minutes. Remove supernatant to Eppendorf labded 1.0 M NaCl. Repeat elution steps.

Receptor Binding Assay:

Bdow is a method for testing the binding affinity of die present polypeptides.

Test for present polypeptides' ability to bind IL8R2 receptors as measured by competition with ^ML-δ.

DNA encoding die receptor was isolated from human genomic DNA by PCR using oligonucleotide primers based on published sequences, as described in Murphy et al.. Science 253: 1280 (1991) and Holmes et aL. Sdence 253: 1278 (1991). DG44, Chinese hamster ovary (CHO) cdls were transfected widi either IL8R1 or IL8R2 cDNA under die control of the cytomegalovirus immediate early promoter and enhancer. A standard caldum phosphate protocol was used. The expression vector included die dihydrofblate reductase gene. Thus, die cells were selected in hypoxantfaine and diymidine deficient medium. Clones expressing die receptor were identified by fluorescent labeling with anti-peptide antibodies and by IL-8 binding assays.

Culture receptor expressing cells were to confluence in 96-well RemσvaWell plates for the receptor binding assay. Plate cells at 1-2 x 10⁵ cells per cm² in 50% Dulbecco's Modified Eagle's Medium (DMEM); 50% Ham's F12, hypoxantfaine,. diymidine, 10% dialyzed fetal calf serum (dFCS). Next incubate the cell monolayers at room temperature for three hours with 0.2 mL Hepes-BSA binding buffer containing 0.2 nM ^,25I-IL-8 and the wanted concentrations of the present polypeptides. The binding buffer 25 mM Hepes, pH 7.0; 150 mM Nad; 5 mM Cad* 5 mM MgC_V, 1 mgmL BSA. Measure non-specific binding in die presence of 1 pg mL of unlabded IL-8. Wash cells once widi Hepes-BSA binding buffer. Determine bound l-IL-8 with a gamma counter.

Neutrophil Chemotaxis Assay:

Test for present polypeptides' ability to induce chemotaxis of neutrophils. The assays essentially as described in DeForge et al.. J Immunol 148: 2133-2141 (1992).

For this assay, freshly isolate neutrophils from whole blood using Neutrophil Isolation Media (NIM), manufactured by Cardinal Assodates, Santa Fe, New Mexico. Isolate the cells essentially as described by Cardinal Associates. Add 17 mL of NIM and 30 mL of blood to 50 mL tubes. Centrifuge tube at 500 g for 50 minutes at room temperature. Remove contaminating red blood cells by lysis in ice cold water.

Label die purified neutrophils by incubation widi 2',7'-bis-(2-carboxvethyl 5(and-6 carboxyfluorescein, acetoxymediyl ester, manufactured by Molecular Probes, Eugene, Oregon. The procedure as described in DeForge et al., J Immunol 148: 2133- 2141 (1992). Suspend die neutrophils at 2 x 10* cells mL in PBS without calcium and magnesium, 0.1% BSA. Add label to the above cells at a final concentration of 2 μM. Incubate cells 30 minute incubation at 37°C. Wash die labeled neutrophils twice with PBS without ca ium and magnesium. Resuspend die cell pdlet in Hanks Balanced Salt solution widi 0.1% BSA.

For die chemotaxis assay, use a Neuroprobe 96-wdl diemotaxis chamber witfa a 10 um duck, 3 nm pore, bonded polycarbonate membrane. To die bottom of the wells, add 30 pL of the Hanks, 0.1% BSA buffer widi die wanted amount of the present polypeptides. To the top of the wells, add suspension 50 nL of labeled cells at a concentration of 5 x 10⁶ cells mL. Incubate the cells at 37°C for 25 minutes. Quantify neutrophil migration by fluorescence reading of tiie filter.

Use to detect for fluorescence, 485 nm exdtation and 530 nm emission detection filters. F-Met-Leu-Phe at 100 nM manufactured by Sigma, St Louis, Missouri, as a positive control for maximal signal on each experiment

Example 2: IL8 Mutants - Altering d e Amino Terminal Binding Domain Polypeptides:

The amino add sequence of the polypeptides as found in SEQ ID NO:l except as follows:

Host Cell: As in Example 1 Polypeptide Isolation: As in Exampte 1 Receptor Binding Assay: As in Example 1 Neutrophil Chemotaxis Assay: As in Example 1

Example 3: GROVIL8R1 Binding Domain Chimeras Polypeptides:

The amino add sequence is described in PCT appln. no. WO92 00326, herein incorporated by reference. The antino acid sequence of the polypeptides as found in SEQ ID NO_2 except as follows:

co

DO CO

rri en a m:

f m≡

CD

CO m

m

CO

CO CO

m

CO -_£ m .

CD

Host Cells: As in Example 1 Polypeptide Isolation: As in Example 1 Receptor Binding Assay: As in Example 1 Neutrophil Chemotaxis Assay: As in Example 1

8BQUENCB LISTING

(1) OTNT INFORMATION:

(i) APPLICANT: Takaap-Olaon, Patricia

Shyaaala, Vankatakriβhna Marnatf-Hawarmd, Mary Elian

(ii) TZTLB OF INVBNTIOW: Polypaptidβa with Intarlaukin 8 Receptor 1 (ΣL8R1) Specific Binding Domain*

(iii) NUMBER OF SEQUENCES: 4

(iT) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Chiron Corporation

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(2) HFOBMATZGH PCR SEQ ZS BOtlx

(i) HBUUBMOt CHARACTERISTICS:

(A) LENOTHJ 72 amino acids

(B) TIPS: amino acid

(C) 8TRAMDBDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TTPB: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

BT Ala lys Glu Leu Arg Cys βln Cys lie Ly* Thr Tyr S»r Lye Pro 1 5 10 15

Phe Bis Pro Lys Phe He lys βln Leu Arg Val Zle Glu Ser ^βly Pro 20 25 30

Bis Cys Ala Asn Thr Glu Zle Zle Val lys Leu Ser Asp ^βly Arg ^βlu 35 40 45

Leu Cys Leu Asp Pro ∑_ys βlu Asn Trp Val βln Arg Val Val ^βlu Lys 50 55 60

Phe Leu Lys Arg Ala βlu Asn 8mτ 65 70

(2) ZBFCRMATIOV FOR SBQ ID N0:2:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 amino acids

(B) TYPE: amino acid

(C) 8TRANDEI-NESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SBQ ID NO:2:

Ala Ser Val Val Thr βlu Leu Arg Cys βln Cys Leu βln Thr Leu Gin 1 5 10 15 βly Zle Bis Leu ys Asn Zle βln Ser Val Asn Val Arg Ser Pro βly 20 25 30

Pro Bis Cys Ala βln Tar βlu Val Zle Ala Thr Leu lys Asn βly lys 35 40 45

Iff* Ala Cys Leu Asn Pro Ala 8er Pro Met Val βln Lys Zle Zle βlu 50 55 60

I-ys zle Leu Asn X-ys βly Ser Thr Asn 65 70

(2) ZBFORMATZOI FOR SBQ ΣD MO:3

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TTPB: amino acid

(C) βTAAHBEI-tJBSS: single (D) TOPOLOGY: linear (ii) MOLECULE TTPB: peptide

(xi) SEQUENCE DESCRIPTION: SBQ ZD NO:3: lys Zaa Tyr Baa Lys

1 5

(2) ZBFORMATZOH FOR SBQ ZD MO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TTPB: amino acid

(C) 8TRAHDBDNBSS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TTPB: peptide

(xi) SEQUENCE DESCRIPTION: SBQ ID NO:4:

Arg βlu Leu Zaa Xaa Zaa Pro 1 5

Claims

WHAT IS CLAIMED:

1. A polypeptide comprising an amino acid sequence capable of binding IL8R1, wherein the polypeptide is not IL8.

2. Th e polypeptide of claim 1 comprising an amino acid sequence that comprises a first IL8R1 specific binding domain, wherein the polypeptide exhibits a chemokine protein structure and the binding domain is spaced to permit IL8R1 binding.

3. The polypeptide of claim 2, wherein the IL8R1 specific binding domain comprises an amino acid sequence that is identical to one selected from the group consisting of

Ser-Ala-Lys-Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His,

(amino acid residues 1 to 18 of SEQ ID NO:1);

Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His

(amino acid residues 4 to 18 of SEQ ID NO:1);

Lys-Thr-Tyr-Ser-Lys, (amino acid residues 11 to 15 of SEQ ID NO:1);

Lys-Xaa-Tyr-Xaa-Lys, (SEQ ID NO:3 );

Gly-Arg-Glu-Leu-Cys-Leu-Asp-Pro, (amino acid residues 46 to 53 of SEQ ID NO:1); Arg-Glu-Leu-Cys-Leu- Asp-Pro, (amino acid residues 47 to 53 of SEQ ID NO:1); and Arg-Glu-Leu-Xaa-Xaa-Xaa-Pro, (SEQ ID NO.4).

4. The polypeptide of claim 1, further comprising a second IL8R1 -pecific binding domain, wherein the binding domains are spaced to permit IL8R1 binding, and the first binding domain is selected from the group consisting of

Ser-Ala-Lys-Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His,

(amino acid residues 1 to 18 of SEQ ID NO:1); Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His,

(amino acid residues 4 to 18 of SEQ ID NO:1);

Lys-Thr-Tyr-Ser-Lys, (amino acid residues 11 to 15 of SEQ ID NO:1); and

Lys-Xaa-Tyr-Xaa-Lys, (SEQ ID NO:3);

and the second binding domain is selected from the group consisting of

Gly-Arg-Glu-Leu-Cys-Leu-Asp-Pro, (amino acid residues 46 to 53 of SEQ ID NO:1); Arg-Glu-Leu-Cys-Leu- Asp-Pro, (amino acid residues 47 to 53 of SEQ ID NO:1); and Arg-Glu-Leu-Xaa-Xaa-Xaa-Pro, (SEQ ID NO:4).

5. The polypeptide of claim 2, wherein the polypeptide further exhibits a functional characteristic of a chemokine other than native IL8.

6. The polypeptide of claim 1, wherein the amino acid sequence is represented by the formula

A - B - C;

wherein B comprises an IL8R1 specific binding domain, and wherein A and C each comprises amino acid sequence effective to prevent the rapid degradation of the binding domain.

7. The polypeptide of claim 6, wherein A and C each comprise fragments of the amino acid sequence of a chemokine other than IL8.

8. The polypeptide of claim 7, wherein the chemokine is selected from the group consisting of platelet factor 4; β-thrombo globulin; GROα, GROβ, GROγ IP-10, mig, ENA-78, macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, monocyte chemoattractant protein- 1/JE, RANTES, HC-14, C10, and I-309.

9. The polypeptide of claim 7, wherein A and C are fragments from different chemokines.

10. The polypeptide of claim 6, wherein B comprises an amino acid sequence having the formula - b₁ - X - b₂ - wherein b₁ and b₂ are IL8R1 specific binding domains, and X represents one or more amino acid residues that are effective to permit the binding of the polypeptide to IL8R1.

11. The polypeptide of claim 1, wherein the amino acid sequence is represented by the formula

A - B - C;

wherein B comprises an IL8R1 specific binding domain, and wherein A and C comprise of an amino acid sequence that comprises a mutant of a fragment of the amino acid sequence of a chemokine.

12. A polypeptide comprising a first amino acid sequence that comprises a functional characteristic of a first altered IL8R1 specific binding domain.

13. The polypeptide of claim 9, further comprising a second amino acid sequence that comprises a functional characteristic of a second altered IL8R1 specific binding domain.

14. The polypeptide of claim 9, wherein the IL8R1 specific binding domain is selected from the group consisting of

Ser-Ala-Lys-Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His,

(amino acid residues 1 to 18 of SEQ ID NO:1);

Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tys-Ser-Lys-Pro-Phe-His,

(amino acid residues 4 to 18 of SEQ ID NO:1); Lys-Thr-Tyr-Ser-Lys, (amino acid residues 11 to 15 of SEQ ID NO:1); and

Lys-Xaa-Tyr-Xaa-Lys, (SEQ ID NO:3);

Gly-Arg-Glu-Leu-Cys-Leu-Asp-Pro, (amino acid residues 46 to 53 of SEQ ID NO:1); Arg-Glu-Leu-Cys-Leu-Asp-Pro, (amino acid residues 47 to 53 of SEQ ID NO:1); and Arg-Glu-Leu-Xaa-Xaa-Xaa-Pro, (SEQ ID NO:4).

15. The polypeptide of claim 13, wherein the first IL8R1 binding domain before alteration is selected from the group consisting of

Ser-Ala-Lys-Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His,

(amino acid residues 1 to 18 of SEQ ID NO:1);

Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His,

(amino acid residues 4 to 18 of SEQ ID NO:1);

Lys-Thr-Tyr-Ser-Lys, (amino acid residues 11 to 15 of SEQ ID NO:1); and

Lys-Xaa-Tyr-Xaa-Lys, (SEQ ID NO:3);

and the second binding domain is selected from the group consisting of

Gly- Arg-Glu-Leu-Cys-Leu-Asp-Pro, (amino acid residues 46 to 53 of SEQ ID NO:1); Arg-Glu-Leu-Cys-Leu-Asp-Pro, (amino acid residues 47 to 53 of SEQ ID NO:1); and Arg-Glu-Leu-Xaa-Xaa-Xaa-Pro, (SEQ ID NO:4).

16. A polynucleotide comprising a polynucleotide sequence that encodes a polypeptide comprising an amino acid sequence that comprises a first IL8R1 specific binding domain, wherein the polypeptide exhibits a chermokine protein structure and is other than a native IL8 polypeptide.

17. A host cell comprising a polynucleotide that comprises a polynucleotide sequence that encodes a polypeptide comprising an amino acid sequence that comprises a first IL8R1 specific binding domain, wherein the polypeptide exhibits a chemokine proton structure and is other than a native IL8 polypeptide.

18. A method of producing a polypeptide comprising an IL8R1 binding domains, wherein the method comprises:

(a) providing a host cell comprising a polypeptide comprising an amino acid sequence that comprises a first IL8R1 specific binding domain, wherein the polypeptide exhibits a chemokine protein structure and is other than a native IL8 polypeptide.

(b) culturing the host cell under conditions that induce expression of the polypeptide.

19. The polypeptide of claim 2, wherein at least one of amino acid residues corresponding to amino acid residues 11, 13, 15, 47, 48, 49, or 53 of SEQ ID NO:1 is substituted or deleted to alter the IL8R1 binding affinity of the polypeptide.

20. An polypeptide comprising an amino acid sequence that comprises native human IL8 (SEQ ID NO:1), wherein at least one of the amino acid residues 11, 13, 15, 47, 48, 49, or 53 of SEQ ID NO:1 is substituted or deleted to alter the IL8R1 binding affinity of the polypeptide.

21. The polypeptide of claim 13, wherein the amino acid sequence comprises a fragment of native human IL8.

22. A polypeptide comprising an amino acid sequence that comprises an IL8R1 specific binding domain, wherein the bindmg domain comprises an amino acid sequence that is identical to one selected from the group consisting of

Ser-Ala-Lys-Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His,

(amino acid residues 1 to 18 of SEQ ID NO.1);

Glu-Leu-Arg-Cys-Gln -Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His,

(amino acid residues 4 to 18 of SEQ ID NO:1); Lys-Thr-Tyr-Ser-Lys, (amino acid residues 11 to 15 of SEQ ID NO:1);

Lys-Xaa-Tyr-Xaa-Lys, (SEQ ID N0:3);

Gly- Arg-Glu-Leu-Cys-Leu-Asp-Pro, (amino acid residues 46 to 53 of SEQ ID NO:1); Arg-Glu-Leu-Cys-Leu-Asp-Pro, (amino acid residues 47 to 53 of SEQ ID NO:1); and Arg-Glu-Leυ-Xaa-Xaa-Xaa-Pro, (SEQ ID NO:4), wherein the polypeptide is not native IL8 and is capable of binding IL8R1.

23. A method for modulating an IL8R1 mediated biological response comprising contacting cells capable of a IL8R1 mediated biological response with a modulating amount of a polypeptide comprising an amino acid sequence that comprises a first IL8R1 specific landing domain, wherein the polypeptide exhibits a chemokine protein structure and is other than a native IL8 polypeptide.

24. A method of inhibiting IL8 binding to IL8R1 by contacting the cells producing IL8R1 with an inhibiting amount of a polypeptide comprising an amino acid sequence that comprises a first IL8R1 specific binding domain, wherein the polypeptide exhibits a chemokine protein structure and is other than a native IL8 polypeptide.