WO1995035376A2 - Polypeptides a domaines de liaison du recepteur 1 de l'interleukine 8 (il8r1) - Google Patents

Polypeptides a domaines de liaison du recepteur 1 de l'interleukine 8 (il8r1) Download PDF

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Publication number
WO1995035376A2
WO1995035376A2 PCT/US1995/007895 US9507895W WO9535376A2 WO 1995035376 A2 WO1995035376 A2 WO 1995035376A2 US 9507895 W US9507895 W US 9507895W WO 9535376 A2 WO9535376 A2 WO 9535376A2
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amino acid
polypeptide
lys
seq
il8r1
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PCT/US1995/007895
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WO1995035376A3 (fr
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Patricia Tekamp-Olson
Shyamala Venkatakrishna
Mary Ellen Wernette-Hammond
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Chiron Corporation
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Priority to JP8502584A priority Critical patent/JPH10501700A/ja
Priority to EP95924033A priority patent/EP0766737A2/fr
Priority to MX9606720A priority patent/MX9606720A/es
Priority to AU34602/95A priority patent/AU3460295A/en
Publication of WO1995035376A2 publication Critical patent/WO1995035376A2/fr
Publication of WO1995035376A3 publication Critical patent/WO1995035376A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5421—IL-8
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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
  • polypeptides of the present invention can thus act as either antagonists or agonists of IL8 for IL8R1 or IL8R2 binding.
  • cytokines Cells utilize diffusible mediators, called cyto kines, to signal one another.
  • a superfamily of cytokines are the chemo-rines, which includes IL8.
  • 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.
  • 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 ⁇ sheet in the form of a Greek key; and (3) an C-terminal ⁇ helix, which lies over the ⁇ -sheet.
  • the first two cysteine residues are separated by an amino acid residue; the first two cysteine residues in the CC family are not
  • seventeen chemokines have been described.
  • Six are members of the CXC family and include, platelet factor 4 (PF4); ⁇ - thromboglobulin; NAP-1/IL8; gro ⁇ , ⁇ , and ⁇ , ff-10; mig; ENA-78.
  • PF4 platelet factor 4
  • ⁇ - thromboglobulin include, platelet factor 4 (PF4); ⁇ - thromboglobulin; NAP-1/IL8; gro ⁇ , ⁇ , and ⁇ , ff-10; mig; ENA-78.
  • the CXC family is also known as the ⁇ family.
  • the remaining chemokines are part of the CC family:
  • MEP-1 ⁇ and MEP-1 ⁇ macrophage inflammatory proteins
  • MCP-1/JE monocyte chemoattractant protein-1/JE
  • RANTES HC-14; C10, and I-309. This family has also been designated as the ⁇ family.
  • 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, ⁇ -glucuronidase and elastase.
  • IL8 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.
  • EU8R1 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 GRO ⁇ , GRO ⁇ , GRO ⁇ .
  • 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.
  • the polypeptides other than IL8 that posses a chemokine protein structure includes, for example, PF4, ⁇ -thromboglobulin, GRO ⁇ , GRO ⁇ , GRO ⁇ , IP- 10, mig, ENA-78, MlP-1 ⁇ , MlP-1 ⁇ , 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.
  • 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.
  • a chemokine other than IL8 that is provided with a functional characteristic of IL8, i.e., banding to IL8R1
  • 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.
  • 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.
  • 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.
  • such other amino acid sequences are effective to preclude rapid degradation of the polypeptide.
  • binding domains are linked with amino acid sequences derived from polypeptides of the superfctmily of proteins called the chemokines.
  • chemokines amino acid sequences derived from polypeptides of the superfctmily of proteins.
  • 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.
  • chemokines include PF4, ⁇ -thromboglobulin, GRO ⁇ , GRO ⁇ , GRO ⁇ , IP-10, mig, ENA-78, MIP-1 ⁇ , MlP-1 ⁇ , 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:
  • polypeptides other than native IL8, comprising at least one IL8R1 specific binding domain
  • 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.
  • 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 ⁇ sheet (Greek key), and a carboxy-terminal ⁇ helix.
  • native human IL8 forms a ho modimer with a 2-fold axis of symmetry, a six-stranded ⁇ sheet with a pair of ⁇ helices lying atop the ⁇ sheet
  • the placement of the cysteines and the size of the ⁇ 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.
  • TL8R1 specific binding domains These domains are found in the amino-terminal loop and in strand 3 of the ⁇ 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 GRO ⁇ , an IL8R2 agonist
  • 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- Ly
  • 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.
  • 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).
  • binding domains of ⁇ tiier 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.
  • the present polypeptides contain two IL8R1 specific binding domains "spaced within the polypeptide to permit IL8R1 binding.”
  • 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.
  • 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.
  • 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 GRO ⁇ .
  • 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 IC 50 concentration is the concentration that inhibits 50% of the maximal receptor binding of the native IL8; the smaller the IC 50 , the greater the binding affinity. Therefore, a polypeptide is considered to bind to IL8R1 if, for example, its IC 50 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.
  • 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.
  • IL8 receptors are G-coupled proteins which, when proper signal transduction activity occurs, triggers an increase of intracellular Ca 2+ and an activation of phospholipase C.
  • Signal transduction can be measured by observing the levels of inositol triphosphate (IP 3 ) and diacylglycerol (DAG), which are increased due to phospholipase C activation and cyclic AMP (cAMP).
  • IP 3 inositol triphosphate
  • DAG diacylglycerol
  • Conventional assays can be used to measure the intracellular levels of Ca 2+ , IP 3 , and DAG to determine whether the IL8 receptor-mediated response has been modulated.
  • Assays for measuring levels of free cytosolic Ca 2+ 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.
  • chemokine refers to a superfamily of naturally occurring proteins, which are diffusible mediators that cells use to signal one another.
  • 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.
  • chemokines include platelet factor 4 (PF4); ⁇ -thromboglobulin; NAP-1/IL8; gro ⁇ , ⁇ , and ⁇ ; IP-10; mig; ENA-78; macrophage inflammatory proteins (MEP-1 ⁇ and MlP-1 ⁇ ); monocyte chemoattractant protein- 1/JE (MCP-1/JE); RANTES; HC-14; C10, and I-309.
  • PF4 platelet factor 4
  • ⁇ -thromboglobulin include NAP-1/IL8; gro ⁇ , ⁇ , and ⁇ ; IP-10; mig; ENA-78; macrophage inflammatory proteins (MEP-1 ⁇ and MlP-1 ⁇ ); monocyte chemoattractant protein- 1/JE (MCP-1/JE); RANTES; HC-14; C10, and I-309.
  • 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.
  • 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.
  • 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 2 ⁇ 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
  • 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.
  • 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.
  • 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 ⁇ sheet domains as described in greater detail below.
  • 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.
  • 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 (res
  • Another group of IL8R1 binding domains is the group of ⁇ sheet binding domains.
  • the amino acid sequences of these domains are based on the sequence of the third strand of the ⁇ 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).
  • binding domains of other native IL8 can be determined by sequence alignment of the native human IL8 to other native IL8.
  • 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.
  • 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.
  • 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:
  • B represents an IL8R1 specific binding domain.
  • B can represent a sequence containing more than one IL8R1 specific binding domain, such as represented by the formula -b 1 - X - b 2 - where b 1 and b 2 each represent an IL8R1 specific binding domain and X represents one or more amino acid residues.
  • the amino acid sequence of b 1 is selected from a group of amino terminal binding domains
  • the amino acid sequence of b 2 is selected from the group of ⁇ sheet binding domains as disclosed above.
  • 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 ⁇ sheet in the form of a Greek key; and a C-terminal ⁇ helix that lies over the ⁇ sheet.
  • the three stranded ⁇ sheet of the polypeptides of the present invention is preferably of similar size to those found in chemokines.
  • the strands of the ⁇ 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 ⁇ 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 ⁇ sheet
  • the C-terminal ⁇ helix of the polypeptides of the present invention having a chemokine protein structure lies over the ⁇ sheet
  • the length of the ⁇ helix is not critical and may or may not overhang the edge of the ⁇ sheet
  • the length of the ⁇ -helix is from about 9 to 25 residues; more usually, from about 12 to 22; even more usually 15 to about 19 residues.
  • the ⁇ 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.
  • 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.
  • 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.
  • 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.
  • constructing a chimeric chemokine is one means of constructing a polypeptide of the present invention having binding domains appropriately to permit IL8R1 binding.
  • the IL8R1 specific binding domains can spaced to permit IL8R1 binding by substituting the domains for the homologous regions in the GRO ⁇ polypeptide.
  • the C-terminal ⁇ helix of GRO ⁇ can be substituted into the native human IL8 polypeptide. Therefore, the binding domains retain their native configuration.
  • the polypeptide can exhibit non-native IL8 biological activity due to the presence of the GRO ⁇ ⁇ helix.
  • 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.
  • 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
  • fragments of the amino acid sequences of the chemokines can be assembled together to construct a polypeptide of the present invention.
  • the present polypeptide may possess the amino acid sequence of the amino terminal of native human IL8, the first two strands of the ⁇ sheet structure of NAP-2, the third strand of the ⁇ sheet of IL8, and the ⁇ helix of GRO ⁇ .
  • 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.
  • 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.
  • amino acid sequence for the present polypeptides can also be chosen for their ability to confer functional characteristics.
  • the ⁇ helix sequence of GRO ⁇ may be chosen for the present polypeptide to confer a biological activity of GRO ⁇ .
  • sequences of the present polypeptide can be altered to reduce or enhance the biological activities.
  • amino acid sequence a GRO ⁇ /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.
  • amino acid residues of 1 to 18 of SEQ ID NO:1 amino acid residues of 1 to 18 of SEQ ID NO:1;
  • amino acid residues correspond to residues 11, 13, 15, 47, 48, 49, and 53 of SEQ ID NO:1.
  • 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.
  • 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.
  • 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.
  • 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.
  • yeast alcohol dehydrogenase promoter which has an upstream activator sequence (UAS) that modulates expression in the absence of a readily available source of glucose.
  • UAS upstream activator sequence
  • 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.
  • effective promoters can contain a regulatory region linked with a heterologous expression initiation region.
  • 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.
  • ADH yeast alcohol dehydrogenase
  • GPDH yeast glyceraldehyde-3-phosphate-dehydrogenase
  • CMV cytomegalovirus
  • 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.
  • 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
  • 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.
  • 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.
  • viral terminators are also operable in certain host cells; for instance, the SV40 terminator is functional in CHO cells.
  • selectable markers 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.
  • markers may render the host cell immune to drugs such as ampicillin, chloramphenicol, erythromycin, neomycin, and tetracycline.
  • 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
  • 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 2 ⁇ 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.
  • 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
  • the expression vector comprising a polynucleotide encoding the present polypeptide is inserted into the host cell.
  • Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and typically protocol includes either treating th e bacteria with CaCl 2 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.
  • 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.
  • heterologous polynucleotides 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.
  • 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).
  • 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).
  • an insect cell can be infected with a virus containing an polynucleotide encoding the present polypeptides.
  • the virus When the virus is replicating in the infected cell, the present polypeptides will be expressed if operably linked to a suitable promoter.
  • 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;
  • 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.
  • NPV nucleopolyhedrosis viruses
  • GV granulosis viruses
  • baculovirus strain Autographa californica NPV AcNPV
  • Rachiplusia ou NPV Galleria mellonella NPV
  • 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).
  • insect cells Spodoptera frugiperda type 9 are infected with baculovirus strain Autographa californica NPV (AcNPV) containing a polynucleotide encoding the present polypeptides.
  • AcNPV Autographa californica NPV
  • Such a baculovirus is produced by homologous recombination between a transfer vector containing the coding sequence and baculovirus sequences and a genomic baculovirus DNA.
  • 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.
  • 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.
  • another means for determining if coding sequence was incorporated into the baculovirus genome is to sequence the recombinant baculovirus genomic DNA.
  • expression of the present polypeptide by cells infected with the recombinant baculovirus is another verification means.
  • polypeptide of the present invention 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.
  • Receptor binding assays herein may utilize cells that naturally produce the IL8R1 or IL8R2 receptors, such as human neutrophils.
  • a polynucleotide encoding either the IL8R1 or IL8R2 can be introduced into a cell to produce the desired receptor.
  • the assay dther whole cells or membranes can be used to determine receptor binding.
  • 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
  • One means of measuring the biological activity of the present polypeptide is by a signal transduction assay.
  • Typical signal transduction assays measure Ca 2+ , IP 3 , and DAG levels as described in more detail below.
  • Most cellular Ca 2+ 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 Ca 2+ ions in the cytoplasm.
  • fluorescent dyes such as fura-2
  • 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 Ca 2+ 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 Ca 2+ concentrations.
  • the rise of free cytosolic Ca 2+ 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 (IP 3 ). Binding of IL8 or IL8 agonists will increase the concentration of DAG and IP 3 . Thus, signal transduction activity can be measured by monitoring the concentration of these hydrolysis products.
  • DAG 1,2-diacylglycerol
  • IP 3 water-soluble inositol 1,4,5-triphosphate
  • radioactively labelled ⁇ -inositol is added to the media of host cells expressing IL8R1 or IL8R2.
  • the 3 H-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.
  • Amersham provides an inositol 1,4,5-triphosphate assay system.
  • Amersham provides tritylated inositol 1,4,5-triphosphate and a receptor capable of distinguishing the radioactive inositol from other inositol phosphates.
  • a receptor capable of distinguishing the radioactive inositol from other inositol phosphates.
  • an effective and accurate competition assay can be performed to determine the inositol triphosphate levels.
  • 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).
  • Example 1 IL8 Mutants - Altering the ⁇ Sheet Binding Domain
  • 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
  • a protocol for purification of the present polypeptide is set forth bdow.
  • 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.
  • DMEM Dulbecco's Modified Eagle's Medium
  • dFCS dialyzed fetal calf serum
  • NIM Neutrophil Isolation Media
  • chemotaxis assay For the chemotaxis assay, use a Neuroprobe 96- well chemotaxis chamber witfa a 10 ⁇ m duck, 3 ⁇ m pore, bonded polycarbonate membrane. To the bottom of the wells, add 30 ⁇ L of the Hanks, 0.1% BSA buffer with the wanted amount of the present polypeptides. To the top of the wells, add suspension 50 ⁇ L of labeled cells at a concentration of 5 x 10 6 cells/mL. Incubate the cells at 37°C for 25 minutes. Quantify neutrophil migration by fluorescence reading of the filter.
  • Host Cell As in Example 1
  • amino acid sequence is described in PCT appln. no. WO92/00326, herein incorporated by reference.

Abstract

L'invention concerne un procédé destiné à conférer des domaines de liaison spécifiques d'IL8R1 sur un polypeptide, ainsi que des polypeptides contenant des domaines de liaison spécifiques d'IL8R1 modifiés ou non modifiés. L'invention concerne également un procédé d'utilisation de ces polypeptides en tant qu'agonistes ou antagonistes, et un procédé de production desdits polypeptides.
PCT/US1995/007895 1994-06-20 1995-06-20 Polypeptides a domaines de liaison du recepteur 1 de l'interleukine 8 (il8r1) WO1995035376A2 (fr)

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JP8502584A JPH10501700A (ja) 1994-06-20 1995-06-20 インターロイキン８レセプター１（ｉｌ８ｒ１）結合ドメインを有するポリペプチド
EP95924033A EP0766737A2 (fr) 1994-06-20 1995-06-20 Polypeptides a domaines de liaison du recepteur 1 de l'interleukine 8 (il8r1)
MX9606720A MX9606720A (es) 1994-06-20 1995-06-20 Polipeptidos con dominios de enlace al receptor 1 de la interleucina 8(il8r1).
AU34602/95A AU3460295A (en) 1994-06-20 1995-06-20 Polypeptides with interleukin 8 receptor 1 (il8r1) binding domains

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Publication number Priority date Publication date Assignee Title
WO1998014581A1 (fr) * 1996-10-02 1998-04-09 Schering Corporation Chemokines mammaliennes
US5877276A (en) * 1994-09-23 1999-03-02 The Board Of Regents Of The University Of Nebraska Polypeptide agonists for human interleukin-8
US6204024B1 (en) 1997-09-12 2001-03-20 Akzo Nobel N.V. CCR5 RNA transcription based amplification assay
EP1536834A2 (fr) * 2002-09-13 2005-06-08 Chemokine Therapeutics Corp. Conception d'analogues de chimiokines permettant de traiter des maladies humaines

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US5079228A (en) * 1990-02-05 1992-01-07 Board Of Regents, The University Of Texas System Peptide inhibitors of neutrophil activating factor induced chemotaxis
WO1993011159A1 (fr) * 1991-12-04 1993-06-10 The Biomedical Research Centre Limited Analogues de l'interleukine-8 humaine

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US5079228A (en) * 1990-02-05 1992-01-07 Board Of Regents, The University Of Texas System Peptide inhibitors of neutrophil activating factor induced chemotaxis
WO1993011159A1 (fr) * 1991-12-04 1993-06-10 The Biomedical Research Centre Limited Analogues de l'interleukine-8 humaine

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FEBS LETTERS, vol. 341, no. 2-3, March 1994 AMSTERDAM NL, pages 187-192, P. LOETSCHER ET AL 'Both Interleukin-8 receptors independently mediate chemotaxis. Jurkat cells transfected with IL-8R1 or IL-8R2 migrate in response to IL-8, GROalpha and NAP-2 ' *
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 266, no. 28, 5 October 1991 MD US, pages 18989-18994, C.A. HEBERT ET AL 'Scanning mutagenesis of Interleukin-8 identifies a cluster of residues required for receptor binding' *
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 23, 10 June 1994 MD US, pages 16075-16081, I. CLARK-LEWIS ET AL 'Structural requirements for Interleukin-8 function identified by design of analogs and CXC chemokine hybrids' *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5877276A (en) * 1994-09-23 1999-03-02 The Board Of Regents Of The University Of Nebraska Polypeptide agonists for human interleukin-8
WO1998014581A1 (fr) * 1996-10-02 1998-04-09 Schering Corporation Chemokines mammaliennes
US6204024B1 (en) 1997-09-12 2001-03-20 Akzo Nobel N.V. CCR5 RNA transcription based amplification assay
EP1536834A2 (fr) * 2002-09-13 2005-06-08 Chemokine Therapeutics Corp. Conception d'analogues de chimiokines permettant de traiter des maladies humaines
EP1536834A4 (fr) * 2002-09-13 2007-11-07 Chemokine Therapeutics Corp Conception d'analogues de chimiokines permettant de traiter des maladies humaines

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EP0766737A2 (fr) 1997-04-09
AU3460295A (en) 1996-01-15

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