KR20050020749A - Compositions useful as ligands for the formyl peptide receptor like 1 receptor and methods of use thereof - Google Patents

Abstract

We have identified the CKβ8-1 truncated variant, CKβ8-1 (25-116), as a bifunctional ligand for two different GPCRs: chemokine receptor CCR1 and formyl peptide receptor like 1 receptor (FPRL1). In addition, the inventors confirmed that in addition to functional activity in CCR1, CKβ8-1 (25-116) is a functional ligand for GPCR receptor FPRL1 involved in inflammatory response and immunity by mobilizing monocytes and neutrophils. In addition, the inventors have identified an alternative cleaved exon of CKβ8-1, SHAAGtide. In combination with mochemokine CKβ8-1 (25-116), SHAAGtide works completely in both monocytes and neutrophils known to express FPRL1.

Description

COMPOSITIONS USEFUL AS LIGANDS FOR THE FORMYL PEPTIDE RECEPTOR LIKE 1 RECEPTOR AND METHODS OF USE THEREOF}

The present invention relates to compositions useful as ligands for formyl peptide receptor like 1 receptors and methods of use thereof.

Chemokines (chemotactic cytokines) function as molecular markers for the recruitment and activation of T lymphocytes, neutrophils, and macrophages and signal the pathogen's battleground. The recruitment of leukocytes, which lead to resistance to infection, depends on the gradient of chemokines. Chemokines are a family of small molecules (8-10 KD) that mediate various biological processes, including leukocyte trafficking, homing, immunoregulation, hematopoiesis, and angiogenesis. to be. Chemokines play an essential role in innate immunity and inflammatory responses (Baggiolini et al. (1994); Baggiolini et al. (1997); Rollins (1997)). Four subfamilies of chemokines were reported based on the distance between the first two conserved cysteine residues: C, CC, CXC, CX3C. All known chemokines are signaled via four groups of seven transmembrane receptors belonging to the G protein-coupled receptor and the pertussis toxin-sensitive heterotrimeric G protein of the G _i line: XCR, CCR, CXCR, CX3CR (Murphy et. al. (2000)). Extracellular binding can activate specific signal transduction pathways that induce a variety of responses, such as chemotaxis. In a chemokine system, multiple chemokines can activate a single chemokine receptor; For example, receptor CCR1 ligates regulated on activation normal T tell expressed (RANTES), macrophage inflammatory protein (MIP-1α), and MIP-1β chemokines. Similarly, a single chemokine can activate several receptors (Mantovani (1999)).

Monocytes and neutrophils, which play an important role in the pathogenesis of inflammation and antigen presentation, respond to chemokines (Lee et al. (2000)). Monocytes express chemokine receptors CCR1, CCR2, CCR5, CCR8, CXCR2, CXCR4 (Uguccioni et al. (1995); Weber et al. (2000)). Ligand MIP-1α and monocyte chemoattractant protein 1 (MCP1) have been reported to be potent monocyte activators in vitro (Fantuzzi et al. (1999)). Neutrophils are critical for a variety of acute inflammatory responses and play a role in directing immunity to the Th1 response (Bonecchi et al. (1999)). They respond to some CXC chemokines but do not migrate to most CC chemokines. Human neutrophils express two highly substituted IL-8 receptors, CXCR1 and CXCR2.

CCL23; hmrp-2a; Myeloid progenitor inhibitor factor 1 (MPIF-1); Chemokine CKβ8, known as SCYA23 (current nomenclature and Genome ID system), is a 99-amino acid CC chemokine with six cysteines. It is expressed essentially in the liver, lungs, pancreas and bone marrow. CKβ8 exhibits chemotactic activity in monocytes, dendritic cells, dormant lymphocytes (Forssmann et al. (1997)) and inhibits colony formation in bone marrow-derived low proliferative potential colony-forming cells (Patel et al. (1997). )). CKβ8-1, an alternative cleavage form of 116-amino acid CKβ8, has been reported. CKβ8 and CKβ8-1 mature forms have been designated ligands for the CCR1 receptor (Youn et al. (1998)). Cross-desensitization studies in monocytes and eosinophils suggest that CKβ8-1 binds predominantly to CCR1. Further processing at the NH ₂ -terminus of CKβ8 results in 76-75 residue proteins showing more pronounced activity in CCR1 expressing cells (Macphee et al. (1998), Berkhout et al. (2000)).

In addition to chemokine receptors, neutrophils and monocytes also express G protein-bound N-formyl peptide receptor (FPR) and homologous N-formyl peptide receptor like 1 (FPRL1). FPRL1 was initially defined as an orphan receptor because FPRL1 was not known when it was first cloned (Bao et al. (1992); Murphy et al. (1992); Ye et al. (1992)). Since it binds to lipoxin A ₄ , it has been designated as an LXA ₄ receptor (Fiore et al. (1994)). In addition, several different peptides / proteins have been reported to bind FPRL1 with low affinity (see FIG. 1). Serum amyloid A, a protein secreted during acute inflammation, has been reported as a functional ligand of moderate affinity (Su et al. (1999)). β amyloid fragment (1-42) and neurotoxic prion peptide 106-126 are also low affinity ligands, suggesting that FPRL1 plays a role in neurodegenerative diseases (Le et al. (2001)). Some other low affinity ligands are: peptides derived from HIV envelope proteins (Su et al. (1999), Deng et al. (1999)); Helicobacter pylori peptide, Hp (2-20). Some synthetic peptides such as Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ (WKYMVm) and Trp-Lys-Tyr-Met-Val-Met-NH ₂ (WKYMVM) (“W peptide 1 and 2 ") have been reported as strong ligands for these receptors (Christophe et al. (2001); Baek et al. (1996)). However, these non-naturally occurring peptides derived from random hexapeptide libraries were physiologically independent.

summary

We have identified that CKβ8-1 truncated variant, CKβ8-1 (25-116), is involved in inflammatory response and innate immunity through its role as a functional ligand for formyl peptide receptor like 1 receptor (FPRL1). In addition, we have shown that in addition to CKβ8-1 (25-116), alternatively cleaved exons of CKβ8-1, ie SHAAGtide and other truncated variants of SHAAGtide, act on both cells known to express FPRL1. Confirmed. Functional SHAAGtide induces calcium flux immediately after receptor-ligand binding in leukocytes and attracts monocytes, neutrophils, mature dendritic cells (mDCs) and immature dendritic cells (iDCs).

In one embodiment, the invention relates to proteins and peptides including SHAAGtide and SHAAGtide except CKβ8-1 (25-116). In addition, the present invention relates to a nucleic acid encoding SHAAGtide, a protein containing SHAAGtide and a nucleic acid encoding a peptide, an antibody specifically binding to SHAAGtide, a fusion protein comprising SHAAGtide.

In another embodiment, the invention is directed to a composition comprising SHAAGtide or a protein or peptide comprising a SHAAGtide sequence. Such compositions are suitable for administration to enhance FPRL1 activity in a subject.

In another embodiment, the present invention is directed to a kit comprising such a composition. Such kits are, for example, assembled to facilitate administration of the pharmaceutical composition.

In another embodiment, the invention relates to a method of treating a disease in a patient, said method comprising administering a compound comprising SHAAGtide or a protein or peptide comprising a SHAAGtide sequence to modulate the activity of the FPRL1 receptor do.

In another embodiment, the present invention relates to methods and kits useful for identifying such antagonists. Such methods consist of contacting a composition comprising a biologically active SHAAGtide or a protein or peptide comprising a SHAAGtide sequence with a FPRL1 receptor in the presence of a candidate antagonist molecule.

1 is a table depicting reported FPRL1 endogenous low affinity ligands and non-natural ligands.

FIG. 2 is a table showing amino acid sequence alignment of CCL23 / CKβ8 variants with human CCL15 / MIP-1α and CCL3 / MIP-1δ.

We have identified the CKβ8-1 truncated variant, CKβ8-1 (25-116), as a bifunctional ligand for two different GPCRs: chemokine receptor CCR1 and formyl peptide receptor like 1 receptor (FPRL1). In addition, the present inventors confirmed that in addition to being active as a CCR1 ligand, CKβ8-1 (25-116) is involved in the inflammatory response and immunity by mobilizing monocytes and neutrophils through its role as a functional ligand for FPRL1. CKβ8 attracts cells including monocytes, dendritic cells, dormant lymphocytes via OCR1, but lacks the alternatively-cleaved exon found in CKβ8-1 (25-116) (SHAAGtide sequence). CKβ8-1 (1-116) (116 amino acids), an alternatively-cleaved form of CKβ8, is a functional ligand for CCR1 receptors such as CKβ8. However, CKβ8-1 (1-116) does not function through the SHAAGtide sequence.

In addition, the present inventors have found that the novel variant of the peptide (the variant of SHAAGtide peptide and SHAAGtide peptide-then "SHAAGtide"), the truncated variant of the cleavage exon of CC chemokine CCL23, CKp8-1 (25-116), is highly resistant to the FPRL1 receptor. It was confirmed to be an effective ligand. These peptides induce calcium flux in white blood cells expressing FPRL1. In addition, SHAAGtide effectively attracts monocytes, neutrophils, mature dendritic cells (mDC), immature dendritic cells (iDC), and other leukocyte subsets. Together with the Mochemokine CKβ8-1 (25-116), the SHAAGtide peptide (SEQ ID NO: 1) and certain SHAAGtide variants act on both monocytes and neutrophils known to express FPRL1. Functional SHAAGtide induces calcium flux immediately after receptor-ligand binding in leukocytes and attracts monocytes, neutrophils, mature dendritic cells (mDC) and immature dendritic cells (iDC) in chemotaxis assays.

The present invention relates to proteins and peptides including SHAAGtide and SHAAGtide except CKβ8-1 (25-116) and CKp8-1 (1-116). In addition, the present invention includes nucleic acids encoding SHAAGtide except nucleic acids encoding CKβ8-1 (25-116) and CKp8-1 (1-116) and nucleic acids encoding proteins and peptides including SHAAGtide. Regarding The present invention also relates to compositions comprising SHAAGtide, as well as proteins and peptides comprising SHAAGtide, including CKβ8-1 (25-116). Such compositions are suitable for administration to enhance FPRL1 activity in a subject.

The invention also relates to kits comprising such compositions. Such kits are, for example, assembled to facilitate administration of the pharmaceutical composition.

The present invention also relates to a method for treating a subject in need of stimulation of an inflammatory response and innate immunity. Stimulation of this activity is directed to individuals suffering from diseases, such as infectious diseases (and vaccinations, patent application "Methods and Compositions for Inducing an Immune Response", filed May 7, 2000, reference no. 10709/23). helpful. This method consists in stimulating the FPRL1 receptor by administering a composition comprising SHAAGtide, a protein or peptide comprising SHAAGtide, or other stimulatory molecule.

The present invention also relates to methods of treating individuals in need of downregulation of inflammatory responses and innate immunity. Downregulation of this activity is beneficial for individuals suffering from neurodegenerative diseases such as Alzheimer's disease or Creutzfeldt-Jakob disease. This method consists in administering a composition comprising an antagonist to FPRL1 receptor function to downregulate the FPRL1 receptor.

The invention also relates to methods and kits useful for identifying such antagonists. Such methods consist of contacting a composition comprising a biologically active SHAAGtide or a protein or peptide comprising an active SHAAGtide with a FPRL1 receptor in the presence of a candidate antagonist molecule. Antagonists for FPRL1 receptor function are compounds that reduce receptor activity compared to the results observed in the absence of candidate compounds. This method is carried out in vitro or in vivo. In addition, the kits are assembled to facilitate such in vitro or in vivo testing.

Molecules Containing SHAAGtide and SHAAGtide

Polypeptides Containing SHAAGtide Peptides and SHAAGtide

Table 1 shows the SHAAGtide polypeptide sequence (SEQ ID NO: 1) and the polypeptide sequences of the SHAAGtide truncated variant and other variants.

FIG. 2 shows the SHAAGtide polynucleotide sequence (SEQ ID NO: 12) and the polynucleotide sequence of the variant different from the SHAAGtide truncated variant. Table 3 shows the human CKβ8-1 (25-116) nucleotide sequence (SEQ ID NO: 20). 2, human CCL15 / MIP-1α (SEQ ID NO: 19); CCL3 / MIP-1δ (SEQ ID NO: 17); Leukotactin (SEQ ID NO: 18); The amino acid sequence of the human CCL23 / CKβ8 variant is shown [CKβ (1-99) -SEQ ID NO: 13; CKβ (25-99) -SEQ ID NO: 14; CKβ (1-116) -SEQ ID NO: 15; Amino acid sequence alignment of CKβ (25-116) -SEQ ID NO: 16]. Four conserved cysteine residues are observed in the box and two additional cysteines are observed in the dashed box which are not normally observed in the CC chemokine lineage. Alternatively cleaved exons of CCL23 / CKβ8-1 are underlined.

Table 1: SHAAGtide and various other truncated variants-amino acid sequences

Table 2: SHAAGtide and various other truncated variants-polynucleotide sequences

Table 3: Human CKβ8-1 (25-116) Nucleotide Sequence (SEQ ID NO: 12)

SHAAGtide molecules, derivatives, analogs

SHAAGtide of the present invention includes the molecules described in Table 1. In addition, various other derivatives of SHAAGtide peptides and nucleotides can be synthesized by standard techniques. Derivatives are nucleic acid sequences or amino acid sequences that are formed from native compounds directly or indirectly by modification or partial substitution. An analog is a nucleic acid sequence or amino acid sequence that has a structure similar to the native compound but is inconsistent and distinct in terms of a particular component or side chain. Analogs can be synthesized from different evolutionary origins.

Derivatives and analogs may or may not be full length, if they carry modified nucleic acids or amino acids. For example, SEQ ID NO: 3 contains only the first N-terminal 15 amino acids of the SHAAGtide molecule (SEQ ID NO: 1). Derivatives or analogs of SHAAGtide nucleic acids or peptides include regions substantially homologous to SHAAGtide nucleic acids or peptides with at least 70%, 80% or 95% identity in a nucleic acid or amino acid sequence of identical size or in a sequence aligned by homology algorithms. Molecules, or molecules that can be hybridized with complementary sequences encoding the above-described peptide sequences under stringent, moderately stringent or lowly stringent conditions, include, but are not limited to (Ausubel et al., 1987). Complementary nucleic acid molecules are molecules in which a stable double helix is produced by the formation of hydrogen bonds that are substantially complementary to the sequence and that are nearly mismatched. "Complementarity" means Watson-Crick or Hoogsteen base pairs between nucleotides.

The specificity of the single stranded DNA hybridizing with the complementary fragment is determined by the "stringency" of the reaction conditions. Hybridization stringency increases with decreasing tendency to form DNA double helices. In nucleic acid hybridization reactions, the stringency can be chosen to favor the particular hybridization (high stringency) used to identify full-length clones from, for example, libraries. A less specific hybridization (low stringency) can be used to identify related but inaccurate DNA molecules (same but not identical) or segments.

DNA double helix is a specific organic solvent that reduces (1) the number of complementary base pairs, (2) type of base pairs, (3) salt concentration (ion strength) of the reaction mixture, (4) reaction temperature, and (5) double helix stability. , For example, by the presence of DNA formamide. In general, the longer the probe, the higher the temperature required for proper annealing. The usual way is to change the temperature: relatively high temperatures result in more rigorous reaction conditions. Ausubel et al., 1987 provide a clear interpretation of the stringency of hybridization reactions.

"Under stringent conditions" refers to hybridization protocols in which nucleotide sequences that are at least 60% homologous to each other hybridize. In general, stringent conditions are selected approximately 5 ° C. below the thermal melting point (Tm) for a particular sequence at defined ionic strength and pH. Tm is the temperature at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium (under defined ionic strength, pH, nucleic acid concentration). In general, since the target sequence is present in excess at Tm, 50% probe is occupied at equilibrium.

"Dense hybridization conditions" are conditions under which a probe, primer or oligonucleotide hybridizes only to a target sequence. Stringency conditions are sequence-dependent and vary from situation to situation. Stringent conditions include (1) low ionic strength and high temperature scrubbing (eg, 15 mM sodium chloride, 50 mM sodium citrate, 0.1% sodium dodecyl sulfate at 50 ° C.); (2) denaturant during hybridization (e.g. 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5) at 42 ° C), 750 mM sodium chloride, 75 mM sodium citrate); Or (3) 50% formamide. Typically, the rinse is at 5 ° C. 5 × SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt solution, sonicated salmon sperm DNA (50 μg). / Ml), 0.1% SDS, 10% dextran sulfate; 0.2 × SSC (sodium chloride / sodium citrate) at 42 ° C. and 50% formamide at 55 ° C .; It is then composed of 0.1 x SSC high rigor wash liquid containing EDTA at 55 ° C. Suitably, these conditions are those under which sequences which at least 65%, 70%, 75%, 85%, 90%, 95%, 98% or 99% homology with each other hybridize. These conditions are given by way of example and not limitation.

"Intermediate stringency conditions" are conditions that hybridize with a wash solution in which the polynucleotide hybridizes to the entire fragment, derivative or analog of SEQ ID NOS: 7-12, 14 with slightly less stringency (Sambrook, 1989). One example is hybridization in 6X SSC, 5X Denhardt solution, 0.5% SDS, 100 mg / ml denatured salmon sperm DNA at 55 ° C .; This is followed by one or more washes in IX SSC, 0.1% SDS at 37 ° C. Temperature, ionic strength and the like can be adjusted to match experimental factors such as probe length. Other medium stringency conditions have been reported in the literature (Ausubel et al., 1987; Kriegler, 1990).

"Low stringency conditions" are conditions that hybridize with a wash solution in which the polynucleotide hybridizes with the entire fragment, derivative or analog of SEQ ID NOS: 7-12, 14, with a slightly less stringent than the medium stringency (Sambrook, 1989). Unlimited examples of low stringency hybridization conditions include 35% formamide, 5X SSC, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg at 40 ° C. / Ml denatured salmon sperm DNA, hybridized at 10% (wt / vol) dextran sulfate; This is followed by one or more washes at 50 ° C. in 2 × SSC, 25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.1% SDS. Other low stringency conditions, such as low stringency conditions for heterogeneous hybridization, are well reported in the literature (Ausubel et al., 1987; Kriegler, 1990; Shilo and Weinberg, 1981).

In addition to the naturally-occurring alleles of SHAAGtide, mutational changes can be introduced in SEQ ID NO: 1 to modify the amino acid sequence of encoded SHAAGtide where the SHAAGtide function is not significantly altered. For example, amino acid substitutions at the C-terminal amino acid residues were performed in the sequence of SEQ ID NO: 6. "Non-essential" amino acid residues are residues that can be changed from the wild-type sequence of SHAAGtide without a change in biological activity, while "essential" amino acids are necessary for biological activity. For example, amino acid residues conserved in the SHAAGtide of the present invention are not expected to be modified. Amino acids for which conservative substitutions can be made are known in the art.

Useful conservative substitutions are shown in Table 4, “Preferred Substitutions”. Conservative substitutions in which one amino acid is replaced with another amino acid of the same type are within the scope of the present invention unless such substitution physically alters the biological activity of the compound.

Table 4: Preferred Substitutions

Non-conservative substitutions that affect (1) the structure of the polypeptide backbone, eg in the form of β-sheets or α-helices, (2) charges, (3) hydrophobicity, or (4) the size of the target site side chain, are particularly SHAAGtide. SHAAGtide function can be altered if the sequence forms part of a larger polypeptide molecule. The residues are subdivided into several groups based on common side chain properties, as shown in Table 5. Non-conservative substitutions replace members of these kinds with members of other species. Substitutions may be introduced into conserved substitution sites, more preferably non-conserved sites.

Table 5: Amino Acid Types

Kinds amino acid Hydrophobic Norleucine, Met, Ala, Val, Leu, Ile Neutral hydrophilic Cys, Ser, Thr acid Asp, Glu Basic Asn, Gln, His, Lys, Arg Disturbed Chain Mode Gly, Pro Aromatic Trp, Tyr, Phe

Variant polypeptides can be made by methods known in the art, oligonucleotide-mediated (specific site) mutagenesis, alanine scanning, PCR mutagenesis. Specific site mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction screening mutagenesis (Wells et al., 1985) or other known techniques can be performed on cloned DNA to make SHAAGtide variant DNA. (Ausubel et al., 1987; Sambrook, 1989).

"Isolated" or "purified" SHAAGtide of the present invention consists of a polypeptide, protein or biologically active fragment isolated or recovered from a component of the native environment. Isolated SHAAGtides include those that are heterologous or in vitro expressed in genetically engineered cells.

Contaminating components include substances that would typically interfere with the diagnostic or therapeutic use of the polypeptides according to the invention. To be substantially separated, the formulation contains less than 30%, preferably less than 20%, more preferably less than 10%, most preferably less than 5% dry weight of non-SHAAGtide contaminants.

The polypeptides and fragments can be produced by any method known in the art, for example, expression through vectors such as bacteria, viruses, eukaryotic cells. In addition, in vitro synthesis, such as peptide synthesis, may also be used.

An "active polypeptide or polypeptide fragment" retains biological and / or immunological activity similar to that of the SHAAGtide polypeptide shown in Table 1, but need not necessarily be the same. In the context of discussing this polypeptide itself, rather than the actual biological role of SHAAGtide in inducing or enhancing FPRL1 activity, immunological activity is an aspect of the SHAAGtide polypeptide in that certain antibodies against the SHAAGtide antigenic epitope bind to SHAAGtide. Biological activity refers to the inhibitory or promoting function induced by the native SHAAGtide polypeptide. Biological activity of the SHAAGtide polypeptide is binding to the FPRL1 receptor, chemotaxis or induction of calcium flux immediately after FPRL1 receptor binding. Dose-dependent or non-dose-specific biological assays can be used to determine SHAAGtide activity. Nucleic acid fragments encoding a biologically active portion of SHAAGtide isolate a polynucleotide sequence encoding a polypeptide having SHAAGtide biological activity and express an encoded portion of SHAAGtide (eg, by in vitro recombinant expression) of the SHAAGtide polypeptide. It can be made by evaluating the activity of the encoded portion.

In general, SHAAGtide polypeptide variants that preserve SHAAGtide polypeptide-like function include any variant in which residues are substituted with other amino acids at specific sites in the sequence, and the possibility of inserting additional residues between two residues of the parent protein and the parent. The possibility of deleting one or more residues from the sequence. Any amino acid substitutions, insertions or deletions belong to the present invention. In a positive environment, the substitution is a conservative substitution as defined above.

Table 1 shows that deletion of amino acids at the C-terminus of the SHAAGtide sequence is less likely to cause loss of FPRL1 activity than deletions at the N-terminus (see Example 9). For example, SEQ ID NO: 8, consisting of the 11 N-terminal amino acids of the SHAAGtide sequence, maintains intermediate FPRL1 activity. However, deletion of three N-terminal amino acids (SEQ ID NO: 2) results in very low FPRL1 activity. However, deletion of the terminal amino acid at the N-terminus (SEQ ID NO: 11) does not result in complete loss in FRPL1 activity.

"SHAAGtide variant" means an active SHAAGtide polypeptide that retains (1) 60% amino acid sequence identity with the full length native SHAAGtide polypeptide sequence or (2) any fragment of the full length SHAAGtide polypeptide sequence. For example, SHAAGtide polypeptide variants include SHAAGtide polypeptides that add or delete one or more amino acid residues at the N- or C-terminus of the full length native amino acid sequence, except for fragments identical to CKβ8 and CKβ8-1. The SHAAGtide polypeptide variant is at least 80%, preferably at least 81%, more preferably at least 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the full length native sequence SHAAGtide polypeptide sequence. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably at least 99% amino acid sequence identity. Typically, SHAAGtide variant polypeptides have at least 10 amino acids, preferably at least 20 amino acids, more preferably 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or 300 amino acids, or their Have an amino acid length of at least.

“Percent amino acid sequence identity” refers to the amino acid residues vs. amino acids in a candidate sequence when the two sequences are aligned. It is defined as the ratio of amino acid residues in the same SHAAGtide. Sequences are aligned to determine% amino acid identity and, if necessary, intervals are introduced to obtain maximum% sequence identity; Conservative substitutions are not considered part of the sequence identity. Amino acid sequence alignment procedures to determine percent identity are known to those of skill in the art. The peptide sequences are aligned using publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software.

Once the amino acid sequence is aligned, the percent sequence identity of a constant amino acid sequence A (or a constant amino acid sequence A having a specific amino acid sequence identity to a certain amino acid sequence B) for a certain amino acid sequence B can be calculated as follows: amino acid sequence identity % = X / Y100

Where X is the number of amino acid residues recorded in A and B as the same match by sequence alignment program or algorithm alignment;

Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length of amino acid sequence B, then% amino acid sequence identity of A to B is not equivalent to% amino acid sequence identity of B to A.

Fusion polypeptides are useful for expression studies, cell-localization, biopsies, and SHAAGtide purification. SHAAGtide A "chimeric protein" or "fusion protein" consists of a SHAAGtide fused to a non-SHAAGtide polypeptide. Non-SHAAGtide polypeptides are not substantially homologous to SHAAGtide polypeptides. SHAAGtide fusion proteins include any portion of the entire SHAAGtide, including many biologically active moieties. For example, SHAAGtide can be fused to the C-terminus of the GST (glutathione S-transferase) sequence. Such fusion proteins allow purification of recombinant SHAAGtide. In certain host cells (eg, mammals), heterologous signal sequence fusions improve SHAAGtide expression and / or secretion.

Antibodies specific for SHAAGtide and SHAAGtide variant sequences also belong to the invention. Methods of producing polyclonal and monoclonal antibodies, including binding fragments (eg, F _{(ab) 2} ) and single chain variants, are well known. Thus, polyclonal or monoclonal antibodies can be prepared by standard techniques.

The chemotactic composition of the present invention contains one or more polynucleotides or polypeptides having a SHAAGtide sequence. In one embodiment, the composition contains SHAAGtide as an isolated or recombinant polynucleotide or polypeptide. In one embodiment, SHAAGtide is the dominant (ie, greater than 50 wt%, preferably greater than 80 wt%) species in the composition (eg, polypeptides, polynucleotides, lipids, carbohydrates). The chemotactic composition of the present invention contains SHAAGtide without materials normally associated with the in situ environment.

The isolated SHAAGtide nucleic acid molecule is purified from the environment found in nature and separated from at least one contaminating nucleic acid molecule. Isolated SHAAGtide molecules are distinguished from certain SHAAGtide molecules present in the cell.

Use of SHAAGtide composition in the treatment of disease

The present invention provides a prophylactic and therapeutic method for treating an individual at risk of or suffering from a disease associated with abnormal FPRL1 receptor or FPRL1 ligand activity.

Diseases or abnormalities of humans or other species that can be treated with proteins or peptides comprising SHAAGtide or SHAAGtide, or with inhibitors or agonists of FPLR1-SHAAGtide interactions, are peripheral-chronic inflammation-related diseases, such as chronic inflammation, thrombosis, Atherosclerosis, restenosis, chronic venous insufficiency, recurrent bacterial infections, sepsis, skin infections, kidney disease, glomerulonephritis, fibrotic lung disease, allergic diseases, IBS, rheumatoid arthritis, acute bronchitis, etc. Do not. CNS-related diseases such as neurodegenerative diseases, Alzheimer's disease, multiple sclerosis, Parkinson's disease, neuritis, HIV-associated neurological diseases, HIV-associated dementia, CNS bacterial infections, brain toxoplasmosis Gaciameba infection, Listeria infection, prion disease, subacute spongiform encephalopathy, macular degeneration can also be treated.

Diseases characterized by elevated FPRL1 levels or biological activity can be treated with therapeutic agents that antagonize (ie, decrease or inhibit) activity. Antagonists can be administered in a therapeutic or prophylactic manner. Therapeutic agents that can be used include (1) inactive SHAAGtide peptides, or analogs, derivatives, fragments or homologs thereof; (2) SHAAGtide antisense nucleic acid; (3) an antibody against a SHAAGtide peptide, or analog, derivative, fragment or homologue thereof; (4) modulators that antagonize the activity of the FPRL1 receptor (ie, inhibitors and antagonists).

 Diseases characterized by reduced FPRL1 levels or biological activity can be treated with therapeutic agents that increase (or increase) activity. A therapeutic agent that upregulates activity can be administered therapeutically / prophylactically. Therapeutic agents that can be used include (1) a molecule comprising a SHAAGtide peptide, or an analog, derivative, fragment or homologue thereof; (2) SHAAGtide nucleic acid; (3) modulators that enhance the activity of the FPRL1 receptor.

The present invention provides a method for preventing a disease or condition associated with abnormal FPRL1 receptor expression or activity in an individual by administering a drug that modulates FPRL1 activity. Individuals at risk of disease due to abnormal FPRL1 activity can be identified, for example, by diagnostic or prophylactic analysis or a combination thereof. Administration of a prophylactic agent may be performed prior to symptoms characteristic of FPRL1 abnormalities to prevent or delay the progression of the disease or condition. Depending on the type of FPRL1 abnormality, for example FPRL1 agonists or FPRL1 antagonists can be used to treat the patient. Appropriate drugs can be determined based on screening assays.

Another aspect of the invention relates to a method of modulating FPRL1 activity for therapeutic purposes. The modulating method is contacting the cell with a drug that modulates one or more FPRL1 activities associated with the particular cell. Drugs that modulate FPRL1 activity may be nucleic acids or proteins, naturally occurring cognate ligands of FPRL1, peptides, SHAAGtide peptide mimetics, or other small molecules. These drugs can stimulate FPRL1 activity. These drugs can inhibit FPRL1 activity. Modulation methods can be performed in vitro (eg, by culturing the cells with the drug) or in vivo (eg, by administering the drug to the subject). For example, the method consists in administering SHAAGtide or nucleic acid molecules as a therapy that compensates for reduced or abnormal FPRL1 or FPRL1 ligand expression or activity.

Stimulation of FPRL1 activity is preferred if, for example, an FPRL1 or FPRL1 ligand has abnormally down-regulated or increased FPRL1 or FPRL1 ligand activity has a positive effect, for example in the treatment or infection of an infection. In contrast, reduced FPRL1 or FPRL1 ligand activity is preferred if, for example, FPRL1 or FPRL1 ligand activity is abnormally up-regulated or reduced FPRL1 or FPRL1 ligand activity exhibits a positive effect, for example in the treatment of chronic inflammation.

Appropriate in vitro or in vivo assays may be performed to determine the effectiveness of a particular therapeutic agent and whether its administration is suitable for the treatment of diseased tissue.

In various specific embodiments, in vitro assays are performed with representative cell types associated with a patient's disease to determine whether a particular therapeutic agent exerts the desired effect on that cell type. Modalities used for treatment may be examined in appropriate animal models, including but not limited to rats, mice, chicks, cattle, monkeys, rabbits, dogs, etc., prior to testing in human subjects.

Diseases and disorders associated with inflammation and infection can be treated by the methods of the present invention. In this disease or condition, it inhibits or promotes the action of the FPRL1 ligand on the FPRL1 receptor to modulate the immune response.

The compositions of the present invention may be administered orally, externally (intramuscularly, intraperitoneally, intravenously, ICV, intraventricular injection or infusion, subcutaneous injection, or implant), inhalation spray, nasal cavity, vaginal, rectal, sublingual or topical route. And may be prepared in suitable unit dosage forms containing conventionally non-toxic pharmaceutically acceptable carriers, adjuvants, excipients suitable for each route of administration. The composition of the present invention is effective in humans, in addition to the treatment of warm-blooded animals such as mice, rats, horses, cattle, sheep, dogs, cats, monkeys and the like.

  Combination therapies that modulate FPRL1 or FPRL1 ligand activity and therapies for preventing and treating infectious or infectious diseases are described as a combination of a compound according to the invention and other compounds known for such use.

For example, in the treatment or prevention of inflammation, the compounds of the present invention may be anti-inflammatory or analgesic agents (e.g., anesthetic anesthetics), lipoxygenase inhibitors (e.g. 5-lipoxygenase inhibitors), interleukin inhibitors (e.g. TNFα ), Interleukin-1 inhibitors, NMDA agonists, nitric oxide inhibitors or nitric oxide synthesis inhibitors, non-steroidal anti-inflammatory agents, or cytokine-inhibiting anti-inflammatory agents, for example acetaminophen. It is used in combination with aspirin, codeine, fentanyl, ibuprofen, indomethacin, ketorolac, morphine, naproxen, phenacetin, pyricampam, steroidal analgesics, sufentanil, sullindac, and tenidab. Similarly, the compounds of the present invention may be used as pain relief agents; Reinforcing materials such as caffeine, H2-antigens, simethicone, aluminum hydroxide or magnesium; Decongestants such as phenylephrine, phenylpropanolamine, pseudopedrine, oxymethazolin, epinephrine, naphzoline, xylomethazolin, propylhexerine or levo-desoxephedrine; Anti-sea water agents such as codeine, hydrocodone, caramifen, carbetapentane or dextramethorphan; steroid; Cyclosporin A; Methotrexate; IL-10; diuretic; It can be administered with sedative or non-sedative antihistamines.

Pharmaceutical composition

Anti- or antagonists of the FPRL1 receptor may be incorporated into pharmaceutical compositions. Typically, such compositions consist of an anti-inflammatory or antagonist and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" includes all solvents, dispersion media, coatings, antibiotics and antibacterials, isotonic and absorption delaying agents, and the like suitable for pharmaceutical administration (Gennaro (2000)). Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, and the like. Liposomes and non-aqueous excipients such as nonvolatile fixed oils may also be used. The use of these compositions is contemplated, except where conventional media or agents are not suitable for the active compound. Supplementary active compounds can also be incorporated into the compositions.

Pharmaceutical compositions of anti-nausea or antagonists are prepared for the intended route of administration, including intravenous, intradermal, subcutaneous, oral (eg inhalation), transdermal (ie surface), transmucosal, rectal administration. Solutions or suspensions suitable for extra-intestinal, intradermal or subcutaneous administration contain the following components: sterile diluents such as water for injection, saline solution, nonvolatile perfume oils; Antibiotics such as benzyl alcohol or methyl parabens; Antioxidants such as ascorbic acid or sodium bisulfite; Chelating agents such as ethylenediamineetheracetic acid (EDTA); Buffers such as acetates, citrates or phosphates; Agents that modulate tonicity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, for example hydrochloric acid or sodium hydroxide. Extra-intestinal preparations may be placed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection contain sterile aqueous solutions or dispersions and sterile powders for the instant preparation of sterile injection solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, CREMOPHOREL (BASF, Parsippany, N. J.) or phosphate buffer (PBS). In all cases, the composition must be sterile and fluid enough to be administered by syringe. Such compositions must be stable during manufacture and storage and must be protected from contamination by microorganisms such as bacteria and fungi. The carrier can be, for example, water, ethanol, polyols (eg glycerol, propylene glycol, liquid polyethylene glycols), solvents or dispersions containing suitable mixtures. Proper fluidity can be maintained, for example, using a coating such as lecithin, to maintain the required particle size in the case of dispersions or using surfactants. Various antibiotics and antimicrobials, such as parabens, chlorobutanol, phenol, ascorbic acid and chimerosal, can inhibit microbial contamination. Isotonic agents, for example, sugars, polyalcohols (eg mannitol), sorbitol, sodium chloride may be included in the composition. The composition contains agents that can delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by mixing the desired amount of active compound with a suitable component or a combination thereof in a suitable solvent and sterilizing. Generally, dispersions are prepared by mixing the active compound with a sterile excipient containing a basic dispersion medium and the required other ingredients. Methods of preparing sterile powders for the preparation of sterile injectable solutions include vacuum drying and freeze-drying which yield powders containing the active and necessary ingredients from sterile solutions.

Generally, oral compositions contain an inert diluent or an edible carrier. These can be encased in gelatin capsules and compressed into tablets. The active compound for oral administration can be mixed with excipients and used in the form of tablets, troches or capsules. Oral compositions may also be prepared as mouthwashes using a fluid carrier, wherein the compound dissolved in the fluid carrier is administered orally. It may contain a pharmaceutically suitable binder and / or adjuvant material. Tablets, pills, capsules, troches and the like may be used as compounds of the following components or similar properties: binders such as microcrystalline cellulose, gum tragacanth or gelatin; Excipients such as starch or lactose; Disintegrants such as alginic acid, PRIMOGEL or corn starch; Lubricants such as colloidal silicon dioxide; Sweeteners such as sucrose or saccharin; Or flavors such as peppermint, methyl salicylate or orange flavor.

For inhalation administration, the compounds of the present invention are delivered to the aerosol spray from a nebulizer or pressure vessel containing a suitable propellant, for example a gas such as carbon dioxide.

Systemic administration may be transmucosal or transdermal. For transmucosal or transdermal administration, penetrants are selected that can penetrate the target barrier. Transmucosal penetrants are detergents, bile acids, fusidic acid derivatives and the like. Nasal sprays or suppositories can be used for transmucosal administration. For transmucosal administration, the active compounds are prepared in ointments, plasters, gels or creams.

The compounds of the present invention may also be prepared in the form of suppositories (e.g., with bases such as cocoa butter or other glycerides) or tablet enemas for rectal delivery.

In one embodiment, the active compound is a carrier that blocks the rapid removal of the compound from the body, for example in a sustained release formulation including an implant and a microencapsulated delivery system. Biodegradable or biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid. Polyethylene glycols such as PEG are also excellent carriers. Such materials can be purchased commercially from ALZA Corporation (Mountain View, Calif.) And NOVA Pharmaceuticals, Inc. (Lake Elsinore, Calif.) Or can be prepared by those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be made according to methods known to those skilled in the art (Eppstein et al. US Patent No. 4,522, 811.1985).

Unit dosage forms or extragranular compositions can be made to facilitate administration and dosing uniformity. A unit dosage form refers to physically discrete units containing a therapeutically effective amount of the active compound and the required pharmaceutical carrier and suitable for single administration to the treated individual. Unit dosage forms in the present invention directly depend on the unique properties of the active compound, the particular therapeutic effect required, and the limitations inherent in the preparation of the active compound.

The nucleic acid molecule of SHAAGtide can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors can be delivered to an individual, for example, by intravenous injection, topical administration (Nabel and Nabel, US Patent No. 5,328,470, 1994), or stereotactic injection (Chen et al. (1994)). Pharmaceutical formulations of gene therapy vectors may be composed of a delayed release matrix containing an acceptable diluent or containing a gene delivery excipient. Alternatively, where a complete gene delivery vector, such as a retroviral vector, can be produced without damage from recombinant cells, the pharmaceutical agent may comprise one or more cells producing a gene delivery system.

In one aspect, SHAAGtide is delivered to DNA so that the polypeptide can be produced in situ . In one embodiment, the DNA is “nacin” (Ulmer et al. (1993); Cohen, (1993)). Nasal DNA can increase DNA by coating it with a biodegradable bead that is effectively delivered to a carrier, such as a cell. In such vaccines, DNA is present in a variety of delivery systems known to those of skill in the art, including nucleic acid expression systems, bacterial expression systems or viral expression systems.

Vectors used to transfer genetic material from organisms to organisms can be subdivided into two general categories: Cloning vectors are not essential for proliferation in suitable host cells and replicate plasmids or phage into regions where foreign DNA can be inserted. do; Foreign DNA constituting the vector is replicated and propagated. Expression vectors (plasmids, yeast, or animal viral genomes) are used to introduce foreign genetic material into host cells or tissues for the transcription and translation of foreign DNA, such as SHAAGtide. Some promoters are particularly useful, for example inducible promoters that regulate gene transcription in response to certain factors. An operably linking SHAAGtide polynucleotide to an inducible promoter can modulate the expression of the SHAAGtide polypeptide or fragment. Examples of classical inducible promoters are promoters that respond to α-interferon, thermal shock, heavy metal ions and steroids such as glucocorticoids (Kaufman, 1990. Methods Enzymol 185: 487-511) and tetracycline. Another preferred inducible promoter is a promoter that is not endogenous to the cell into which the construct is inserted but reacts in these cells when an inducing factor is supplied from the outside. However, other forms of expression vectors are contemplated, for example viral vectors (eg, replication defective retroviruses, adenoviruses, adeno-associated viruses).

Vector selection depends on the fate of the organism or cell and the vector used. The vector may be replicated once in the target cell or may be a "suicide" vector. In general, vectors include signal sequences, origins of replication, marker genes, enhancer elements, promoters, transcription termination signals.

The pharmaceutical composition may further contain other therapeutically active compounds conventionally used for the treatment of FPRL1-associated diseases.

In the treatment or prophylaxis of diseases requiring FPRL1 regulation, the appropriate dose level of a single or multiple doses of the anti-inflammatory or antagonist is approximately 0.01-500 mg / kg body weight of the patient. The dose level is preferably approximately 0.1 to 250 mg / kg per day, more preferably approximately 0.5 to 100 mg / kg per day. Suitable dosage levels are approximately 0.01 to 250 mg / kg per day, approximately 0.05 to 100 mg / kg per day, or approximately 0.1 to 50 mg / kg per day. Doses within this range are 0.05 to 0.5, 0.5 to 5, or 5 to 50 mg / kg per day. Compositions for oral administration may vary from 1.0 to 1000 mg of active ingredient, in particular 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, depending on the condition of the patient being treated. , 600.0, 750.0, 800.0, 900.0, 1000.0 mg in the form of tablets containing the active ingredient. The compounds of the invention are administered in a regimen of 1 to 4 times daily, preferably 1-2 times daily.

However, the dose level and frequency of dosing for any particular patient may vary depending on the activity of the particular compound used, metabolic stability and duration of action, age, weight, overall health, sex, diet, mode and time of administration, rate of release, drug It depends on the combination, the severity of the particular disease, and the various procedures the host is undergoing.

Kit

In one aspect, the present invention provides a package or container comprising (1) a biologically active composition or FPRL1 anti-inflammatory agent of the present invention; (2) pharmaceutically acceptable adjuvant or excipient; (3) excipients for administration, such as syringes; (4) Present a kit containing instructions for administration. Also contemplated are embodiments in which two or more of these components (1)-(4) are found in the same vessel.

Once the kit is supplied, the different components of the composition can be packaged in separate containers and mixed just before use. Individual packaging of these components allows for long term storage without loss of function of the active ingredient.

The reactants included in the kit are supplied in containers, so that the lifetimes of the different components are preserved and are not absorbed or modified by the container materials. For example, sealed glass ampoules contain lyophilized SHAAGtide polypeptide or polynucleotide, or a buffer packaged under a neutral, non-reactive gas such as nitrogen. The ampoule is composed of any suitable material, such as glass, organic polymers such as polycarbonate, polystyrene, etc., ceramics, metals or any other material typically used to maintain a similar reaction. Another example of a suitable container is a simple bottle made from a similar material such as an ampoule and a lid holding a foiled interior such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. The container is a bottle that holds a stopper that can be pierced by a sterile access port, for example a hypodermic needle. The other container has two compartments separated by a membrane that are easily removable and allow the components to mix immediately after removal. Removable membranes are glass, plastic, rubber, and the like.

You can also provide how-to materials in the kit. Usage may be printed on paper or other substrate or provided on an electronic-readable medium such as floppy disk, CD-ROM, DVD-ROM, Zip disk, videotape, audiotape or the like. Detailed usage may not be physically attached to the kit; Instead, the user can directly visit an Internet web site specifically designated or e-mailed by the kit's manufacturer or distributor.

Screening and Detection Methods

SHAAGtide (and SHAAGtide nucleotides used to express SHAAGtide) can be used as reactants in methods of selecting compounds that modulate FPRL1 receptor activity. Such compounds are useful for treating diseases characterized by the lack or excessive production of FPRL1 receptor or FPRL1 receptor ligands, or the production of FPRL1 receptor or FPRL1 receptor ligand forms with abnormal activity compared to wild-type molecules. In general, such compounds can be used to modulate biological functions associated with FPRL1 receptor / FPRL1 receptor ligands.

The present invention identifies modalities affecting FPRL1 receptors or FPRL1 receptor ligands, that is, candidates or test compounds or agents (eg, peptides, mimetics, small molecules or other drugs), foods, combinations thereof, and the like. We present a method (screening assay). This is a stimulating or inhibitory effect. In addition, the present invention relates to compounds identified in such screening assays.

Tests for compounds that increase or decrease FPRL1 receptor activity in response to or independent of a ligand are preferred. Compounds can modulate FPRL1 receptor activity by increasing or decreasing the activity of the FPRL1 receptor itself (antigenics and antagonists).

Test compounds may be combined library methods: biological libraries; Spatially addressable parallel solid phase or liquid phase libraries; Synthetic library methods requiring deconvolution; "One bead, one compound" library method; It can be obtained in a number of combinations in a synthetic library method using affinity chromatography screening. Biological library approaches are limited to peptides, but the other four approaches are carried out in small molecule libraries of peptides, non-peptide oligomers or compounds (Lam, 1997).

"Small molecule" means a composition having a molecular weight of less than about 5 kD, more preferably less than about 4 kD, most preferably less than 0.6 kD. Small molecules may be nucleic acids, peptides, polypeptides, peptidomimetic, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungi, bacteria or agar extracts, are known in the art and can be selected by the assays of the present invention. Typical synthetic methods of molecular libraries have been reported in the literature (Carell et al., 1994a; Carell et al., 1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994; Zuckermann et. al., 1994).

Compound libraries include solutions (Houghten et al., 1992), beads (Lam et al., 1991), chips (Fodor et al., 1993), bacteria, spores (Ladner et al., US Patent No. 5,223,409, 1993). , Plasmid (Cull et al., 1992) or phage (Cwirla et al., 1990; Devlin et al., 1990; Felici et al., 1991; Ladner et al., US Patent No. 5,223,409,1993; Scott and Smith , 1990).

Many assays are available that screen candidates or test compounds that bind to or modulate the activity of the FPRL1 receptor. Cell-free assays, for example, contact an FPRL1 receptor or biologically active fragment with a SHAAGtide compound that binds to the FPRL1 receptor to produce an assay mixture, contact the assay mixture with a test compound, and test the ability of the test compound to interact with the FPRL1 receptor. Wherein the step of measuring the ability of the test compound to interact with the FPRL1 receptor consists of measuring the ability of the FPRL1 receptor to preferentially bind to or modulate its activity. Cell-based assays include, for example, calcium flow assays, binding assays, cell migration assays, and the like described in the Examples.

The immobilization of a molecule comprising a SHAAGtide sequence or one of its counterparts (eg, FPRL1) facilitates separation of complexed forms from a single or uncomplexed form of both proteins and facilitates high power analysis. The binding of the test compound to the SHAAGtide molecule or FPRL1 receptor molecule in the presence or absence of a candidate compound, or the interaction of the SHAAGtide molecule with the FPRL1 receptor molecule, may be any container suitable for containing the reactants, eg, microtiter plates, test tubes, micro- In a centrifuge tube. Fusion proteins may be provided that add domains that allow one or both proteins to bind to the matrix. For example, GST (glutathione S-transferase) -SHAAGtide fusion protein or GST-target fusion protein is absorbed into glutathione Sepharose beads (SIGMA Chemical, St. Louis, MO) or glutathione induced microtiter plates, and then tested Or, the test compound and unabsorbed FPRL1 receptor or SHAAGtide molecule are combined and the mixture is incubated under conditions that induce complex formation (eg, physiological salts and pH conditions). After incubation, the beads or microtiter plates are washed to remove unbound components from the matrix immobilized on the beads and the complexes are measured directly or indirectly. Alternatively, the complex is separated from the matrix and standard levels of SHAAGtide binding or activity are measured.

Other techniques for immobilizing proteins on the matrix can also be used for screening assays (United States Patent Application Serial Number 09 / 721,902). SHAAGtide molecules or FPRL1 receptor molecules can be immobilized using a biotin-avidin or biotin-streptavidin system. Biotinylation is achieved using many reactants, for example biotin-NHS (N-hydroxy-succinimide; PIERCE Chemicals, Rockford, IL) and streptavidin-coated 96 well plates (PIERCE Chemical) Can be fixed). Alternatively, antibodies or antibody fragments that react with SHAAGtide molecules or FPRL1 receptor molecules but do not interfere with the binding of SHAAGtide and FPRL1 receptor molecules can be induced in the wells of the plate, and antibody conjugates can capture the FPRL1 receptor molecules or SHAAGtide in the wells. . In addition to those described for GST-immobilized complexes, methods of detecting such complexes rely on immunodetection of complexes with antibodies that react with FPRL1 receptor molecules or SHAAGtide molecules and detection of enzymatic activity associated with FPRL1 receptor molecules or SHAAGtide molecules. Enzyme-linked assays.

To demonstrate that compounds are anti-inflammatory agents of the FPRL1 receptor, one can assess whether they inhibit the activity of SHAAGtide at the receptor.

Suitably, such compounds possess at least one of the following characteristics:

(1) potent inhibition of binding of a molecule comprising a SHAAGtide or SHAAGtide sequence to the FPRL1 receptor;

(2) significant inhibition of the Ca ²⁺ response of a molecule comprising a SHAAGtide or SHAAGtide sequence that binds FPRL1;

(3) defined non-specific Ca ²⁺ reactions; or

(4) inhibition of chemotactic activity.

Standard in vitro binding assays can be used to demonstrate the affinity of the compound for the FPRL1 receptor (and therefore inhibit the activity of SHAAGtide by competitive interaction with the receptor). Suitably, the active compound exhibits an IC ₅₀ value of <10 μM, more preferably <5 μM, most preferably <1 μM.

Compounds that inhibit SHAAGtide activity affect intracellular Ca ²⁺ levels in SHAAGtide stimulated cells. Ligand binding to the FPRL1 receptor results in G-protein induced activation of phospholipase C, which results in the conversion of phosphatidylinositol phosphate to inositol phosphate and diacylglycerol. Inositol phosphate binds to receptors located at intracellular sites and releases Ca ²⁺ into the cytoplasm. In addition to increasing Ca ²⁺ concentrations due to release from intracellular scales, receptor binding of inositol phosphate increases the flow of extracellular calcium across the membrane into the cell.

Thus, activation of the FPRL1 receptor by SHAAGtide and inhibition of this activation by the compounds according to the invention can be measured by analyzing the increase in free intracellular Ca ²⁺ levels. Typically, this can be achieved using calcium-sensitive fluorescent probes such as quin-2, fura-2, indo-1. The effectiveness of active compounds to block Ca ²⁺ reactions depends on the content of active compounds and chemokines present. In general, when 10 nM chemokine is present, 10 μM of the active compound induces 20-100% inhibition of Ca ²⁺ response.

To confirm that the active compound induces a non-specific Ca ²⁺ response, cells bearing multiple receptors, including the receptor to which the active compound targets, were incubated with the compound. The cells were then stimulated with ligands for the target receptor and then with ligands for other receptors found in the same cell. The equivalent response of the non-target receptor to the ligand in the absence of the compound suggests that the active compound is specific for the target receptor.

To identify chemotaxis, cell migration assay formats such as the ChemoTx® system (NeuroProbe, Rockville, MD) or any other suitable device or system (Bacon et al., 1988; Penfold et al., 1999) can be used. Can be. In short, these cell migration assays are performed as follows. After collecting and preparing cells bearing the active target chemokine receptors, these cells are mixed with candidate antagonists. The mixture is placed in the upper chamber of the cell transfer device. In the lower chamber, a stimulatory concentration of chemokine ligands is added. Thereafter, migration assays are conducted and terminated, and cell migration is assessed.

The present inventors confirmed the SHAAGtide activity on FPRL1 receptor expressed in monocytes, neutrophils, immature dendritic cells, mature dendritic cells. Thus, such cells can be used in in vitro assay methods. Abundant or substantially purified cell populations can be used for in vitro chemotaxis assays. These cell populations can be prepared by a variety of methods known in the art, depending on the particular cell type desired. Typically, the substantially purified cell population is cultured under specific conditions; Physical characteristics such as behavior in density gradients; Fluorescent activated cell sorting (FACS), immunoprecipitation with characteristic markers (eg, antibodies against cell-surface proteins (preferably monoclonal antibodies)); Or prepare in another way.

Cells can be identified by methods similar to histology (Luna, 1968) and immunological staining (Harlow et al. 1998; Coligan et al., 1991). Methods of preparing substantially purified cell compositions for use in in vitro chemotaxis assays are briefly described in the Examples below. However, the present invention does not require a specific purification method as long as it can obtain the desired cells; Many variations and alternative methods are known to those skilled in the art. In addition, many other purification and detection methods, including those suitable for cells not specifically described herein, are known in the art or may be readily developed. In addition, cloned cell lines derived from immune system tissue can be used in the chemotaxis assays described herein. Overall immunization, purification and cell culture methods are described in Coligan et al. (1991). Unless otherwise specified, cultured cells are cultured at 5% CO ₂ , 37 ° C.

Suitable methods for monocyte purification are described by Bender et al., 1996 and US Pat. No. 5,994,126. In brief, monocytes are isolated from PBMCs by depleting T cells with immobilized antibodies to the pan T cell surface marker CD2. Conveniently, a commercial source of CD2 antibodies attached to magnetic beads (Dynal; Lake Success, NY) is used. PBMCs isolated from a buff coat (typically 35 mls with 400 x 10 ⁶ PMBC) by traditional Ficoll gradient centrifugation method were treated with MACS buffer [1% BSA (Sigma) at 20 x 10 ⁶ cells per ml. Containing DPBS (HyClone; Logan, UT)]. DPBS is Dulbecco phosphate buffer _{[CaCl 2 (0.1 g / ℓ} ), KCl (0.2 g / ℓ), KH 2 PO 4 (0.2 g / ℓ), MgCl 2 -6H 2 0 (0.1 g / ℓ), NaCl (8.0 g / l), Na ₂ HP0 ₄ (2.16 g / l)]. Appropriate amount of fixed CD ²⁺ magnetic beads (typically 10 μl / 10 ⁶ cells) is added to the cells. The mixture is incubated at 4 ° C. for 15 minutes with gentle rotation. Magnetic labeled T cells are transferred from unlabeled cells in a magnetic cell sorter (Dynal), according to the manufacturer's protocol. Unlabeled cells are mainly monocytes and B cells.

B cells in this formulation are removed using different adhesion properties. In brief, PBMC depleted T cells are allowed to adhere to the plastics of T-175 tissue culture flasks (100 × 10 ⁶ cells / flask; Costar; Acton, MA) at 3 ° C. for 3 hours. Non-adherent cells (mainly B cells) are aspirated. To completely remove non-adherent cells, the flask is washed three times with DPBS. The resulting cells are rich in monocytes (ie> 90%).

Monocytes can also be isolated by positive selection of the CD14 antigen. In brief, PBMCs are isolated from peripheral blood such as smokescreens by standard Ficoll gradient centrifugation and resuspended at 1 × 10 ⁶ cells / ml in MACS buffer. Immobilized antibodies to CD14 surface antigens such as CD14 + magnetic beads (Milteyni) are added (1 μL beads / 1 × 10 ⁶ cells) and the mixture is incubated at 4 ° C. for 15 minutes. Monocytes are separated from other cell populations by passing the mixture through a positive selection column in a magnetic cell sorter (Miltenyi Biotech; Auburn, CA) according to the manufacturer's protocol. Monocytes remaining in the column elute with MACS after detaching the column from the MACS instrument. Cells are then pelleted by centrifugation and resuspended at 10 ⁶ cells / ml in RMPI + 10% FCS medium. Monocytes isolated in this way are cultured in essentially the same manner as in monocytes isolated by the CD2 + depletion method.

Methods suitable for the purification of dendritic cells, including individual maturation and immature populations, are known in the art. Substantially purified dendrites (including subpopulations of mature or immature cells) can be prepared under selective in vitro culture conditions.

Dendritic cells are widely distributed in all tissues in contact with potential pathogens (eg, skin, gastrointestinal and respiratory, T cell-rich areas of secondary lymphoid tissue). In the skin and upper respiratory tract they form a lattice of highly dendritic cells (langerhans cells in the skin). After capturing the antigen, the dendritic cells migrate to the T cell site of the draining lymphatic vessels in peripheral tissues such as the skin or intestine, where they present the underlying antigen. Immature dendrites function to absorb and process antigens. While traveling to the draining lymph nodes, the DC matures. Mature dendritic cells function as key APCs that initiate an immune response by inducing pathogen specific cytotoxicity and proliferation of helper T cells.

Substantially pure populations of dendrites can be produced in in vitro culture. In addition, marked changes in the expression of chemokine receptors during dendritic cell maturation occur, which can be used to identify cell stages (Campbell et al. 1998; Chan et al. 1999; Dieu et al. 1998; Kellermann et. al. 1999). For example, immature dendritic cells mainly express CCR1, CCR5, CXCR4. Immediately after maturation, these receptors are down regulated, with the exception of CXCR4.

Immature forms of dendritic cells in culture undergo maturation, which is thought to be similar to the phenomenon that occurs during the migration of dendritic cells from the point of antigen contact to secondary lymphoid tissue. Humans or macaque dendritic cells at various stages of development can be generated by culturing from CD14 ⁺ blood progenitor cells using specific cytokines. Independent lineage dendritic cells can be differentiated from umbilical cord blood or bone marrow CD34 + progenitor cells. In one embodiment of the present invention, a subpopulation of dendritic cells is generated for in vitro analysis (ie, evaluation of chemotaxin potency and selectivity for defined DC subtypes) for identification of chemotactic compositions. Typical subpopulations of dendrites include (1) immature peripheral blood monocyte derived cells; (2) mature peripheral blood monocyte derived cells; (3) cells derived from CD34 + progenitor cells. Subpopulations are isolated or produced by a variety of methods known in the art. For example, immature and mature dendritic cells from PBMC are described in Bender et al. Produce according to supra.

In short, PBMCs deplete T cells using immobilized antibodies against the cell surface marker CD2 (present in all T cells). Commercial CD2 + dynabeads (Dynal) can be used according to the manufacturer's protocol. T-cell depleted mixtures are separated into adherent vs non-adherent fractions by incubating the cells in tissue culture grade plastic at 37 ° C. for 3 hours. Non-adherent cells are gently removed and adherent cells (typically CD14 ⁺ monocytes) are culture media supplemented with 1000 U / ml GM-CSF and IL-4 (R & D Systems, Minneapolis, MN) (e.g., RMPI + 10% FCS) (“1 day”). Between 3-7 days, the cells begin to show an unclear morphology and cytokines are given on days 2, 4 and 6, at which point the cells can be harvested as immature dendrites. In one embodiment, cells of this in vitro step are isolated and used for the assay. Typically, approximately 10 × 10 ⁶ dendrites are obtained from 400 × 10 ⁶ PBMCs.

The 7-day immature dendrites show a typical dendritic cell morphology with an extended cell body and several processes. Cell size is markedly increased compared to progenitor monocytes. Immature dendrites can be phenotyped by monitoring the expression of cell surface markers.

Immature dendrites (generated from peripheral blood monocytes or bone marrow-derived CD34 + progenitor cells) can be further activated and differentiated into mature dendritic cells. Two methods are mainly used: MCM (macrophage condition medium) and double-stranded RNA-poly (I: C) stimulation (Cella et al, 1999; Verdijk et al. 1999).

In the MCM method, 6-day immature dendrites are harvested by centrifugation and resuspended at 10 ⁶ cells / ml in mature medium (eg, MCM diluted up to 1: 1 with RPMI containing 10% FCS). Cells are incubated for an additional 3 days without the addition of GM-CSF (1000 U / mL) and IL-4. Day 9 cells are used as mature dendritic cells.

In the poly (I: C) method, 6-day immature dendrites were harvested and standard culture supplemented with 20 μg / ml poly (I: C) (Sigma), 1000 U / ml GM-CSF, IL-4 Resuspend in medium (RPMI + 10% FCS). The cells are incubated for an additional 3 days without the addition of cytokines. Day 9 cells are used as mature dendritic cells.

Mature dendritic cells generated by these two different methods exhibit phenotypic and functional characteristics that are distinct from those of immature dendritic cells or progenitor monocytes. Mature dendritic cells from each agent are thoroughly characterized with FACS to ensure that the desired cell type is achieved.

Note that the mature dendritic cells generated express markedly higher levels of type II MHC than immature cells at the cell surface. Expression of CD80, CD83, CD86 is also up-regulated. Chemokine receptor expression also changes dramatically during the maturation process. For example, CCR1, CCR5 are drastically down-regulated in mature cells, while CCR7 is up-regulated and appears on the cell surface within hours after addition of MCM. Functionally, mature dendritic cells cannot effectively absorb antigen but have the ability to promote proliferation of native T and B cells. In addition, mature dendritic cells change their behavior; They no longer respond to ligands for CCR1, CCR2, CCR5, for example MIP-1α, RANTES, MIP-1β. Instead, they respond to the CCR7 ligands SLC and ELC.

MCM is described in Romani et al. Prepare slightly modified in the process described in 1996. Briefly, Petri dishes (100 mm, Falcon) are coated with 5 ml of human Ig (10 mg / ml) for 30 minutes at 37 ° C. and washed 2-3 times with PBS immediately before use. 50 × 10 ⁶ PBMCs in 8 ml are applied to human Ig-coated plates for 1-2 hours. Non-adsorbed cells are washed and discarded. Adherent cells are cultured in fresh complete medium (RPMI + 10% normal human serum) at 37 ° C. and the resulting medium (MCM) is collected after 24 hours. TNF-α concentration in MCM is measured by standard ELISA methods (eg, TNF-α ELISA kit (R & D Systems, Minneapolis, MN)). Final TNF-α levels in MCM are adjusted to 50 U / mL by mixing with an appropriate amount of MCM containing RPMI / 10% fetal bovine serum.

Suitable methods for neutrophil purification are known in the art. According to one suitable method, fresh whole blood (WB) is diluted 1: 1 with 3% dextran in a 50 ml centrifuge tube and allowed to settle for 30-45 minutes at room temperature. 25 ml WB + 25 ml dextran produced approximately 35 ml supernatant after 30 min settling. The supernatant is applied to 12-15 ml Ficoll and centrifuged at 400 x g for 30-40 minutes at 18-20 ° C. The plasma / platelet layer containing mononuclear cells and Ficoll-Plaque is removed by aspiration. Neutrophils are found in the white layer above the erythrocyte (RBC) layer (in some formulations the neutrophil and erythrocyte layer are mixed). In these cases, RBCs are removed by hypotonic lysis: 12.5 ml of cold 0.2% NaCl is added to neutrophils / RBC pellets with stirring. While still stirring, 12.5 mL of cold 1.6% NaCl is added immediately. Cells are centrifuged and recovered at 60-100 × g for 10 minutes. If necessary, the dissolution step is repeated. The resulting neutrophils are> 95% pure (the remaining cells are predominantly eosinophils).

Prognosis Analysis

The diagnostic methods described herein can also be used to identify individuals at risk of developing or at risk of developing a disease or condition associated with abnormal FPRL1 receptor or FPRL1 ligand expression or activity. For example, the assays described above can be used to identify individuals suffering from or at risk of diseases such as neurodegenerative diseases. Typically, a test sample is obtained from an individual and the FPRL1 receptor or FPRL1 ligand is detected or assayed for activity. For example, the test sample may be a biological fluid (eg serum), cell sample or tissue.

Prognosis assays may be administered to a subject in some form (eg, anti-inflammatory, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, food, etc.) to treat a disease or condition associated with abnormal FPRL1 receptor or FPRL1 ligand expression or activity. It can be used to determine if it can be. Such methods can be used to determine whether an individual can be effectively treated with a disease agent. The present invention can be used to determine whether an individual can be effectively treated with agents for diseases associated with abnormal FPRL1 receptor or FPRL1 ligand expression or activity. Such an assay obtains a test sample and detects SHAAGtide or nucleic acid, where the presence of SHAAGtide or nucleic acid diagnoses that the subject may be administered a therapeutic agent for a disease associated with abnormal FPRL1 receptor or FPRL1 ligand expression or activity.

The following examples are intended to illustrate the present invention, but are not limited thereto.

Example 1 CKβ8-1 (25-116) stimulates intracellular calcium flow in CCR1 expressing cells, similar to other CKβ8 variants.

Novel NH ₂ -terminal truncations of human recombinant chemokine, rucotaxin, three known CKβ8 variants [CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116)] and CKβ8-1 Form, CKβ8-1 (25-116), was obtained from R & D Systems (Minneapolis, MN). CKβ8-1 (25-116) compared the ability to induce intracellular calcium flux in stable human CCR1 transfected HEK239 cells compared to three other variants. Human HEK293-CCR1 cells were prepared using Fugena 6 (Roche, IN) according to the manufacturer's protocol. HEK-293 cell lines were maintained in DMEM containing 10% FBS supplemented with 800 μg / ml G-418.

Stable expression of human chemokine receptor CCR1 in HEK293 cells was achieved as follows: Full length cDNA encoding CCR1 was cloned by polymerase chain reaction (PCR) from genomic DNA isolated from peripheral blood cells. PCR products were cloned into pcDNA3.1 (Invitrogen, Carlsbad, Calif.) Using standard molecular cloning procedures and fully sequenced to confirm identity. HEK293 cells were transfected using two microorganisms of the CCRl / pcDNA3.1 construct as follows. FuGENE: DNA complex was prepared by mixing 6.0 μl FuGENE 6 reactant (Roche Molecular Biochemicals, CA) with 2 μg CCRl / pcDNA3.1 in 100 μl serum free medium (Hyclone, CO). After 30 minutes of incubation at room temperature, these complexes were added to 60 mm culture plates containing 0.5-1 × 10 ⁶ cells in 10 ml DMEM medium supplemented with 10% FBS (Hyclone, CO). After mixing, the cells were returned to the incubator and incubated at 37 ° C. for 2 days. 48 hours after transfection, Genetinin (G418) (Mediatech, Herndon, VA) was added at a final concentration of 800 μg / ml. The cells were then plated in 96-well plates at a concentration of 20,000 cells / well. After 2-3 weeks under G418 selection, stable geneticin-resistant CCR1 expressing cells evaluated the ability to flow calcium in response to MIP-1α at a concentration of 1-500 nM.

Ca ²⁺ flow reaction was performed using an intracellular radiation fluorescent dye, Indo-1. Cells were loaded with Indo-1 / AM (3 μM; Molecular Probes, Eugene, OR) in culture medium (45 min, 20 ° C., 10 ⁷ cells / ml). After dye loading, cells were washed once with 10 ml PBS and resuspended at 10 ⁶ cells / ml in HBSS containing 1% FBS. Cytoplasmic [Ca ²⁺ ] emission was measured by excitation at 350 nm using a Photon Technology International fluorometer (excitation at 350 nm, emission at 400 and 490 nm).

In the HEKCCR1-293 transfectant, CKβ8-1 (25-116) and other CKβ38 variants induced rapid calcium flux at 100 nM. These two truncated variants CKβ8 (25-99) and CKβ8-1 (25-116) induce high calcium responses, while signals produced by variants CKβ8 (25-99) and CKβ8-1 (1-116) Was lower than this. None of these chemokines induced signals in untransfected parental HEK293 cells, suggesting that the activity is due to CCR1 and not to exogenous receptors. Maximum receptor stimulation achieved with 100 nM CKβ8 (25-99) and CKβ8-1 (25-116) was equivalent to that obtained with the same concentration of CCR1 agonist, leucotaxin.

Example 2: CCL23 variant CKβ8-1 (25-116) exhibits unique activity independent of CCR1 in human monocytes and neutrophils.

Novel NHs of human recombinant chemokines, rucotaxin, MIP-1α, three known [CKβ8 variants CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116)] and CKβ8-1 _{The two} -terminal truncated form, CKβ8-1 (25-116), was obtained from R & D Systems (Minneapolis, MN). Human monocytes were generated from a buffy coat (Stanford Blood Center, Palo Alto, CA) according to standard protocols. In brief, PBMCs were separated by standard density gradient centrifugation (Ficoll-Paque-Plus, Pharmacia). Monocytes were purified by CD14 Microbeads (Miltenyi, Auburn, CA) magnetic positive selection. Human neutrophils were isolated from fresh peripheral blood of healthy individuals by gradient centrifugation in Ficoll-Hypaque (Hyclone, Calif.).

The activity of the CCL23 variant was tested in freshly prepared human monocytes and neutrophils using the calcium flow assay described in Example 1. Although all chemokines stimulate some calcium release from monocytes, CKβ8 (1-99) and CKβ8-1 (1-116) showed poor activity even at 250 nM. CKβ8 (25-99) showed slightly higher calcium stimulation. However, CKβ8-1 (25-116) showed unique calcium flux due to prolonged calcium release. Maximum receptor stimulation achieved with 100 nM CKβ8-1 (25-116) was at least twice higher than the results achieved with the same concentration of rucotaxin.

In neutrophils, 100 nM rucotaxin induced calcium flow while MIP-1α, CKβ8 (1-99), CKβ8 (25-99), CKβ8 (1-116) did not induce calcium flow. However, CKβ8-1 (25-116) induced distinct calcium release. Its size was much higher than the results observed for the same amount of rucotaxin stimulation.

Example 3: Cross-desensitization assay conducted on HEK293-CCR1 transfectants, monocytes, neutrophils.

In cross-desensitization assay, cells were stimulated sequentially with rucotaxin and chemokines CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116), CKβ8-1 (25-116). . In the HEK293-CCR1 transfectants (Example 1), rucotaxin induced a similar pattern of receptor desensitization to all variants. Pretreatment of cells with 100 nM rucotaxin completely inhibited the calcium flow response to all ligands.

Similar receptor cross-desensitization assays were performed using monocytes and neutrophils (Example 2). In monocytes, rucotaxin completely desensitized CCL23 variants, CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116). In contrast, rucotaxin prestimulation did not desensitize CKβ8-1 (25-116) activity in monocytes. In neutrophils, CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116) were inactive and prestimulation with rucotaxin had no effect. However, rucotaxin prestimulation did not desensitize stimulation with CKβ8-1 (25-116).

Example 4: CCL23 Variant for Binding with CCR1-expressing Cells ¹²⁵ Competes with 1-MIP-1α.

The binding properties of CCL23 variants were compared in human CCR1 expressing cells. ^The ability of the MIP-1α and CCL23 variants CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116), CKβ8-1 (25-116) to compete for ¹²⁵ 1-MIP-1α binding It was investigated in HEK293-CCR1 cells (Example 1). Cells were incubated with ¹²⁵ I-labeled MIP-1α (final concentration ˜0.05 nM) in the presence of unlabeled chemokines (3 h at 4 ° C .: 25 mM HEPES, 140 mM NaCl, 1 mM CaCl ₂ , 5). mM MgCl ₂ , 0.2% BSA, adjusted to pH 7.1). The reaction mixture was aspirated with a PEI-treated GF / B glass filter using a cell harvester (Packard). The filter was washed twice (25 mM HEPES, 500 mM NaCl, 1 mM CaCl ₂ , 5 mM MgCl ₂ , adjusted to pH 7.1). Scintillant (MicroScint-10; 35 μl) was added to the dried filter and the filter was calculated on a PackardTopcount scintillation counter. Data was analyzed and plotted using Prism software (GraphPad Software, San Diego, Calif.).

Competition curves were observed with increasing concentrations of MIP-1α or CCL23 variants. MIP-1α showed an IC ₅₀ value of 0.54 nM. CCL23 variants showed IC ₅₀ values of 64 nM, 1.34 nM, 206 nM, and 112 nM, respectively. CKβ8-1 (1-116) showed 3-4 times less potency than CKβ8 (1-99) on the displacement of binding ¹²⁵ I-MIP-1α from CCR1 in these transfectants. This is in line with relatively weak affinity. As expected, the truncated CKβ8 (1-99), CKβ8 (25-99), showed a 40-fold increase in IC ₅₀ . However, the IC ₅₀ for the same amino acid truncated variant of CKβ8-1 (1-116), CKβ8-1 (25-116), was increased by 1-fold.

Similar binding competition tests were performed in monocytes and neutrophils. These cells were prepared in the same manner as in Example 2. The binding competition between MIP-1α in neutrophils could not be investigated because MIP-1α does not bind to neutrophils. In monocytes, MIP-1α is holding the IC ₅₀ of 0.27 nM and, CCL23 variants pictures of each 10 nM, 0.25 nM, 55 nM , IC 50 of 5 nM. Overall, CKβ8 (1-99) and CKβ8 (25-99) showed IC ₅₀ similar to that observed in HEK293-CCR1 cells. However, CKβ8-1 (1-116) and CKβ8-1 (25-116) showed higher MIP-1α displacement activity in monocytes, especially CKβ8-1 (25-116) more than 10 times higher. ¹²⁵ I-MIP-1α binding-competition data (IC ₅₀ ) is shown in Table 6. IC ₅₀ for each interaction was inferred from non-linear least squares curve fitting.

Table 6 - HEK293-CCR1 transfected ¹²⁵ I-MIP-1α binding competition data for the body and the infected monocytes

Example 5: Variant CKβ8-1 (25-116) induces human monocytes and neutrophil migration with novel migration properties.

Migration analysis was performed in monocytes and neutrophils. Human monocytes and neutrophils (Example 2) were collected and resuspended in chemotactic medium (CM). CM consisted of Hank buffered saline solution (Gibco, MA) containing CaCl ₂ (1 mM) and MgSO ₄ (1 mM) and added 0.1% BSA (Sigma, St. Louis, MO). The assay was performed on 96-well ChemoTx® microplates (Neuroprobe, Mass.). Rucotaxin, MIP-1α, chemokines [CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116) or CKβ8-1 (25-116), Example 1) were transferred to lower wells ( Add to a final volume of 29 L) and add 20 μl cell suspension (5 × 10 ⁶ cells / ml for monocytes; 2.5 × 10 ⁶ cells / ml for neutrophils) to a polycarbonate filter (5 μm pore for monocytes). Size; 3 μm pore size for neutrophils). After 90 minutes of incubation (37 ° C., 100% humidity, 5% CO ₂ ), cells were scraped from the top surface of the filter. Cells moving to the lower chamber were quantified with Quant Cell Proliferation Assay Kit (Molecular Probes, OR). In monocytes, CKβ8-1 (25-99), CKβ8-1 (1-99), CKβ8-1 (1-116) showed moderate activity at all concentrations up to 100 nM. CKβ8-1 (25-116) showed almost similar activity with other variants at 1 and 10 nM, but showed significantly higher activity than three other variants at 100 nM.

The same test was performed on human neutrophils. In general, human neutrophils lacked a strong response to CCR1 ligands. None of the known CCR1 ligands, including rucotaxin and MIP-1α, showed activity, which is in line with calcium flow results. However, CKβ8-1 (25-116) induced a strong response at 100 nM. Its size is comparable to other potent CXCR1 and CXCR1 ligands, including IL-8 and GRO-α.

Example 6: CKβ8-1 (25-116) can induce intracellular calcium flux and chemotaxis in formyl peptide receptor like 1 (FPRL1) expressing cells.

Functional activity of CKβ8-1 (25-116) was investigated in human FPRL1-L1.2 transfectants and native L1.2 cells. Stable expression of formyl peptide-like receptor 1 (FPRL1) in L1.2 cells was achieved as follows. Full length cDNA encoding FPRL1 was cloned by polymerase chain reaction (PCR) from genomic DNA isolated from undifferentiated HL-60 cells. The polymerase chain reaction product was cloned into pcDNA3.1 (Invitrogen, Carlsbad, Calif.) Using standard molecular cloning procedures and fully sequenced to confirm identity. 20 mg of FPRL1 / pcDNA3.1 construct was cut and linearized with Bsm1 (New England Biolabs, Beverly, Mass.) And transfected murine B cell line LI.2 as follows. 2.5 × 10 ⁶ cells were washed twice and resuspended in 0.8 ml PBS. Cells were incubated with linearized FPRL / pcDNA3.1 construct DNA for 10 minutes at room temperature, transferred to 0.4-cm cuvettes and subjected to a single electroporation pulse at 250 V, 960 μF. Electroporated cells were incubated for 10 minutes at room temperature, and the cultures were transferred to RPMI supplemented with 10% FCS at 37 ° C. Geneticin (G418) was added at a final concentration of 800 μg / ml 48 hours after transfection and cells were plated in 96-well plates at 25,000 cells / well. After 2-3 weeks under drug screening, stable genetisine-resistant FPLR1 expressing cells were evaluated for their ability to stabilize Ca ⁺⁺ in response to SHAAGtide or CKbeta 8-1 at concentrations of 1 to 1000 nM.

Calcium flow tests were performed on these transfectants using the method described in Example 1. Among the CCL23 variants, only CKβ8-1 (25-116) stimulated calcium release in FPRL1 expressing cells. Synthetic peptides Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ (WKYMVm) and Trp-Lys-Tyr-Met-Val-Met-NH ₂ (WKYMVM) which are known non-natural ligands for FPRL1 ( "W Peptides 1 and 2") (Phoenix Pharmaceuticals (Belmont, Calif.) Induced rapid calcium flux. CKβ8-1 (25-116) induced calcium release was caused by transfection of parent cells or other chemokine receptors. CKβ8-1 (25-116) induced calcium flow dose response analysis revealed 10-20 nM of EC50 in these cells CKβ8-1 (1-116) was found in FPRL1 expressing cells. No activity was seen even at 200 nM.

The ability of CKβ8-1 (25-116) to induce migration of FPRL1 expressing cells was examined. Inspection conditions were the same as in Example 5. Parental L1.2 cells did not migrate during this assay, but cells expressing FPRL1 migrated in a species dose-dependent manner in response to CKβ8-1 (25-116) ranging from 1 nM to 1 μM. Half-maximal cell migration was observed at 30 nM. The magnitude of the maximum response was higher than that observed for the synthetic peptides WKYMVm and WKYMVM. In general, CKβ8-1 (25-116) showed a broader bell curve in FPRL1-mediated migration compared to other chemokines. Thus, CKβ8-1 (25-116) acts through the receptor FPRL1 expressed in monocytes and neutrophils in addition to being active in CCR1.

Example 7: CKβ8-1 (25-116) was expressed in human monocytes and FPRL1 expressing cells ¹²⁵ I-labeled WKYMVm binding can be eliminated.

The binding of CKβ8-1 (25-116) to FPRL1 resulted in ¹²⁵ I-labeled WKYMVm ( ¹²⁵ I-labeled Trp-Lys-Tyr-Met-Val-D- from human FPRL1-L1.2 transfectants and human monocytes). Met-NH ₂ , PerkinElmer Life Science (Boston, Mass.) Was determined by measuring the ability of CKβ8-1 (25-116) to remove. Cells were incubated with 0.01 nM ¹²⁵ I-WKYMVm for 3 hours at 4 ° C. in the presence of increasing concentrations of unlabeled WKYMVm or CKβ8-1 (25-116). The IC 50 for each concentration was inferred from non-linear least squares curve fitting of data using Prism software (GraphPad Software).

For FPRL I-L1.2 transfectants and monocytes, competition curves were observed with increasing concentrations of WKYMVm or CKβ8-1 (25-116) (Table 7). This curve was not observed in other CCL23 variants.

Table 7 - ¹²⁵ I- labeled WKYMVm competition curves from competition curves observed in WKYMVm or CKβ8-1 (25-116)

IC50 Human monocytes L1.2 FPRL1 Cells WKYMVm 1.5 nM 80 nM CKβ8-1 31 nM 196 nM

Example 8: Chemokine or SHAAGtide induced calcium flow by immature dendritic cells, mature dendritic cells, monocytes or neutrophils.

Human recombinant CKβ8-1 (25-116) chemokines were obtained from R & D Systems (Minneapolis, MN). Peptide SHAAGtide SEQ ID NO: 1 and SEQ ID NO: 6 and control protein (inverse of SEQ ID NO: 1) were synthesized and HPLC-purified by conventional techniques described in Sambrook et al., 1989 and Ausubel et al., 1999.

Human monocytes were generated from a buffy coat (Stanford Blood Center, Palo Alto, Calif.) Or fresh blood from healthy individuals according to standard protocols. In brief, PBMCs were separated by Ficoll-Paque gradient centrifugation (Ficoll-Paque-Plus, Pharmacia). Monocytes were purified by CD14 Microbeads (Miltenyi) magnetic positive selection. Human neutrophils were isolated from fresh blood from healthy individuals by dextran sedimentation and gradient centrifugation in Ficoll-Hypaque (Hyclone, Calif.). All cells were washed and resuspended in RPMI medium containing 10% FBS (1 × 10 7 / ml).

Immature DCs were induced by culturing CD14 + monocytes in the presence of GM-CSF and IL4. In brief, monocytes were cultured in T-175 flasks at 10 ⁶ cells / ml in RPMI / 10% FCS. Recombinant human GM-CSF and IL4 were added on days 0, 2, 4 and 6 at final concentrations of 1000 u / ml and 500 u / ml, respectively. Cells were harvested for 7 days of immature DC and characterized for surface protein expression by FACS analysis. DC maturation was performed by culturing 6-day immature DCs in macrophage-conditioning medium (MCM). In brief, 6-day immature DCs were collected by centrifugation and resuspended in MCM at 10 ⁶ cells / ml. Medium was supplemented with 1000 u / ml GM-CSF and 500 u / ml IL4. After an additional 3 days of culture, cells were harvested with mature DCs and characterized by surface protein expression flow cytometry.

MCM was made as follows: PBMCs isolated from a buffy coat were incubated for 30 minutes at 37 ° C. in a plastic flask pre-coated with 10 mg / ml human IgG (Sigma, St Louis, MO). After 30 minutes, non-adherent cells were removed, adherent cells were washed three times with DPBS and then incubated in RPMI / 10% human serum. Condition-medium was collected after 24 hours. TNF-α concentrations important for DC maturation were measured with the TNF-α ELISA kit (R & D Systems, MN). Final TNF-α levels in MCM were adjusted to 50 u / ml by mixing with RPMI / 10% human serum and stored in a -80 ° C. freezer until use.

Ca ²⁺ flow reaction was performed using an intracellular radiation fluorescent dye, Indo-1. Cells were loaded with Indo-1 / AM (3 μM; Molecular Probes, Eugene, OR) in culture medium (45 min, 20 ° C., 10 ⁷ cells / ml). After dye loading, cells were washed once with 10 ml PBS and resuspended at 10 ⁶ cells / ml in HBSS containing 1% FBS. Cytoplasmic [Ca ²⁺ ] emission was measured by excitation at 350 nm using a Photon Technology International fluorometer (excitation at 350 nm, emission at 400 and 490 nm).

SHAAGtide SEQ ID NO: 1, SEQ ID NO: 6, and CKβ8-1 (25-116) induced rapid flow in human monocytes and neutrophils and showed partial activity in immature dendrites and mature dendritic cells. No significant calcium flow was observed in the control peptide. Since immature dendritic cells express high levels of CCR1, CKβ8-1 (25-116) induces Ca ²⁺ release in these cells.

Example 9: chemokine, SHAAGtide, SHAAGtide truncated variant induced calcium flow by stably expressed FPRL1 cells.

Stable expression of human FPRL1 in L1.2 cells was achieved in the same manner as in Example 6. Calcium flow testing was performed on the transfectants by the method described in Example 1. Chemokines ((CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116), CKβ8-1 (25-116)) were prepared in the same manner as in Example 1. W Peptide 1 And 2 were obtained in the same manner as in Example 6. In addition, the following SHAAGtide sequences and truncated variants (Example 8) were examined:

CCXP1 SEQ ID NO: 1

CCXP2 SEQ ID NO: 2

CCXP3 SEQ ID NO: 3

CCXP4 SEQ ID NO: 4

CCXP5 SEQ ID NO: 5

CCXP6 SEQ ID NO: 6

CCXP7 SEQ ID NO: 7

CCXP8 SEQ ID NO: 8

CCXP9 SEQ ID NO: 9

CCXP10 SEQ ID NO: 10

All ligands were added in a dose response manner and the highest calcium flow response was measured. Table 8 shows CKβ8 (25-116) (SEQ ID NO: 16) and specific SHAAGtide induced calcium flow in FPRL1 transfectants. However, CKβ8 (1-116) without free SHAAGtide N-terminus showed no significant flow. These results suggest that the N-terminus of SHAAGtide is important for activity in FPRL1 transfectants. These SHAAGtide with truncated N-terminus or substituted C-terminus showed no loss of activity as observed after truncation of the N-terminus.

Table 8-Induction of calcium flux in FPRL1 L1.2 cells (sequences for which no IC50-IC50 values are listed showed little (no) significant activity)

Example 10 Chemotactic Activity of Chemokine, SHAAGtide, SHAAGtide Truncated Variants in FPRL1-L1.2 Cells.

Chemotactic Activity of Chemokines [CKβ8 (1-99) and CKβ8 (25-99)], SHAAGtide (SEQ ID NO: 1 and SEQ ID NO: 2), W Peptides 1 and 2 (Example 6) in FPRL1-L1.2 Cells Was measured by migration analysis. Chemokines were prepared in the same manner as in Example 1. SHAAGtide sequences (SEQ ID NO: 1 and SEQ ID NO: 2) were prepared in the same manner as in Example 8. FPRL1-L1.2 cells were prepared in the same manner as in Example 6.

Migration analysis was performed on 96-well ChemoTx® microplates (Neuroprobe) using the protocol described in Example 5. SEQ ID NO: 1 and CKβ8-1 (25-116) migrate transfectants, suggesting that they act on the receptor.

Example 11 Chemotactic Activity of Chemokine, SHAAGtide, SHAAGtide Truncated Variants in Human Monocytes and Neutrophils

In human monocytes and neutrophils chemokines [CKβ8 (1-99), CKβ8 (25-99), CKβ8 (1-116), CKβ8 (25-116)], W peptides 1 and 2, SHAAGtide sequence (SEQ ID NO: 1 and Chemotactic activity of SEQ ID NO: 2) was determined by migration analysis. The peptide was prepared in the same manner as in the previous example. Migration analysis was performed on 96-well ChemoTx® microplates (Neuroprobe) using the protocol described in Example 5. SHAAGtide SEQ ID NO: 1 and CKβ8-1 (25-116) induced migration of both neutrophils and monocytes.

references

US Patent No. 4,522, 811. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides. 1985

US Patent No. 5,223, 409. Directed evolution of novel binding proteins. 1993.

US Patent No. 5,328, 470. Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor. 1994.

U.S. Pat. No. 5,994, 126. Method for in vitro proliferation of dendritic cell precursors and their use to produce immunogens. 1999.

Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, et al. 1987. Current protocols in molecular biology. John Wiley & Sons, New York.

Baek SH, Seo JK, Chae CB, Suh PG, Ryu SH. 1996. Identification of the peptides that stimulate the phosphoinositide hydrolysis in lymphocyte cell lines from peptide libraries. J Biol Chem 271 (14). W8170-5.

Baggiolini, M., and C. A. Dahinden. 1994.CC chemokines in allergic inflammation. Immunol Today 15: 127.

Baggiolini, M., B. Dewald, and B. Moser. 1997. Human chemokines: an update. Annu Rev Immunol 15: 675.

Bender et al., 1996, J Immunol. Methods 196: 121-35

Berkhout, T. A., J. Gohil, P. Gonzalez, C. L. Nicols, K. E. Moores, C. H. Macphee, J. R. White, and P. H. Groot. 2000. Selective binding of the truncated form of the chemokine CKbeta8 (25-99) to CC chemokine receptor l (CCRI). Biochem Pharmacol 59: 591.

Bonecchi, R., N. Polentarutti, W. Luini, A. Borsatti, S. Bernasconi, M. Locati, C. Power, A. Proudfoot, T. N. Wells, C. Mackay, A. Mantovani, and S. Sozzani. 1999.Up-regulation of CCRI and CCR3 and induction of chemotaxis to CC chemokines by IFN-gamma in human neutrophils. J Immunol 162: 474.

Bao L, Gerard NP, Eddy RL Jr, Shows TB, Gerard C. 1992. Mapping of genes for the human C5a receptor (C5AR), human FMLP receptor (FPR), and two FMLP receptor homologue orphan receptors (FPRH1, FPRH2) to chromosome 19. Genomics 13 (2). 43 7-40.

Campbell et al., 1998, J Cell Biol 141: 1053

Carell, T., E. A. Wintner, and J. Rebek Jr. 1994a. A novel procedure for the synthesis of libraries containing small organic molecules. Angewandte Chemie International Edition. 33: 2059-2061.

Carell, T., E. A. Wintner, and J. Rebek Jr. 1994b. A solution phase screening procedure for the isolation of active compounds from a molecular library. Angewandte Chemie International Edition. 33: 2061-2064.

Carter, P. 1986. Site-directed mutagenesis. Biochem J. 237: 1-7.

Cella et al, 1999, J Exp Med. 189: 821-9.

Chan et al., 1999, Blood 93: 3610.

Chen, S. H., H. D. Shine, J. C. Goodman, R. G. Grossman, et al. 1994. Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo. Proc Natl Acad Sci US A. 91: 3054-7.

Cho, C. Y., E. J. Moran, S. R. Cherry, J. C. Stephans, et al. 1993. An unnatural biopolymer. Science. 261: 1303-5.

Christophe, T., Karlsson, A., Dugave, C, Marie-Josephe Rabiet, Francois Boulay, F., and Dahlgren, C. (2001). The Synthetic Peptide Trp-Lys-Tyr-Met-Val-Met-NH2 Specifically Activates Neutrophils through FPRL1 / lipoxin A ₄ Receptors and Is an Agonist for the Orphan Monocyte-expressed Chemoattractant Receptor FPRL2. J. Biol. Chem., Vol. 276, Issue 24, 21585-21593.

Cohen. 1993. Science 259: 1691-1692.

Coligan, 1991. Current Protocols in Immunology, Wiley / Greene, NY.

Cull, M. G., J. F. Miller, and P. J. Schatz. 1992. Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor. Proc Natl Acad Sci USA. 89: 1865-9.

Cwirla, S. E., E. A. Peters, R. W. Barrett, and W. J. Dower. Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci USA. 87: 6378-82.

Deng, X., Ueda, H., Su, SB, Gong, W., Dunlop, NM, Gao, J.-L., Murphy, PM, and Wang, JM (1999) A Synthetic Peptide Derived From Human Immunodeficiency Virus Type 1 gpl20 Downregulates the Expression and Function of Chemokine Receptors CCR5 and CXCR4 in Monocytes by Activating the 7-Transmembrane G-Protein-Coupled Receptor FPRL1 / LXA4R. Blood 94, 1165-1173.

Devlin, J. J., L. C. Panganiban, and P. E. Devlin. 1990. Random peptide libraries: a source of specific protein binding molecules. Science. 249: 404-6.

DeWitt, S. H., J. S. Kiely, C. J. Stankovic, M. C. Schroeder, et al. 1993. "Diversomers": an approach to nonpeptide, nonoligomeric chemical diversity. Proc Natl Acad Sci U S A. 90: 6909-13.

Dieu et al., 1998, JExp Med 188: 373.

Fantuzzi, L., P. Borghi, V. Ciolli, G. Pavlakis, F. Belardelli, and S. Gessani. Loss of CCR2 expression and functional response to monocyte chemotactic protein (MCP-1) during the differentiation of human monocytes: role of secreted MCP-1 in the regulation of the chemotactic response. Blood 94: 875

Felici, F., L. Castagnoli, A. Musacchio, R. Jappelli, et al. 1991. Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J Mol Biol. 222: 301-10.

Fodor, S. P., R. P. Rava, X. C. Huang, A. C. Pease, et al. 1993. Multiplexed biochemical assays with biological chips. Nature. 364: 555-6.

Forssmann, U., M. B. Delgado, M. Uguccioni, P. Loetscher, G. Garotta, and M. Baggiolini. 1997. CKbeta8, a novel CC chemokine that predominantly acts on monocytes. In FEBS Lett, Vol. 408, p. 211.

Gallop, M. A., R. W. Barrett, W. J. Dower, S. P. Fodor, et al. 1994. Applications of combinatorial technologies to drug discovery. Background and peptide combinatorial libraries. J Med Chem. 37: 1233-51.

Gennaro, A. R. 2000. Remington: The science and practice of pharmacy. Lippincott, Williams & Wilkins, Philadelphia, PA.

Harlow, E., and D. Lane. 1988. Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 726 pp.

Harlow, E., and D. Lane. 1999. Using antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Houghten, R. A., J. R. Appel, S. E. Bondelle, J. H. Cuervo, et al. 1992. The use of synthetic peptide combinatorial libraries for the identification of bioactive peptides. Biotechniques. 13: 412-21.

Kaufman, R. J. 1990. Vectors used for expression in mammalian cells. Methods Enzymol. 185: 487-511.

Kellermann et al., 1999, J Immunol 162: 3859.

Kriegler, M. 1990. Gene transfer and expression: A laboratory manual. Stockton Press, New York. 242 pp

Lam, K. S. 1997. Application of combinatorial library methods in cancer research and drug discovery. Anticancer Drug Design. 12: 145-167.

Lam, K. S., S. E. Salmon, E. M. Hersh, V. J. Hruby, et al. 1991. General method for rapid synthesis of multicomponent peptide mixtures. Nature. 354: 82-84.

Le, Y., Oppenheim J. J., and Wang, J. M. 2001. Survey: Pleiotropic roles of formyl peptide receptors, Cytokine and Growth Factor Reviews 12: 91-105.

Lee, S. C., M. E. Brummet, S. Shahabuddin, T. G. Woodworth, S. N. Georas, K. M. Leiferman, S. C. Gilman, C. Stellato, R. P. Gladue, R. P. Schleimer, and L. A. Beck. 2000. Cutaneous injection of human subjects with macrophage inflammatory protein-1 alpha induces significant recruitment of neutrophils andmonocytes. J Immunol 164. 3392

Luna, L. G. 1968. The Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology ", McGraw-Hill, 3rd edition.

Macphee, CH, ER Appelbaum, K. Johanson, KE Moores, CS Imburgia, J. Fornwald, T. Berkhout, M. Brawner, PH Groot, K. O'Donnell, D. O'Shannessy, G. Scott, and JR White. 1998. Identification of a truncated form of the CC chemokine CK beta-8 demonstrating greatly enhanced biological activity. J lmmunol 161: 6273.

Mantovani, A. 1999. The chemokine system: redundancy for robust outputs. Immunol Today 20: 254.

Murphy, P. M., Ozcelik, T., Kenney, R. T., Tiffany, H. L., McDermott, D., and Francke, U. 1992. A structural homologue of the N-formyl peptide receptor. Characterization and chromosome mapping of a peptide chemoattractant receptor family J. Biol. Chem. 267,7637-7643

Murphy, P. M., M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, K. Matsushima, L. H. Miller, J. J. Oppenheim, and C. A. Power. 2000. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev 52. 145.

Patel, VP, BL Kreider, Y. Li, H. Li, K. Leung, T. Salcedo, B. Nardelli, V. Pippalla, S. Gentz, R. Thotakura, D. Parmelee, R. Gentz, and G. Garotta. 1997. Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors. J Exp Med 185: 1163.

Romani et al., 1996, J. Immunol. Methods 196: 137

Rollins, B. J. 1997. Chemokines. Blood 90: 909.

Sambrook, J. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor.

Scott, J. K., and G. P. Smith. 1990. Searching for peptide ligands with an epitope library. Science. 249: 386-90.

Shao Bo Su, Ji-liang Gao, Wang-hua Gong, Nancy M. Dunlop, Philip M. Murphy, Joost J. Oppenheim and Ji Ming Wang. T21 / DP107, A Synthetic Leucine Zipper-Like Domain of the HIV-1 Envelope gp41, Attracts and Activates Human Phagocytes by Using G-Protein-Coupled Formyl Peptide Receptors. Journal of Immunology, 162: 5924-5930.

Su, SB, Gao, J.-L., Gong, W., Dunlop, NM, Murphy, PM, Oppenheim, JJ, and Wang, JM 1999.T21 / DP107, A Synthetic Leucine Zipper-Like Domain of the HIV- 1 Envelope gp41, Attracts and Activates Human Phagocytes by Using G-Protein-Coupled Formyl Peptide Receptors. J Immunol. 162,5924- 5930.

Uguccioni, M., M. D'Apuzzo, M. Loetscher, B. Dewald, and M. Baggiolini. 1995. Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1 alpha and MIP-1 beta on human monocytes. Eur JImmunol 25. 64.

Ulmer et al., (1993) Science 259: 1745-1749

Verdijk et al., 1999, Immunol. 1: 57-61

Weber, C., K. U. Belge, P. von Hundelshausen, G. Draude, B. Steppich, M. Mack, M. Frankenberger, K. S. Weber, and H. W. Ziegler-Heitbrock. 2000. Differential chemokine receptor expression and function in human monocyte subpopulations. J Leukoc Biol 67: 699.

Wells, J. A., M. Vasser, and D. B. Powers. 1985.Casette mutagenesis: an efficient method for generation of multiple mutations at defined sites. Gene. 34: 315-23. Ye, R. D., Cavanagh, S. L., Quehenberger, O., Prossnitz, E. R., and Cochrane, C. G. 1992. Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide receptor. Biochem. Biophys. Res. Commun. 184, 582-589.

Youn, B. S., S. M. Zhang, H. E. Broxmeyer, S. Cooper, K. Antol, M. Fraser, Jr., and B. S. Kwon. Characterization of CKbeta8 and CKbeta8-1: two alternatively spliced forms of human beta-chemokine, chemoattractants for neutrophils, monocytes, and lymphocytes, and potent agonists at CC chemokine receptor 1.Blood 91: 3118.

Zoller, M. J., and M. Smith. 1987. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. Methods Enzymol. 154: 329-50.

Zuckermann, R. N., E. J. Martin, D. C. Spellmeyer, G. B. Stauber, et al. 1994. Discovery of nanomolar ligands for 7-transmembrane G-protein-coupled receptors from a diverse N- (substituted) glycine peptoid library. J Med Chem. 37: 2678-85.

SEQUENCE LISTING <110> Miao, Zhenhua Premack, Brett Schall, Thomas J. <120> Compositions useful as ligands for the formyl peptide receptor like 1 receptor and methods of use <130> 10709/25 <140> US 10 / 141,620 <141> 2002-05-07 <150> US 60 / 328,241 <151> 2001-10-09 <160> 30 <170> PatentIn version 3.1 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> SHAAGtide <400> 1 Met Leu Trp Arg Arg Lys Ile Gly Pro Gln Met Thr Leu Ser His Ala 1 5 10 15 Ala gly <210> 2 <211> 15 <212> PRT <213> Artificial sequence <220> <223> SHAAGtide variant <400> 2 Arg Arg Lys Ile Gly Pro Gln Met Thr Leu Ser His Ala Ala Gly 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial sequence <220> <223> SHAAGtide variant <400> 3 Met Leu Trp Arg Arg Lys Ile Gly Pro Gln Met Thr Leu Ser His 1 5 10 15 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <220> <223> SHAAGtide variant <400> 4 Ile Gly Pro Gln Met Thr Leu Ser His Ala Ala Gly 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial sequence <220> <223> SHAAGtide variant <400> 5 Met Leu Trp Arg Arg Lys Ile Gly Pro Gln Met Thr 1 5 10 <210> 6 <211> 18 <212> PRT <213> Artificial sequence <220> <223> SHAAGtide variant <400> 6 Met Leu Trp Arg Arg Lys Ile Gly Pro Gln Met Thr Leu Ser His Ala 1 5 10 15 Ala tyr <210> 7 <211> 16 <212> PRT <213> Artificial sequence <220> <223> SHAAGtide variant <400> 7 Trp Arg Arg Lys Ile Gly Pro Gln Met Thr Leu Ser His Ala Ala Gly 1 5 10 15 <210> 8 <211> 11 <212> PRT <213> Artificial sequence <220> <223> SHAAGtide variant <400> 8 Met Leu Trp Arg Arg Lys Ile Gly Pro Gln Met 1 5 10 <210> 9 <211> 9 <212> PRT <213> Artificial sequence <220> <223> SHAAGtide variant <400> 9 Trp Arg Arg Lys Ile Gly Pro Gln Met 1 5 <210> 10 <211> 6 <212> PRT <213> Artificial sequence <220> <223> SHAAGtide variant <400> 10 Trp Arg Arg Lys Ile Gly 1 5 <210> 11 <211> 14 <212> PRT <213> Artificial sequence <220> <223> SHAAGtide variant <400> 11 Leu Trp Arg Arg Lys Ile Gly Pro Gln Met Thr Leu Ser His 1 5 10 <210> 12 <211> 279 <212> DNA <213> Homo sapiens <400> 12 atgctctgga ggagaaagat tggtcctcag atgacccttt ctcatgctgc aggattccat 60 gctactagtg ctgactgctg catctcctac accccacgaa gcatcccgtg ttcactcctg 120 gagagttact ttgaaacgaa cagcgagtgc tccaagccgg gtgtcatctt cctcaccaag 180 aaggggcgac gtttctgtgc caaccccagt gataagcaag ttcaggtttg catgagaatg 240 ctgaagctgg acacacggat caagaccagg aagaattga 279 <210> 13 <211> 99 <212> PRT <213> Homo sapiens <400> 13 Arg Val Thr Lys Asp Ala Glu Thr Glu Phe Met Met Ser Lys Leu Pro 1 5 10 15 Leu Glu Asn Pro Val Leu Leu Asp Arg Phe His Ala Thr Ser Ala Asp 20 25 30 Cys Cys Ile Ser Tyr Thr Pro Arg Ser Ile Pro Cys Ser Leu Leu Glu 35 40 45 Ser Tyr Phe Glu Thr Asn Ser Glu Cys Ser Lys Pro Gly Val Ile Phe 50 55 60 Leu Thr Lys Lys Gly Arg Arg Phe Cys Ala Asn Pro Ser Asp Lys Gln 65 70 75 80 Val Gln Val Cys Met Arg Met Leu Lys Leu Asp Thr Arg Ile Lys Thr 85 90 95 Arg lys asn <210> 14 <211> 75 <212> PRT <213> Homo sapiens <400> 14 Arg Phe His Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr Thr Pro Arg 1 5 10 15 Ser Ile Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn Ser Glu 20 25 30 Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg Arg Phe 35 40 45 Cys Ala Asn Pro Ser Asp Lys Gln Val Gln Val Cys Met Arg Met Leu 50 55 60 Lys Leu Asp Thr Arg Ile Lys Thr Arg Lys Asn 65 70 75 <210> 15 <211> 116 <212> PRT <213> Homo sapiens <400> 15 Arg Val Thr Lys Asp Ala Glu Thr Glu Phe Met Met Ser Lys Leu Pro 1 5 10 15 Leu Glu Asn Pro Val Leu Leu Asp Met Leu Trp Arg Arg Lys Ile Gly 20 25 30 Pro Gln Met Thr Leu Ser His Ala Ala Gly Phe His Ala Thr Ser Ala 35 40 45 Asp Cys Cys Ile Ser Tyr Thr Pro Arg Ser Ile Pro Cys Ser Leu Leu 50 55 60 Glu Ser Tyr Phe Glu Thr Asn Ser Glu Cys Ser Lys Pro Gly Val Ile 65 70 75 80 Phe Leu Thr Lys Lys Gly Arg Arg Phe Cys Ala Asn Pro Ser Asp Lys 85 90 95 Gln Val Gln Val Cys Met Arg Met Leu Lys Leu Asp Thr Arg Ile Lys 100 105 110 Thr Arg Lys Asn 115 <210> 16 <211> 92 <212> PRT <213> Homo sapiens <400> 16 Met Leu Trp Arg Arg Lys Ile Gly Pro Gln Met Thr Leu Ser His Ala 1 5 10 15 Ala Gly Phe His Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr Thr Pro 20 25 30 Arg Ser Ile Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn Ser 35 40 45 Glu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg Arg 50 55 60 Phe Cys Ala Asn Pro Ser Asp Lys Gln Val Gln Val Cys Met Arg Met 65 70 75 80 Leu Lys Leu Asp Thr Arg Ile Lys Thr Arg Lys Asn 85 90 <210> 17 <211> 92 <212> PRT <213> Homo sapiens <400> 17 Gln Phe Ile Asn Asp Ala Glu Thr Glu Leu Met Met Ser Lys Leu Pro 1 5 10 15 Leu Glu Asn Pro Val Val Leu Asn Ser Phe His Phe Ala Ala Asp Cys 20 25 30 Cys Thr Ser Tyr Ile Ser Gln Ser Ile Pro Cys Ser Leu Met Lys Ser 35 40 45 Tyr Phe Glu Thr Ser Ser Glu Cys Ser Lys Pro Gly Val Ile Phe Leu 50 55 60 Thr Lys Lys Gly Arg Gln Val Cys Ala Lys Pro Ser Gly Pro Gly Val 65 70 75 80 Gln Asp Cys Met Lys Lys Leu Lys Pro Tyr Ser Ile 85 90 <210> 18 <211> 68 <212> PRT <213> Homo sapiens <400> 18 Ser Phe His Phe Ala Ala Asp Cys Cys Thr Ser Tyr Ile Ser Gln Ser 1 5 10 15 Ile Pro Cys Ser Leu Met Lys Ser Tyr Phe Glu Thr Ser Ser Glu Cys 20 25 30 Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg Gln Val Cys 35 40 45 Ala Lys Pro Ser Gly Pro Gly Val Gln Asp Cys Met Lys Lys Leu Lys 50 55 60 Pro Tyr Ser Ile 65 <210> 19 <211> 66 <212> PRT <213> Homo sapiens <400> 19 Ser Leu Ala Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser Tyr Thr Ser 1 5 10 15 Arg Gln Ile Pro Gln Asn Phe Ile Ala Asp Tyr Phe Glu Thr Ser Ser 20 25 30 Gln Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Arg Ser Arg Gln 35 40 45 Val Cys Ala Asp Pro Ser Glu Glu Trp Val Gln Lys Tyr Val Ser Asp 50 55 60 Leu Glu 65 <210> 20 <211> 54 <212> DNA <213> Artificial sequence <220> <223> SHAAGtide polynucleotide variant <400> 20 atgctctgga ggagaaagat tggtcctcag atgacccttt ctcatgctgc agga 54 <210> 21 <211> 45 <212> DNA <213> Artificial sequence <220> <223> SHAAGtide polynucleotide variant <400> 21 aggagaaaga ttggtcctca gatgaccctt tctcatgctg cagga 45 <210> 22 <211> 45 <212> DNA <213> Artificial sequence <220> <223> SHAAGtide polynucleotide variant <400> 22 atgctctgga ggagaaagat tggtcctcag atgacccttt ctcat 45 <210> 23 <211> 36 <212> DNA <213> Artificial sequence <220> <223> SHAAGtide polynucleotide variant <400> 23 attggtcctc agatgaccct ttctcatgct gcagga 36 <210> 24 <211> 36 <212> DNA <213> Artificial sequence <220> <223> SHAAGtide polynucleotide variant <400> 24 atgctctgga ggagaaagat tggtcctcag atgacc 36 <210> 25 <211> 54 <212> DNA <213> Artificial sequence <220> <223> SHAAGtide polynucleotide variant <400> 25 atgctctgga ggagaaagat tggtcctcag atgacccttt ctcatgctgc atat 54 <210> 26 <211> 48 <212> DNA <213> Artificial sequence <220> <223> SHAAGtide polynucleotide variant <400> 26 tggaggagaa agattggtcc tcagatgacc ctttctcatg ctgcagga 48 <210> 27 <211> 33 <212> DNA <213> Artificial sequence <220> <223> SHAAGtide polynucleotide variant <400> 27 atgctctgga ggagaaagat tggtcctcag atg 33 <210> 28 <211> 27 <212> DNA <213> Artificial sequence <220> <223> SHAAGtide polynucleotide variant <400> 28 tggaggagaa agattggtcc tcagatg 27 <210> 29 <211> 18 <212> DNA <213> Artificial sequence <220> <223> SHAAGtide polynucleotide variant <400> 29 tggaggagaa agattggt 18 <210> 30 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> SHAAGtide polynucleotide variant <400> 30 ctctggagga gaaagattgg tcctcagatg accctttctc at 42

Claims (24)

An isolated protein or polypeptide comprising an N-terminal sequence having at least 80% identity with SEQ ID NO: 1, except for SEQ ID NO: 16. The isolated protein or polypeptide of claim 1, which is a ligand for FPRL1. The isolated protein or polypeptide of claim 1, wherein the N-terminal sequence has at least 90% sequence identity with SEQ ID NO: 1. The isolated protein or polypeptide of claim 1, wherein the N-terminal sequence has at least 95% sequence identity with SEQ ID NO: 1. The isolated protein or polypeptide of claim 1, wherein the N-terminal sequence has 100% sequence identity with SEQ ID NO: 1. The isolated protein or polypeptide of claim 1, wherein the N-terminal sequence is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6. 6. An isolated polypeptide having at least 80% identity with SEQ ID NO: 1. 8. The isolated polypeptide of claim 7 having at least 90% identity with SEQ ID NO: 1. 8. The isolated polypeptide of claim 7 having at least 95% identity with SEQ ID NO: 1. 8. The isolated polypeptide of claim 7 having a sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6. An isolated nucleic acid consisting of a nucleic acid sequence having at least 80% identity with SEQ ID NO: 20. The isolated nucleic acid of claim 11 having at least 90% identity with SEQ ID NO: 20. The isolated nucleic acid of claim 11 having at least 95% identity with SEQ ID NO: 20. 12. The isolated nucleic acid of claim 11 having at least 99% identity with SEQ ID NO: 20. An isolated nucleic acid having a sequence complementary to the nucleic acid of claim 11. 12. The isolated nucleic acid of claim 11 consisting of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 30. 16. The isolated nucleic acid of claim 15 consisting of SEQ ID NO: 20. The isolated nucleic acid of claim 15 consisting of SEQ ID NO: 25. An antibody that specifically binds to the peptide of claim 1. A fusion protein consisting of a non-SHAAGtide polypeptide fused to the peptide of claim 1. A kit comprising a pharmaceutical composition containing the polypeptide of claim 1, a pharmaceutically acceptable carrier, a syringe. In a method for identifying a FPRL1 receptor antagonist, Contacting a cell expressing the FPRL1 receptor with a protein or polypeptide comprising an N-terminal sequence having at least 80% identity with SEQ ID NO: 1, wherein the receptor is stimulated; Contacting the receptor with a candidate antagonist compound; Detecting the FPRL1 receptor. The method of claim 22, wherein the candidate compound is an antibody, peptide, nucleic acid or small molecule. The method of claim 22, wherein the cells are neutrophils, monocytes, T-lymphocytes or dendritic cells.