WO1995035376A2

WO1995035376A2 - Polypeptides with interleukin 8 receptor 1 (il8r1) binding domains

Info

Publication number: WO1995035376A2
Application number: PCT/US1995/007895
Authority: WO
Inventors: Patricia Tekamp-Olson; Shyamala Venkatakrishna; Mary Ellen Wernette-Hammond
Original assignee: Chiron Corporation
Priority date: 1994-06-20
Filing date: 1995-06-20
Publication date: 1995-12-28
Also published as: AU3460295A; CA2193526A1; WO1995035376A3; EP0766737A2; MX9606720A; JPH10501700A

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

Polypeptides with

Interleu kin 8 Recep 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 encoding the polypeptides of the present invention, a method of using the 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 cyto kines, to signal one another. A superfamily of cytokines are the chemo-rines, which includes IL8. A review aiticle about the chemokine superfamily was written by Miller et al., Crit Rev Im mun 12(1.2): 17-46 (1992) and Baggiolini et al., Adv Immunol 55: 97-179 (1994), herein incorporated by reference. The chemokines 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 chemokines 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; mig; 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 MEP-1β); monocyte chemoattractant protein-1/JE (MCP-1/JE); RANTES; HC-14; C10, and I-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. J EXP Med 167: 1364-1376; Ribero et al., Immnnology 73: 472-477 (1991). The molecule also activates the release of superoxide anions and elicits release of the primary granule constituents of neutrophils, including mydoperoxidase, 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 "EU8R1" and "IL8R2." The amino acid sequence of these polypeptides are described in Murphy et al., 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 Ca²⁺. 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-1α, MlP-1β, MCP-1/JE, RANTES, HC-14, C10, 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 IL8, i.e., banding to IL8R1, may be a GR Oγ protein, so that the resulting chim era is an IL8/GROγ polypeptide. The altered domain may be made in the native IL8 or be introduced into another polypeptide, for example, that possesses a c hemokine 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 IL8 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 IL8R1. 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 the chemokines. Thus, tiie IL8R1 binding domains can be linked with fragments derived from other chemokines to construct polypeptides that exhibit the 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, MIP-1α, MlP-1β, MCP-1/JE, RANTES, HC-14, C10, and I-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 with decreased binding affinity to IL8R1 as compared to native IL8 can be effective competitors of native IL8 for the 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 the 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 helix extends over the top of the β sheet Further, native human IL8 forms a ho modimer with a 2-fold axis of symmetry, a six-stranded β sheet with a pair of α helices lying atop the β sheet The placement of the cysteines and the size of the β sheet are also factors in the three dimensional structure. 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 the amino-terminal loop and in strand 3 of the 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 acid residues of 1 to 18 of SEQ ID NO:1); more preferably, the amino acid sequence contains the sequence: Glu-Leu-Arg-Cys-Gln-Cys-Ile- Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His (residues 4 to 18 of SEQ ID NO:1); even more preferably, the amino acid sequence contains the sequence: Lys-Thr-Tyr-Ser-Lys (residues 11 to 15 of SEQ ID NO:1). The amino acid sequence of an IL8R1 specific binding domain can also contain the 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 the sequence of the amino terminal portion of native IL8.

Another example of the amino acid 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, the amino acid sequence contains the sequence Arg-Glu-Leu-Cys- Leo-Asp-Pro (residues 47 to 53 of SEQ ID NO:1). The amino acid sequence of an IL8R1 specific binding domain can contain the 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 the sequence of the 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, the 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 the primary sequence of the polypeptide. Consequently, when the polypeptide assumes its three dimensional conformation, the binding domains are positioned to efficiently interact with the other portions Of the polypeptide and/or the receptor to permit IL8R1 binding. Preferably, the present polypeptide possesses a chemokine protein structure to mimic the 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 acids can be inserted, deleted, or substituted to truncate or excise the binding domain from the polypeptide, such as native IL8. IL8R1 binding domains can be excised from native human IL8 and replaced with amino acid sequence from a corresponding region of other homologous chemokines, such as GROγ. The amino acid 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 the native human IL8 (SEQ ID NO:1). These amino acid residues are maintained in the 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 the art, the receptor binding is usually assessed by the "binding affinity" of the polypeptide. One means of determining binding affinity is to measure the ability of the polypeptide to compete with native IL8 for IL8R1. The IC₅₀ concentration is the concentration that inhibits 50% of the maximal receptor binding of the native IL8; the smaller the IC₅₀, the greater the binding affinity. Therefore, a polypeptide is considered to bind to IL8R1 if, for example, its IC₅₀ 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 the binding of IL8 to its receptor. Modulation occurs when the instant polypeptides compete with the native IL8 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 the 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 the term is applied herein, the 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 Ca²⁺ and an activation of phospholipase C. Signal transduction can be measured by observing the levels of inositol triphosphate (IP₃) and diacylglycerol (DAG), which are increased due to phospholipase C activation and cyclic AMP (cAMP). Conventional assays can be used to measure the intracellular levels of Ca²⁺, IP₃, and DAG to determine whether the IL8 receptor-mediated response has been modulated. Assays for measuring levels of free cytosolic Ca²⁺ are known.

"Native IL8" refers to a polypeptide having an amino acid 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 the amino acid sequence as shown in SEQ ID NO:1. 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-thromboglobulin; NAP-1/IL8; gro α, β, and γ; IP-10; mig; ENA-78; macrophage inflammatory proteins (MEP-1α and MlP-1β); monocyte chemoattractant protein- 1/JE (MCP-1/JE); RANTES; HC-14; C10, and I-309. Other chemokines can be identified by their amino acid homology to the 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 the 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 the 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 the present polypeptide than in the 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 the chimera has the same receptor binding affinity, for example. A composition containing A is "substantially free of B when at least 85% by weight 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 the coding sequence when the promoter is not operably linked to the 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 that 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 cell. Examples of such origins are the 2μ and autonomously replicating sequences, which are effective in yeast; and the 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 the present polypeptides are cultured "under conditions inducing expression." Such conditions allow transcription and translation of the polynucleotide encoding the polypeptide. These conditions include cultivation temperature, oxygen concentration, media composition, pH, etc. For example, if the trp promoter is stilized in the 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 Acid Sequence of IL8R1 Specific Binding Domains

The present inventors have determined the 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 the present invention, the 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 the polypeptide of the present invention contains two binding domains. One domain is selected from the 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 acid sequences are examples of amino terminal binding domains. The group was thusly named because the sequences are based on the amino acid sequence of the amino terminal portion of native IL8. Using the binding domains of native human IL8 as an example, the amino acid sequence of an IL8R1 binding domain is Ser-Ala-Lys-Glu-L eu-Arg-Cys-Gl n-Cys-IIe-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His, (amino acid residues of 1 to 18 of SEQ ID NO:1); more preferably, the amino acid sequence is Glu- Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His (residues 4 to 18 of SEQ ID NO:1); even more preferably, the amino acid sequence is Lys-Thr-Tyr-Ser-Lys (residues 11 to 15 of SEQ ID N0:1). The amino acid 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 the sequence of the third strand of the β sheet of native IL8. An example of the amino acid sequence of such an IL8R1 specific binding domain is Gly-Arg-Glu-Leu-Cys-Leu-Asp-Pro (residues 46 to 53 of SEQ ID NO:1); more preferably, the 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 -b₁- X - b₂ - where b₁ and b₂ each represent an IL8R1 specific binding domain and X represents one or more amino acid residues. Preferably, the amino acid sequence of b₁ 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 the β sheet.

The three stranded β sheet of the polypeptides of the present 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 amino acid 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 the third strand of the β sheet

The C-terminal α helix of the polypeptides of the present invention having a chemokine protein structure lies over the β sheet The length of the 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 acid sequence of the amino terminal IL8R1 binding domains are incorporated in the loop portion. The entire portion including tail and loop is from about 25 to 14 amino acid residues; more preferably, from about 22 to about 18 amino acid residuces. The loop comprises from about 15 to about 6 amino acid residues; more preferably about 12 to about 8 amino acid residues. In addition, preferably, the tail of the amino terminal portion comprises the amino acid sequence Glu-Leu-Arg sequence. This sequence is non-specific sequence for IL8 receptor binding. ln addition, the 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 afrect binding of either IL8 receptors.

More specifically, constructing a chimeric chemokine is one means of constructing a polypeptide of the 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 the native human IL8 polypeptide. Therefore, the binding domains retain their native configuration. In such an embodiment the polypeptide can exhibit non-native IL8 biological activity due to the presence of the GROγ α helix. Thus, in one embodiment of the present invention, the 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 IL8R1 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 acid residues by altering polynucleotide sequences encoding the polypeptide to be altered are conventional in the art

In addition, fragments of the amino acid sequences of the chemokines can be assembled together to construct a polypeptide of the present invention. For example, the present polypeptide may possess the amino acid sequence of the amino terminal of native human IL8, the first two strands of the β sheet structure of NAP-2, the third strand of the β sheet of IL8, and the α helix of GROβ. The amino acid sequences to be utilized to construct the polypeptide of the present invention do not have to be identical to the sequences found in the chemokines to exhibit the desired secondary structure features. For example, the amino acid sequences may be mutants or fusions of the sequences found in the chemokines. Mutants of the chemokines can be constructed by making conservative amino acid substitutions of such. The following are examples of conservative

substitutions:

y Al V l Leu; Asp <→ Glu; Lys g; ; and

Also, insertions and deletions can be made to the amino acid sequences of the chemokines provided that the chemokine protein structure is maintained.

The choice of amino acid sequence for the 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 GROγ/TL8 chimera exhibiting IL8R1 specific binding can be altered to reduce its ability to trigger IL8R1 signal transduction.

The binding domains can be altered to increase or decrease the binding affinity of the present polypeptide to IL8R1. Such polypeptides with 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 the binding affinity of the polypeptide to IL8R1. Other altered landing domains can be made by deleting or inserting residues to the amino acid sequence of the unaltered binding domains so as to alter the binding affinity of the polypeptide. Additional amino acid residues can be incorporated at the N- or C-terminus. In particular, some or all of the amino acid residues of the binding domain can be excised to decrease the binding affinity of the present polypeptide.

The amino acid residues that are of particular interest for IL8R1 specific binding are highlighted in the binding domains below:

Ser-Ala-Lys-Glu-L eu-Arg-Cys-Gl n-Cys-IIe-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His,

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

G lu-Leu-Arg-C ys -Gln-Cys-Ile- Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His,

(residues 4 to 18 of SEQ ID NO:1); Lys-Thr-Tyr-Ser-Lys, (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, (residues 46 to 53 of SEQ ID NO:1);

Arg-Glu-Leu-Cys-Leu-Asp-Pro, (residues 47 to 53 of SEQ ID NO:1); and

Arg-Glu-Leu-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:1. Preferably, these amino acid residues in the binding domains are altered by substitution, deletion, or insertion of another amino acid residue to enhance or decrease the binding affinity of the domain.

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

Once the amino acid sequence of the 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 the present polypeptides can be used to construct an expression vector to produce the polypeptide. At the minimum, an expression vector will contain a promoter which is operable in the host cell and operably linked to the polynucleotide encoding the present polypeptides. Expression vectors may also include signal sequences, terminators, selectable markers, origins of replication, and sequences homologous to host cell 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 the present polypeptide. 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 represser 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 glyceraldehyde-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 polynucleotides encoding the present polypeptides 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 present 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 polynucleotide encoding the present polypeptide. Usually, the terminator of native host cell proteins are operable when attached 3' of the polynucleotide encoding the present polypeptide. 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 optimally 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 biosynth etic 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 depression vector herein to repficate 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 that can be used herein are the 2μ and autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.

Expression vectors herein 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 in the expression vector to integrate the expression cassette. Alternative, the homologous sequences are not linked to the expression vector. 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 herein are known in the art and available from the American Type Culture Collection (ATCC). Bacterial hosts suitable for e xp ressing the present polypeptides include, without limitation: Campylobacter, Bacillus, Escherichia, Lactobacillus, Pseudomonas, Staphylococcus, and Streptococcus. Yeast hosts from the following genera may be utilized: Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomy ces, and Yarrowia. Immortalized mammalian host cells that can be used herein 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 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) in 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 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, for example, are well-known in the art, and typically protocol includes either treating th e 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 as described in 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 CoIE1-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. Biophys. Acta 949:318: Escherichia), (Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus); (Fiedler et al. (1988) Anal. Bi ochem 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 also 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. These methods are described in, 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, 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 Berg et al. (1990) Bio/Technology 8:135. for Kluyveromyces; Cregg et al. (1985) Mol. Cell. Biol. 5:3376: and Kunze et al. (1985) J. Basic Microbiol. 25:141. and U.S. Patent Nos. 4,837,148 and 4,929,555; for Pichia;

Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929. and Ito et al. (1983) J. Bacteriol. 153:163 for Saccharomyces; Beach and Nurse (1981) Nature 300:706 for Schizosaccharomyces; Davidow et al. (1985) Curr. Genet.10:39. and Gaillardin et al. (1985) Curr. Genet.10:49 for 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 polynucleotides) 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 slightiy 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 witit techniques th at 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 an polynucleotide encoding the present polypeptides. When the virus is replicating in the infected cell, the 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 the Class Insecta can be grown in the media of this invention. The orders Diptera and Lepidoptera are preferred. Example of insect spedes 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 that can be used herein are exemplary: Carpocapsa pomeonella (preferably, cell line CP-128); Trichoplusia ni (preferably, cell line TN-368); Autograph californica; Spodoptera frugiperda (preferably, cell fine Sf9); Lymantria dispar, Mamestra brassicae; Aedes albopictus; Orgyia pseudotsugata. Neodiprio sertifer; Aedes aegypti; Autheraea 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;

Attticarsia germmetalia; Apanteles melanoscelu; Arctia caja; and Porthetria dispar. Preferred 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.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 californica 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 Spodoptera frugiperda type 9 (SF9) are infected with baculovirus strain Autographa californica NPV (AcNPV) containing a polynucleotide encoding the present polypeptides. 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 polynucleotide encoding the present polypeptides, as described in 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 a polynucleotide encoding the present polypeptides. Without a functional essential gene the baculovirus genome cannot produce a viable virus. Thus, the viable viruses from the transfection most likely contain the polynucleotide encoding the present polypeptide and the needed essential gene sequences from the transfer vector. Further, lack of occlusion bodies in the infected cells are another verification that the polynucleotide encoding the present polypeptide 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 polynucleotide encoding the 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 the recombinant baculovirus genomic DNA.

Alternatively, expression of the present polypeptide by cells infected with the recombinant baculovirus is another verification means.

Isolation of the Polypeptides

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

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 desired purity. The separation technique selected is not critical to the invention. Many techniques are available. For example, the following are separation techniques differentiating size: dialysis, ultrafiltration, gel filtration, and SDS polyacyr lamide gel electrophoresis. 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

i. 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 the assay, dther 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 for binding to IL8R1. The less the radioactivity measured the less the native EL8 was binding to the receptor. ii. Biological Activity Assays

Signal Transduction Assays

One means of measuring the biological activity of the present polypeptide is by a signal transduction assay. Typical signal transduction assays measure Ca²⁺, IP₃, and DAG levels as described in more detail below.

Most cellular Ca²⁺ ions are sequestered in the mitochondria, endoplasmic reticulum, and other cytoplasmic vesicles, but binding of present polypeptide to the IL8R1 will trigger an 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 culture media of the host cells expressmg IL8R1 or IL8R2 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 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 al., FEBS Lett 311(2): 179-183 (1992) describe some examples of assays measuring free intracellular Ca²⁺ concentrations.

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 (IP₃). 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 Η-inositol is added to the media of host cells expressing IL8R1 or IL8R2. The ³H-inositol taken up by the cells and after stimulation of the cells with the present polypeptide, the resulting inositol triphosphate is separated from the 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 the radioactive inositol from other inositol phosphates. With th ese reagents an effective and accurate competition assay can be performed to determine the inositol triphosphate levels.

Mydoperoxidase Assay

A mydoperoxidase (MPO) assay is an example of another method for measuring the 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 the 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 the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.

Example 1: IL8 Mutants - Altering the β Sheet Binding Domain

Polypeptides:

The amino acid sequence of the polypeptides depicted below is as found in SEQ ID NO:1 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 5.5. in 50 mL plastic screw cap tube and add 400 μL of resin slurry. Rock tube overnight at 4°C. Centrifuge tube at 3,000 rpm at 4ºC for twenty minutes. Pour off supernatant, unbound material.

To wash the resin, transfer the 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 elute the present polypeptides, add 100 μL 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 labeled 1.0 M NaCl. Repeat elution steps.

Receptor Binding Assay:

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

Test for present polypeptides' ability to bind IL8R2 receptors as measured by competition with ¹²⁵I-IL-8.

DNA encoding the 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., Science 253: 1278 (1991). DG44, Chinese hamster ovary (CHO) cells were transfected with either IL8R1 or IL8R2 cDNA under the control of the cytomegalovirus immediate early promoter and enhancer. A standard caldum phosphate protocol was used. The expression vector included the dihydrofolate reductase gene. Thus, the cells were selected in hypoxanthine and thymidine deficient medium. Clones expressing the 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 RemovaWell 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, hypoxanthine, thymidine, 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 ¹²⁵I-IL-8 and the wanted concentrations of the present polypeptides. The binding buffer 25 mM Hepes, pH 7.0; 150 mM NaCl ; 5 mM CaCl₂; 5 mM MgCl₂; 1 mg/mL BSA. Measure non-specific binding in the presence of 1 μg/mL of unlabded IL-8. Wash cells once with Hepes-BSA binding buffer. Determine bound ¹²⁵I-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 the purified neutrophils by incubation with 2',7'-bis-(2-carboxyethyl)- 5(and-6)-carboxyfluorescein, acetoxymethyl ester, manufactured by Molecular Probes, Eugene, Oregon. The procedure as described in DeForge et al., J Immunol 148: 2133- 2141 (1992). Suspend the neutrophils at 2 x 10⁶ 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 the labeled neutrophils twice with PBS without calcium and magnesium. Resuspend the cell pdlet in Hanks Balanced Salt solution with 0.1% BSA.

For the chemotaxis assay, use a Neuroprobe 96- well chemotaxis chamber witfa a 10 μm duck, 3 μm pore, bonded polycarbonate membrane. To the bottom of the wells, add 30 μL of the Hanks, 0.1% BSA buffer with the wanted amount of the present polypeptides. To the top of the wells, add suspension 50 μL 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 the filter.

Use to detect for fluorescence, 485 nm excitation 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 the Amino Terminal Binding Domain

Polypeptides:

The amino acid sequence of the polypeptides as found in SEQ ID NO:1 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: GROγ/IL8R1 Binding Domain Chimeras

Polypeptides:

The amino acid sequence is described in PCT appln. no. WO92/00326, herein incorporated by reference. The amino acid sequence of the polypeptides as found in SEQ ID NO:2 except a follows:

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

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.