ES2368814T3 - USEFUL COMPOSITIONS AS LINKS FOR THE RECEIVER TYPE RECEIVER 1 OF FORMILED PEPTIDES AND METHODS OF USE THEREOF. - Google Patents

Abstract

An isolated protein or polypeptide that modulates FPRL1 receptor activity that comprises a sequence at its N-terminus, the sequence having an identity of at least 90% with SEQ ID No. 1 throughout its length, where the protein or the polypeptide is not one of SEQ ID Nos. 15 and 16.

Description

Compositions useful as ligands for the formylated peptide Receptor 1 receptor and methods of use thereof.

Field of the Invention

The invention relates to compositions useful as ligands for the Formulated Peptide Receptor Type 1 receptor and to the methods of use thereof.

Background

Chemokines (chemotactic cytokines) act as molecular beacons for the recruitment and activation of T lymphocytes, neutrophils, and macrophages, marking battlegrounds with pathogens. The recruitment of leukocytes, the white blood cells responsible for fighting infections, depends on chemokine gradients. Chemokines are a superfamily of small proteins (8-10 KD) that mediate various biological processes including leukocyte trafficking and return, immunoregulation, hematopoiesis, and angiogenesis. To date, 24 chemokine receptors are known. Chemokines play a critical role in innate immunity and inflammatory reactions (Baggiolini et al. (1994); Baggiolini et al. (1997); Rollins (1997).) Four chemokine subfamilies have been described, based on the distance between the first two conserved cysteine residues: C, CC, CXC, and CX3C. All known chemokines signal through four groups of seven transmembrane domain receptors belonging to the Gi protein-coupled heterotrimeric receptors and pertussis toxins sensitive to the Gi family: XCR, CCR, CXCR and CX3CR. (Murphy et al. (2000)). Extracellular binding events can activate specific signal transduction pathways that lead to different responses, such as chemotaxis. In the chemokine system, multiple chemokines can activate a single chemokine receptor; for example, the CCR1 receptor binds RANTES chemokines (regulated in the activation of expressed normal T cells), MIP-1α (a macrophage inflammatory protein) and MIP-1β. Similarly, a single chemokine can activate multiple receptors (Mantovani (1999)).

Monocytes and neutrophils, which play an important role in the pathogenesis of inflammation and in the presentation of antigens, respond to chemokines (Lee et al. (2000)). Monocytes express chemokine receptors CCR1, CCR2, CCR5, CCR8, CXCR2, and CXCR4. (Uguccioni et al. (1995); Weber et al. (2000)). Ligands MIP-1α and Monocyte Chemoattractant Protein 1 (MCP1) have been reported as potent monocyte activators in vitro. (Fantuzzi et al. (1999).) Neutrophils are crucial during many acute inflammatory responses, and may also play a role in targeting immunity toward Th1 responses. (Bonecchi et al. (1999).) Respond mainly to some CXC chemokines but do not migrate for most CC chemokines. Human neutrophils express two high-affinity IL-8 receptors, CXCR1 and CXCR2.

CKβ8 chemokine, also known as CCL23; hmrp-2a; myeloid progenitor inhibitory factor 1 (MPIF-1); SCYA23 (current nomenclature and ID Genome system), is a 99 amino acid CC chemokine containing six cysteines. It is constitutively expressed in the liver, lung, pancreas, and bone marrow. CKβ8 has chemotactic activity on monocytes, dendritic cells, and lymphocytes at rest (Forssmann et al. (1997)) and inhibits colony formation of potential bone marrow-derived low-proliferation colony-forming cells. (Patel et al. (1997)). CKβ8-1, an alternative splicing form of CKβ8 that is 116 amino acids in length, has been reported. Both mature forms CKβ8 and CKβ8-1 have been assigned as ligands for the CCR1 receptor. (Youn et al. (1998)). Cross-desensitization studies on both monocytes and eosinophils indicate that CKβ8-1 binds predominantly to CCR1. Further processing at the NH2 terminus of CKβ8 results in 76 or 75 residue proteins that are significantly more active on cells expressing CCR1 (Macphee et al. (1998), Berkhout et al. (2000)).

In addition to chemokine receptors, neutrophils and monocytes also express the G-protein coupled N-formylated Peptides (FPR) receptor and its homologous N-formylated Peptides (FPRL1) type 1 receptor. Since the ligands for FPRL1 were unknown when it was originally cloned, FPRL1 was initially defined as an orphan receptor. (Bao et al. (1992); Murphy et al. (1992); Ye et al. (1992).) It was assigned as an LXA4 receptor since it binds lipoxin A4 (Fiore et al. (1994).) Furthermore, several different peptides / proteins have been reported to bind FPRL1 with low affinity (see Figure 1). A serum amyloid A, a protein secreted during the acute phase of inflammation, has been reported to be referred to as a medium affinity functional ligand (Su et al. (1999)). A β-amyloid fragment (1-42) and a neurotoxic prion peptide 106-126 are also low affinity ligands, indicating that FPRL1 may play a role in neurodegenerative diseases (Le et al. (2001)). Some other low affinity ligands include: peptides derived from HIV envelope proteins (Su et al. (1999), Deng et al. (1999)); and a peptide from Helicobacter pylori, Hp (2-20). Some synthetic peptides, such as Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm) and Trp-LysTyr-Met-Val-Met-NH2 (WKYMVM) ("W 1 and 2 peptides"), they have been referred to as potent ligands for the receptor. (Christophe et al. (2001); Baek et al. (1996)). However, these non-naturally occurring peptides derived from random hexapeptide libraries have not been shown to be physiologically relevant.

Compendium

The inventors have discovered that the truncation variant of CKß8-1, CKß8- (25-116), is implicated in inflammatory reactions and innate immunity through its role as a functional ligand for the formylated peptide receptor of type 1 (FPRL1). Furthermore, the authors of the present invention have discovered an alternative splicing exon of CKß8-1, called SHAAGtido, and the truncated and other variants of SHAAGtido, together with CKß8-1 (25-116), are functional in both cells that they are known to express FPRL1. Functional SHAAGtides generate calcium flux after receptor-ligand binding in leukocytes and attract monocytes, neutrophils, mature dendritic cells (CDm), and immature dendritic cells (CDi).

In one embodiment, the invention includes SHAAGtides as well as proteins and peptides comprising SHAAGtides, with the exception of CKß8-1 (25-116). In particular, the present invention provides an isolated protein or polypeptide that modulates FPRL 1 receptor activity comprising a sequence at its N-terminus, the sequence having at least 90% identity with SEQ ID No. 1 throughout its length. , where the protein or polypeptide is not one of SEQ ID No. 15 and 16.

In addition, the invention also includes nucleic acids encoding proteins and peptides that comprise SHAAGtides, antibodies that specifically bind SHAAGtides, and fusion proteins that comprise SHAAGtides.

In another embodiment, the invention includes compositions comprising SHAAGtides or proteins or peptides comprising a SHAAGtido sequence. Such compositions include those suitable for administration to a subject to enhance FPRL1 activity.

In a further embodiment, the invention includes kits comprising such compositions. Such kits can be assembled to facilitate the administration, for example, of pharmaceutical compositions.

Also described are methods of treating a subject for a disorder that comprises modulating the activity of an FPRL1 receptor by administering a compound that comprises a SHAAGtido or proteins or peptides that comprise a SHAAGtido sequence.

In a further aspect, the invention includes the methods and kits useful for the identification of such antagonists are also included in the present invention. Such methods comprise the step of contacting an FPRL1 receptor with a composition comprising a biologically active SHAAGtido, or a protein or peptide comprising a SHAAGtido sequence, in the presence of a candidate antagonist molecule. Antagonists for FPRL1 receptor function can be identified as those compounds that reduce receptor activity compared to that observed in the absence of the candidate compound.

DESCRIPTION OF THE DRAWINGS

Figure 1. Table showing referred FPRL1 endogenous low affinity ligands and unnatural ligands. Figure 2. Figure showing amino acid sequence alignment of human CCL23 / CKβ8 variants with human CCL15 / MIP-1α and human CCL3 / MIP-1δ.

DETAILED DESCRIPTION

The authors of the present invention have discovered that a truncation variant of CKβ8-1, CKβ8-1 (25116), is a bifunctional ligand for two different GPCRs: the CCR1 chemokine receptor and the type 1 formylated peptide receptor (FPRL1 ). The authors of the invention also discovered that, in addition to its activity as a CCR1 ligand, CKβ8-1 (25-116) is implicated in inflammatory reactions and immunity by recruiting monocytes and neutrophils through its role as a functional ligand for FPRL1. CKβ8 attracts cells including monocytes, dendritic cells, and resting lymphocytes through OCR1, but lacks the alternative splicing exon found in CKβ8-1 (25-116) (SHAAGtido sequence). CKβ8-1 (1-116), the alternative splicing form of CKβ8 (116 amino acids) is a functional ligand for the CCR1 receptor, like CKβ8. However, CKβ8-1 (1-116) does not exert its functions through the SHAAGtido sequence.

The authors of the present invention have also discovered a class of novel peptides (the SHAAGtido peptide and variants of the SHAAGtido peptide - hereafter collectively known as " SHAAGtidos "), truncation mutants of the CCC23 chemokine splice exon, CKβ8-1 (25-116), which are surprisingly effective and valuable ligands for the FPRL1 receptor. These peptides produce a calcium flux in the leukocytes that express FPRL1. Furthermore, SHAAGtides effectively attract cells including monocytes, neutrophils, mature dendritic cells (CDm), and immature dendritic cells (CDi) and other leukocyte subgroups. The SHAAGtido peptide (SEQ ID NO: 1) and certain variants of SHAAGtido, along with its parental chemokine CKβ8-1 (25-116), are functional on both monocytes and neutrophils known to express FPRL1. Functional SHAAGtides generate calcium flux after receptor-ligand binding in leukocytes and attract monocytes, neutrophils, mature dendritic cells (CDm), and immature dendritic cells (CDi) in chemotactic analyzes. In light of these observations, SHAAGtides represent cryptic functional peptides that are therefore surprisingly effective as ligands for FPRL1.

The invention includes SHAAGtides as well as proteins and peptides comprising SHAAGtides, with the exception of CKβ8-1 (25-116) and CKβ8-1 (1-116). Furthermore, the invention also includes nucleic acids encoding SHAAGtides, as well as nucleic acids encoding proteins and peptides comprising SHAAGtides, with the exception of nucleic acids encoding CKβ8-1 (25-116)) and CKβ8-1 (1- 116). Compositions containing SHAAGtides as well as proteins and peptides comprising SHAAGtides, including CKβ8-1 (25-116) are also included in the invention. Such compositions include those suitable for administration to a subject to enhance FPRL1 activity. Kits comprising such compositions are also included. Such kits can be assembled to facilitate the administration, for example, of pharmaceutical compositions.

Methods for treating a subject in need of stimulation of inflammatory reactions and innate immunity are described herein. Stimulation of such activity may benefit subjects suffering from diseases, for example, infectious diseases (and also vaccination, as described in co-pending patent application " Methods and Compositions for Inducing an Immune Response " filed on May 7, 2002 - agent reference number 10709/23). Such methods comprise stimulating the FPRL1 receptor by administering a composition comprising a SHAAGtido, a peptide or a protein comprising a SHAAGtido, or other stimulatory molecule.

Also described herein are methods of treating a subject in need of down-regulation of inflammatory reactions and innate immunity. Down-regulation of such activity may benefit subjects suffering from diseases including neurodegenerative disorders, such as Alzheimer's disease or Creutzfeldt-Jakob disease. Such methods comprise downregulation of the FPRL1 receptor by administration of a composition comprising an antagonist of FPRL1 receptor function.

The methods and kits for the identification of such antagonists are also included in the present invention. Such methods comprise the step of contacting the FPRL1 receptor with a composition comprising a biologically active SHAAGtido sequence, a peptide or a protein comprising an active SHAAGtido, in the presence of a candidate antagonist molecule. Antagonists of FPRL1 receptor function can be identified as those compounds that reduce receptor activity compared to that observed in the absence of the candidate compound. Such methods can be performed in vitro or in vivo. Furthermore, kits can be assembled to facilitate such in vitro or in vivo testing.

SHAAGtides and molecules comprising SHAAGtides.

SHAAGtido peptides and polypeptides comprising SHAAGtidos

Table 1 shows the SHAAGtido polypeptide sequence (SEQ ID NO: 1) and the polypeptide sequences of certain truncated SHAAGtido variants and other variants. Table 2 shows the SHAAGtido polynucleotide sequence (SEQ ID NO: 12) and the polynucleotide sequences of truncated SHAAGtido variants and other variants. Table 3 shows the Nucleotide Sequence of human CKβ8-1 (25-116) (SEQ ID NO: 20). Figure 2 shows the amino acid sequence alignment of the CCL23 / CKβ8 variants (CKβ (1-99) - SEQ ID NO: 13; CKβ (25-99) - SEQ ID NO: 14; CKβ (1-116 ) - SEQ ID NO: 15; human CKβ (25-116) - (SEQ ID NO: 16) with CCL15 / MIP-1α (SEQ ID NO: 19); CCL3 / MIP-1 S (SEQ ID NO: 17) and human Leukotactin (SEQ ID NO: 18). Four conserved cysteine residues and two additional cysteines, not normally found in the CC chemokine family, are shown in boxes in shaded boxes. The alternately spliced exon of CCL23 / CKβ8- 1 is underlined.

Table 1 SHAAGtido and different truncated variants and other variants - amino acid sequences. Table 2 SHAAGtido and different truncated variants and other variants - polynucleotide sequences

SEQ ID NO:

Designation and Activity FPRL1 Amino acid sequence

one

CCXP1 Native sequence; high activity

2

CCXP2 Low activity

3

CCXP3 High activity

4

CCXP4 Low activity

5

CCXP5 Moderate activity

6

CCXP6 High activity

7

CCXP7 Low activity

8

CCXP8 Moderate activity

9

CCXP9 Low activity

10

CCXP10 Low activity

eleven

CCXP11 Moderate activity

SEQ ID NO:

Polynucleotide sequence

twenty

atgctctgga ggagaaagat tggtcctcag atgacccttt ctcatgctgc agga 54

twenty-one

aggagaaaga ttggtcctca gatgaccctt tctcatgctg cagga

22

atgctctgga ggagaaagat tggtcctcag atgacccttt ctcat Four. Five

2. 3

attggtcctc agatgaccct ttctcatgct gcagga

24

atgctctgga ggagaaagat tggtcctcag atgacc 36

25

atgctctgga ggagaaagat tggtcctcag atgacccttt ctcatgctgc atat 54

26

tggaggagaa agattggtcc tcagatgacc ctttctcatg ctgcagga

27

atgctctgga ggagaaagat tggtcctcag atg 33

28

tggaggagaa agattggtcc tcagatg

29

tggaggagaa agattggt

30

ctctggagga gaaagattgg tcctcagatg accctttctc at 42

5 SHAAGtido molecules, derivatives and analogs

The SHAAGtide peptides of the present invention include those molecules listed in Table 1. In addition, other SHAAGtide peptide and nucleotide derivatives can be synthesized using conventional synthesis techniques. Derivatives are nucleic acid sequences or amino acid sequences formed from

10 native compounds either directly or by modification or partial replacement. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar, but not identical, to that of the native compound but differ from it with respect to certain components or side chains. Analogs can be synthesized or can have a different evolutionary origin.

15 Derivatives and analogs may be complete or may have a length other than complete if the derivative

or the analog contains a nucleic acid or a modified amino acid. For example, SEQ ID NO: 3 contains only the first 15 N-terminal amino acids of the SHAAGtido molecule (SEQ ID NO: 1). Derivatives or analogs of the nucleic acid or SHAAGtido peptide include, molecules comprising regions that are essentially homologous to nucleic acid or SHAAGtido peptide with an identity of at least about 70%, 80%, or 95% along a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is performed by means of a homology algorithm, or whose encoding nucleic acid sequence is capable of hybridizing to a complementary sequence that encodes the above-mentioned peptide sequences under restrictive, moderately restrictive, or less restrictive conditions (Ausubel et al., 1987). A nucleic acid molecule

Complementary is one that is sufficiently complementary to a sequence that hydrogen bonds are formed with few mismatches, forming a stable duplex. " Complementary " refers to Watson-Crick or Hoogsteen base pairings between nucleotides.

The specificity of single-stranded DNA to hybridize with complementary fragments is determined by the "restriction" of the reaction conditions. The restriction of hybridization increases as the propensity to form DNA duplexes decreases. In the nucleic acid hybridization reaction, the restriction can be selected to favor specific hybridizations (high restriction), which can be used to identify, for example, complete clones from a library. Less specific (low restriction) hybridizations can be used to identify related, but not exact (homologous, but not identical) DNA molecules or segments.

DNA duplexes are stabilized by: (1) the number of complementary base pairs, (2) the type of base pairs, (3) the salt concentration (ionic strength) of the reaction mixture, (4) the reaction temperature, and (5) the presence of certain organic solvents, such as formamide that decreases the stability of DNA duplexes. In general, the longer the probe, the higher the temperature required for proper hybridization. A common approach is to vary the temperature: higher relative temperatures result in more restrictive reaction conditions. Ausubel et al., (1987) provide an excellent explanation of the restriction of hybridization conditions.

Hybridization under "restrictive conditions" describes hybridization protocols in which nucleotide sequences homologous to each other at least 60% remain hybridized. Generally, restrictive conditions are selected to be approximately 5 ° C below the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature (at a defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence in equilibrium. Since the target sequences are generally present in excess, at Tm, 50% of the probes are equilibrium occupied.

The " restrictive hybridization conditions " they allow a probe, primer, or oligonucleotide to hybridize only to its target sequence. The restrictive conditions depend on the sequence and will be different. Stringent conditions include: (1) washes at low ionic strength and elevated temperature (eg 15mM sodium chloride, 1.5mM sodium citrate, 0.1% sodium dodecyl sulfate at 50 ° C) ; (2) a denaturing agent during hybridization (eg 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, buffer 50 mM sodium phosphate (pH 6.5; 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C); or (3) 50% formamide. Washes will also typically comprise 5X SSC (0.75M NaCl , 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, salmon sperm DNA sonicated (50 µg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C, with washes at 42 ° C in 0.2 x SSC (sodium chloride / sodium citrate) and 50% formamide at 55 ° C, followed by a highly restrictive wash consisting of 0.1 x EDTA containing SSC at 55 ° C. Preferably, the conditions are such that the homologous sequences are at least about 65%, 70%, 75%, 85%, 90% , 95%, 98%, or 99% with each other typically remain hybridized to each other These conditions are presented as ex are not intended to be limiting.

The "moderately restrictive conditions" they use washing solutions and hybridization conditions that are less restrictive (Sambrook, 1989), such that a polynucleotide will hybridize to all, fragments, derivatives or analogs of SEQ ID NO: 7-12, 14. An example comprises hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg / ml denatured salmon sperm DNA at 55 ° C, followed by one or more washes in 1X SSC, 0.1% SDS at 37 ° C. Temperature, ionic strength, etc. can be adjusted to accommodate experimental factors such as probe length. Other moderately restrictive conditions have been described (Ausubel et al., 1987; Kriegler, 1990).

The "little restrictive conditions" use washing solutions and hybridization conditions that are less restrictive than moderately restrictive (Sambrook, 1989), such that a polynucleotide will hybridize to all, fragments, derivatives, or analogs of SEQ ID NO: 7-12, 14. A Non-limiting example of stringent hybridization conditions are hybridization to 35% formamide, 5X SSC, 50mM Tris-HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficol , 0.2% BSA, 100 mg / ml denatured salmon sperm DNA, 10% dextran sulfate (w / v) at 40 ° C, followed by one or more washes in 2X SSC, Tris-HCl 25 mM (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50 ° C. Other non-restrictive conditions have been described, such as cross-species hybridizations (Ausubel et al., 1987; Kriegler, 1990; Shilo and Weinberg, 1981).

In addition to the naturally occurring allelic variants of SHAAGtido, changes can be introduced by mutations in SEQ ID NO: 1 that cause alterations in the encoded SHAAGtido amino acid sequences that do not significantly alter the function of SHAAGtido. For example, an amino acid substitution can be made at the C-terminal amino acid residue in the sequence of SEQ ID NO: 6. A "non-essential amino acid residue" it is a residue that can be altered from the SHAAGtido wild type sequences without altering biological activity, while an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the SHAAGtides of the invention are predicted to be particularly unlikely to be altered. Amino acids for which conservative substitutions can be made are well known in the art.

Useful conservative substitutions are shown in Table 4, "Preferred Substitutions". Conservative substitutions whereby one amino acid of one class is replaced by another amino acid of the same type are within the scope of the invention so long as the substitution does not materially alter the biological activity of the compound.

Table 4 Preferred substitutions

Original waste

Illustrative substitutions Preferred substitutions

Wing (A)

Val, Leu, Ile Val

Arg (R)

Lys, Gln, Asn Lys

Asn (N)

Gln, His, Lys, Arg Gln

Asp (D)

Glu Glu

Cys (C)

To be To be

Gln (Q)

Asn Asn

Glu (E)

Asp Asp

Gly (G)

Pro, Ala - To

His (H)

Asn, Gln, Lys, Arg Arg

Ile (1)

Leu, Val, Met, Ala, Phe, Norleucine Leu

Leu (L)

Norleucine, Ile, Val, Met, Ala, Phe Ile

Lys (K)

Arg, Gln, Asn Arg

Met (M)

Leu, Phe, Ile Leu

Phe (F)

Leu, Val, Ile, Ala, Tyr Leu

Pro (P)

To To

Be (S)

Thr Thr

Thr (T)

To be To be

Trp (W)

Tyr, Phe Tyr

Tyr (Y)

Trp, Phe, Thr, Ser Phe

Val (V)

Ile, Leu, Met, Phe, Ala, Norleucine Leu,

Nonconservative substitutions that have an effect on (1) the backbone structure of the polypeptide, such as a β-sheet or α-helix conformation, (2) loading, (3) hydrophobicity, or (4) volume of the

The side chain of the target site can modify the function of SHAAGtido, especially when the SHAAGtido sequences comprise a part of a larger polypeptide molecule. The residues are divided into groups based on the properties of the common side chains as indicated in Table 5. Non-conservative substitutions involve changing a member of one of these classes to another class. The substitutions can be introduced at conservative substitution sites or more preferably at non-conserved sites.

Table 5 Classes of amino acids mediated by oligonucleotides (site-directed), alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction selection mutagenesis (Wells et al., 1985) or other known techniques can be performed on cloned DNA to produce the SHAAGtido variant DNA (Ausubel et al., 1987; Sambrook, 1989).

Class

Amino acids

Hydrophobe

Norleucine, Met, Ala, Val, Leu, Ile

Neutral hydrophilic

Cys, Ser, Thr

Acid

Asp, Glu

Alkaline

Asn, Gln, His, Lys, Arg

Disrupts the conformation of the chain

Gly, Pro

Aromatic

Trp, Tyr, Phe

An SHAAGtido " isolated " or " purified " The present invention comprises biologically active polypeptides, proteins or fragments separated and / or recovered from a component of their natural environment. Isolated SHAAGtides include those expressed heterologously in genetically engineered or in vitro expressed cells. Contaminant components include materials that would typically interfere with the diagnostic or therapeutic uses of the polypeptide. To be substantially isolated, the preparations have less than 30% by weight of contaminating material other than SHAAGtide (contaminants), more preferably less than 20%, 10%, and most preferably less than 5% contaminants.

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

An "active polypeptide or polypeptide fragment" it retains a biological, and / or immunological activity similar, but not necessarily identical, to the activity of a SHAAG polypeptide shown in Table 1. Immunological activity, in the context of this study of the polypeptide per se, and not the actual biological role for SHAAGtido by achieving or enhancing FPRL1 activity, it refers to an aspect of a SHAAGtido polypeptide since a specific antibody against a SHAAGtido antigenic epitope binds to a SHAAGtido. Biological activity refers to a function, either inhibitory or stimulatory, caused by a native SHAAGtido polypeptide. A biological activity of a SHAAGtido polypeptide includes, for example, binding to the FPRL1 receptor, or chemotaxis or production of calcium flux upon binding to the FPRL1 receptor. A specific biological analysis (see Examples), with or without dose dependence, can be used to determine SHAAGtido activity. A nucleic acid fragment encoding a biologically active portion of SHAAGtido can be prepared by isolating a polynucleotide sequence encoding a polypeptide having SHAAGtido biological activity, expressing the encoded portion of SHAAGtido (eg, by recombinant expression in vitro) and evaluating the activity of the encoded portion of the SHAAGtido polypeptide.

In general, a variant of a SHAAGtido polypeptide that retains SHAAGtido polypeptide-like function includes any variant in which residues at a particular position in the sequence have been replaced by other amino acids, and additionally includes the possibility of inserting one or more residues additional between two residues of the parental protein as well as the possibility of deleting one or more residues of the parental sequence. Any amino acid substitution, insertion, or deletion is included in the invention. Under favorable circumstances, the substitution is a conservative substitution as defined above.

Table 1 demonstrates that the amino acid deletion at the C-terminus of the SHAAGtido sequence is less likely to cause a loss of FPRL 1 activity than the deletion at the N-terminus (see Example 9). For example, SEQ ID NO: 8, consisting of the 11 N-terminal amino acids of the SHAAGtido sequence, still retains moderate FPRL1 activity. However, the deletion of 3 N-terminal amino acids (SEQ ID NO: 2) results in only low FPRL1 activity. However, the deletion of the amino acid at the N-terminus (SEQ ID NO: 11) does not result in a complete loss of FRPL 1 activity.

" Variant of SHAAGtido " represents an active SHAAGtido polypeptide having at least: (1) an amino acid sequence identity of approximately 80% with a complete native SHAAGtido polypeptide sequence

or (2) any fragment of a complete SHAAGtido polypeptide sequence. Variants of the SHAAGtido polypeptide include SHAAGtido polypeptides in which one or more amino acid residues are added or deleted at the N or C terminus of the entire native amino acid sequence, with the exception of those fragments that are identical to CKβ8 and CKβ8- one. A variant of the SHAAGtido polypeptide will have an amino acid sequence identity of at least about 90%, more preferably an amino acid sequence identity of at least about 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% and most preferably an amino acid sequence identity of at least about 99% with a complete native SHAGGtido polypeptide sequence. Typically, SHAAGtido variant polypeptides are at least about 10 amino acids long, often at least about 20 amino acids long, more often at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids in length,

or more.

The "percentage (%) of sequence identity" is defined as the percentage of SHAAGtido amino acid residues that are identical to the amino acid residues of a candidate sequence when the two sequences are aligned. To determine% amino acid identity, sequences are aligned and if necessary, spaces are entered to achieve maximum% sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures for determining percent identity are well known to those skilled in the art. Often a publicly available computer program such as BLAST, BLAST2, ALIGN2 or the Megalign program (DNASTAR) is used to align the peptide sequences.

When the amino acid sequences are aligned, the% amino acid sequence identity of an amino acid sequence A stops, with, or against a given amino acid sequence B (which can be formulated as a given amino acid sequence A having or comprises a certain% amino acid sequence identity for, with, or against a given amino acid sequence B) can be calculated as:

% amino acid sequence identity = X / Y.100

where

X is the number of amino acid residues scored as identical pairings by the sequence alignment program or the alignment algorithms of A and B e

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

If the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% identity of the amino acid sequence of A with respect to B will not be equal to the% identity of the amino acid sequence of A B with respect to A.

Fusion polypeptides are useful in expression, cell localization, bioassay, and purification studies of SHAAGtides. A " chimeric protein " or a "fusion protein" SHAAGtido comprises a SHAAGtido fused to a polypeptide other than SHAAGtido. A polypeptide other than SHAAGtido is not substantially homologous to a SHAAGtido polypeptide. A SHAAGtido fusion protein can include any portion of the entire SHAAGtido, including any number of biologically active portions. For example, SHAAGtido can be fused to the C-terminus of GST (glutathione S-transferase) sequences. Such fusion proteins facilitate the purification of the recombinant SHAAGtides. In certain host cells, (eg, mammalian), fusions with heterologous signal sequences can improve expression and / or secretion of SHAAGtides.

Antibodies specific for SHAAGtido and variant SHAAGtido sequences are also described herein. Methods for producing polyclonal and monoclonal antibodies, including binding fragments (eg, F (ab) 2) and single chain versions are well known. Accordingly, polyclonal or monoclonal antibodies can be prepared by conventional techniques.

The chemotactic compositions of the invention contain one or more polynucleotides or polypeptides that contain a SHAAGtido sequence. In one embodiment, the composition contains a SHAAGtide that is a polynucleotide or an isolated or recombinant polypeptide. In one embodiment, the SHAAGtide (s) is / are the predominant species (ie, more than about 50%, more often more than about 80% by weight of the total members of the molecule class of composition) of its class (eg, polypeptide, polynucleotide, lipid, carbohydrate) in the composition. The chemotactic compositions of the invention contain SHAAGtides free of the materials normally associated with their environment in situ (if they are of natural origin).

An isolated SHAAGtido nucleic acid molecule is purified from the environment in which it occurs in nature and separated from at least one contaminating nucleic acid molecule. Isolated SHAAGtido molecules are distinguished from the specific SHAAGtido molecule as it exists in cells.

Use of SHAAGtido compositions in the treatment of diseases.

Both therapeutic and prophylactic methods of treating a subject at risk (or susceptible) of a disorder or who have a disorder associated with the aberrant FPRL1 receptor or FPRL1 ligand activity are described herein. Examples include neurodegenerative disorders, such as Alzheimer's Disease.

Diseases or conditions of humans or other species that can be treated with SHAAGtides or proteins or peptides comprising SHAAGtides, or inhibitors or agonists of FPLR1-SHAAGtido interactions, include, but are not limited to, peripheral inflammation-related diseases - chronic, for example: chronic inflammation; thrombosis; atherosclerosis; restenosis; chronic venous insufficiency; recurring bacterial infections; sepsis; skin infections; kidney disease; glomerulonephritis; fibrotic lung diseases; allergic diseases; IBS; rheumatoid arthritis and acute bronquilits. Macroglia and microglia related diseases can also be treated - Central nervous system, for example: neurodegenerative diseases; Alzheimer disease; Multiple sclerosis; Parkinson's disease; neuroinflammation; HIV-associated neurological diseases; dementia associated with HIV; CNS bacterial infections; Toxoplasma gondii in the brain; Acanthamoeba infections; Listeria infections; prion diseases; subacute spongiform encephalopathies and macular degeneration.

Diseases and disorders characterized by increased levels or biological activity of FPRL1 can be treated with therapeutic agents that have an antagonistic effect (that is, reduce or inhibit) activity. Antagonists can be administered in a therapeutic or prophylactic manner. Therapeutic agents that can be used include: (1) molecules comprising inactive SHAAG peptides, or their analogs, derivatives, fragments or homologs; (2) SHAAGtide antisense nucleic acids (3) antibodies to SHAAGtide peptides or their analogues, derivatives, fragments or homologs or (4) modulators (that is, inhibitors and antagonists) that exert an antagonistic effect on the activity of the FPRL1 receptor.

Diseases and disorders characterized by decreased levels or biological activity of FPRL1 can be treated with therapeutic agents that increase (that is, are agonists) activity. Therapeutic agents that upregulate activity can be administered therapeutically or prophylactically. Therapeutic agents that can be used include peptides, or their analogs, derivatives, fragments, or homologs; or an agonist that increases bioavailability. Therapeutic agents that can be used include: (1) molecules comprising SHAAGtide peptides, or their analogues, derivatives, fragments, or homologs; (2) nucleic acids of SHAAGtides; or (3) modulators that exert an agonistic effect on FPRL1 receptor activity.

Also described herein is a method of preventing, in a subject, a disease or condition associated with aberrant FPRL1 receptor expression or activity by administering an agent that modulates FPRL1 activity. Subjects at risk of a disease caused by or to which aberrant activity of FPRL1 contributes can be identified, for example, by any combination of diagnostic tests.

or forecasts. The administration of a prophylactic agent can occur before the manifestation of the characteristic symptoms of FPRL1 aberration, so that the disease or disorder is avoided, or alternatively, its progress is delayed. Depending on the type of aberration of FPRL1, for example, an FPRL1 agonist or an FPRL1 antagonist can be used to treat the subject. The appropriate agent can be determined based on screening analysis.

Also described herein are methods of modulating FPRL1 activity for therapeutic purposes. Modulatory methods involve contacting a cell with an agent that modulates one or more of the FPRL1 activity activities associated with the cell. An agent that modulates the activity of FPRL1 can be a nucleic acid or protein, a naturally occurring cognate ligand of FPRL1, a peptide, a SHAAGtido mimetic peptidomide, or other small molecule. The agent can stimulate the activity of FPRL1. The agent can inhibit an activity of FPRL1. Modulatory methods can be performed in vitro (eg, culturing the cell with the agent) or, alternatively, in vivo (eg, administering the agent to a subject). For example, the method may involve the administration of a SHAAGtido or nucleic acid molecule as therapy to compensate for aberrant or reduced expression or activity of the FPRL1 ligand.

Stimulation of FPRL1 activity is desirable in situations where FPRL1, or the FPRL1 ligand is abnormally down-regulated and / or in which increased FPRL1, or FPRL1 ligand activity is likely to have a beneficial effect; for example, in the treatment of an infection or in a vaccination. Rather, decreased FPRL1, or FPRL1 ligand activity is desired under conditions in which FPRL1, or FPRL1 ligand activity is abnormally upregulated and / or in which decrease is likely FPRL1, or FPRL1 ligand activity has a beneficial effect; for example, in the treatment of chronic inflammation.

In vitro or in vivo analysis can be performed to determine the effect of a specific therapeutic agent and whether its administration is indicated for the treatment of affected tissue.

In different specific embodiments, in vitro analyzes can be performed with cells representative of the type or types involved in the patient's disorder, to determine whether a given therapeutic agent exerts the desired effect on the cell type or types. Modalities for use in therapy can be tested in suitable animal model systems including, but not limited to rats, mice, chickens, cows, monkeys, rabbits, dogs, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model systems known in the art can be used prior to administration to human subjects.

Diseases and conditions associated with inflammation and infection can be treated using the methods of the present invention. The disease or condition is one in which the actions of an FPRL1 ligand on an FPRL1 receptor are to be inhibited or promoted, in order to modulate the immune response.

The compositions may be administered via the oral, parenteral routes of administration (eg, intramuscular, intraperitoneal, intravenous, ICV, intracisternal, subcutaneous injection, or implant injection or infusion), by inhalation, nasal, vaginal, Rectal, sublingual, or topical and can be formulated, alone or together, in suitable dosage unit formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, adjuvants, and carriers suitable for each route of administration. In addition to treating warm-blooded animals such as mice, rats, horses, cattle, sheep, dogs, cats, monkeys, etc., the compositions of the invention are effective for use in humans.

Combination therapy to modulate the activity of FPLR1 or the FPLR1 ligand and thereby prevent and treat infectious diseases or inflammatory disorders and diseases is illustrated by means of the combination of the compounds of this invention and other compounds that are known for such utilities.

For example, in the treatment or prevention of inflammation, the present compounds can be used in conjunction with an anti-inflammatory or analgesic agent such as an opioid agonist, a lipoxygenase inhibitor, such as a 5-lipoxygenase inhibitor, a cyclooxygenase inhibitor, such as a cyclooxygenase-2 inhibitor, an interleukin inhibitor, such as TNFα, an interleukin-1 inhibitor, an NMDA antagonist, a nitric oxide inhibitor, or an inhibitor of nitric oxide synthesis, a non-steroidal anti-inflammatory agent, or a cytokine suppressing anti-inflammatory agent, for example with a compound such as acetaminophen, aspirin, codeine, fentanyl, ibuprofen, indomethacin, cetorolac, morphine, naproxen, phenacetin, piroxicam, a steroidal analgesic, sufentanil, sunlindac, tenidap, and the like. Similarly, the present compounds can be administered with a pain reliever; an enhancer such as caffeine, an H2 antagonist, simethicone, aluminum or magnesium hydroxide; a decongestant such as phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine, nafazolin, xylometazoline, propylhexedrine, or levo-deoxy-ephedrine; an antitussive such as codeine, hydrocodone, caramifene, carbetapentane, or dextramethorphan; a steroid; cyclosporine A; methotrexate; IL-10; a diuretic; and a sedating or non-sedating anti-histamine.

Pharmaceutical compositions

FPRL1 receptor agonists or antagonists can be incorporated into pharmaceutical compositions. Such compositions typically comprise agonists or antagonists and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" includes each and every solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption retardant agent, and the like, compatible with pharmaceutical administration (Gennaro (2000)). Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. Except when a conventional medium or agent is incompatible with an active compound, the use of these compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the agonist or antagonist is formulated to be compatible with its intended route of administration, including intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (i.e., topical), transmucosal, and rectal. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or alkalis, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be included in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (when soluble in water) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid in order to be administered using a syringe. Such compositions must be stable during manufacture and storage and must be preserved against contamination by microorganisms such as bacteria and fungi. The carrier may be a dispersing or solvent medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures. Proper fluidity can be maintained, for example, using a coating such as lecithin, maintaining the required particle size in the case of dispersion, and using surfactants. Different antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain contamination by microorganisms. Isotonic agents can be included in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Compositions that can delay absorption include agents such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into an appropriate solvent with one or a combination of ingredients as required, followed by sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing an alkaline dispersion medium, and the other required ingredients. In sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and lyophilization which produce a powder containing the active ingredient and any desired ingredients from sterile solutions.

Oral compositions generally include an inert diluent or an edible carrier. They can be included in gelatin capsules or formed into tablets. For oral therapeutic administration, the active compound can be incorporated into excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a liquid carrier as a mouthwash, where the compound in the liquid carrier is applied orally. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included. Tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, tragacanth gum, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or STEROTES; an anti-caking agent such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are released in the form of an aerosol spray from a nebulizer or a pressurized container containing a suitable propellant, e.g. eg, a gas such as carbon dioxide.

Generalized administration can also be transmucosal or transdermal. For transmucosal or transdermal administration, penetrating agents are selected that can cross target barriers. Transmucosal penetrating agents include, detergents, bile salts, and fusidic acid derivatives. Nasal sprays or suppositories can be used for transmucosal administration. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams.

The compounds can also be prepared in the form of suppositories (eg, with bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

Active compounds can be prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable or biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poly (glycolic acid), collagen, polyorthoesters, and poly (lactic acid). Polyethylene glycols, p. ex. PEG, they are also good carriers. Such materials can be obtained commercially from ALZA Corporation (Mountain View, CA) and NOVA Pharmaceuticals, Inc. (Lake Elsinore, CA), or prepared by one skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example in (Eppstein et al. US Patent No. 4,522,811, 1985).

Oral formulations or parenteral compositions can be created in a unit dosage form to facilitate administration and uniformity of dosage. The unit dosage form refers to physically discrete units adapted in the form of individual dosages for the subject to be treated, containing a therapeutically effective amount of active compound associated with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention are dictated by, and directly depend on, the unique characteristics of the active compound and the specific desired therapeutic effect, and the inherent limitations of the composition of the active compound.

SHAAGtido nucleic acid molecules can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be released into the subject, for example, by intravenous injection, local administration (Nabel and Nabel, US Patent No. 5,328,470, 1994), or by stereotactic injection (Chen et al. ( 1994)). The pharmaceutical preparation of a gene therapy vector may include an acceptable diluent, or it may comprise a slow release matrix into which the gene delivery vehicle is embedded. Alternatively, when the complete gene release vector can be produced intact from recombinant cells, e.g. For example, retroviral vectors, the pharmaceutical preparation may include one or more cells that produce the gene delivery system.

In one aspect, SHAAGtido is released as DNA so that polypeptides are released in situ. In one embodiment, the DNA is "naked" as described, for example, by Ulmer et al. (1993) and reviewed by Cohen, (1993).

Absorption of naked DNA can be increased by coating the DNA with a carrier, e.g. ex. biodegradable beads, which is effectively transported in cells. In such vaccines, DNA can be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems.

Vectors, used to launch genetic material from organism to organism, can be divided into two general classes: Cloning vectors are replicating plasmids or phages with regions that are not essential for propagation in an appropriate host cell and in which can insert foreign DNA; foreign DNA is replicated and propagated as if it were a component of the vector. An expression vector (such as a plasmid, a yeast, or an animal virus genome) is used to introduce foreign genetic material into a host cell or tissue in order to transcribe and translate the foreign DNA, such as a SHAAGtido . In expression vectors, the introduced DNA is operably linked to elements such as promoters that signal the host cell to transcribe the inserted DNA. Some promoters are exceptionally useful, such as inducible promoters that control gene transcription in response to specific factors. By operably connecting a SHAAGtido polynucleotide to an inducible promoter, the expression of a SHAAGtido polypeptide or its fragments can be controlled. Examples of classic inducible promoters include those that are sensitive to interferon α, heat shock, heavy metal ions, and steroids such as glucocorticoids (Kaufman, 1990. Methods Enzymol 185: 487-511) and tetracycline. Other desirable inducible promoters include those that are not endogenous in the cells into which the construct is being introduced, but are nevertheless sensitive in those cells in which the induction agent is exogenously delivered. In general, useful expression vectors are often plasmids. However, other forms of expression vectors are contemplated, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

The choice of the vector is dictated by the organism or cells that are being used and the desired destination of the vector. Vectors can replicate once in target cells, or they can be "suicide" vectors. In general, vectors comprise signal sequences, origins of replication, marker genes, enhancer elements, promoters, and transcription termination sequences.

The pharmaceutical composition may further comprise other therapeutically active compounds as indicated herein that are normally applied in the treatment of conditions related to FPRL1.

In the treatment or prevention of conditions requiring modulation of FPRL1 the appropriate dosage level of an agonist or antagonist will generally be approximately 0.01 to 500 mg per kg of patient body weight per day, which can be administered in a single dose or in multiple doses. Preferably, the dosage level will be from about 0.1 to about 250 mg / kg per day; more preferably from about 0.5 to about 100 mg / kg per day. A suitable dosage level can be from about 0.01 to 250 mg / kg per day, from about 0.05 to 100 mg / kg per day, or from about 0.1 to 50 mg / kg per day. In this range the dosage can be from 0.05 to 0.5, from 0.5 to 5 or from 5 to 50 mg / kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing from 1.0 to 1000 milligrams of the active ingredient, namely 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, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for symptomatic adjustment of dosage to the patient being treated. The compounds can be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.

However, the specific dosage level and frequency of dosing for a particular patient can be varied, and will depend on a variety of factors including the activity of the specific compound used, metabolic stability, and duration of action of that compound, age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, severity of the specific condition, and the host experiencing the therapy .

Kits

In one aspect, the invention provides kits that contain one or more of the following in a package or container:

(1) a biologically active composition of the invention or an FPRL1 antagonist; (2) a pharmaceutically acceptable adjuvant or excipient; (3) a vehicle for administration, such as a syringe; (4) instructions for administration. Embodiments in which two or more of the components (1) - (4) are found are also contemplated.

When a kit is supplied, the different components of the composition can be packaged in separate containers and mixed immediately before use. Such packaging of the components separately can allow long-term storage without loss of the functions of the active components.

The reagents included in the kits can be supplied in containers of any kind so that the life of the different components is preserved and is not adsorbed or altered by the materials of the container. For example, sealed glass ampoules may contain lyophilized SHAAG polypeptides or polynucleotides,

or buffers that have been packaged in a neutral, non-reactive gas, such as nitrogen. The ampoules can consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc .; ceramic, metal or any other material typically used to contain similar reagents. Other examples of suitable containers include simple bottles that may be made of similar substances in the form of ampoules, and envelopes, which may comprise foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. The containers may have a sterile access port, such as a bottle that has a stopper that can be pierced by a hypodermic needle for injection. Other containers may have two compartments that are separated by an easily removable membrane that after removal allows the components to mix. Removable membranes can be made of glass, plastic, rubber, etc.

Kits may also be provided with instructions. The instructions may be printed on paper or other substrate, and / or may be supplied in the form of an electronic readable medium, such as a floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, an audio tape, etc. Detailed instructions may not be physically associated with the kit; Instead, the user can be referred to a website specified by the kit manufacturer or distributor, or provided in the form of an email address.

Screening and detection methods

SHAAGtides (and nucleotides of SHAAGtides used to express SHAAGtides) can be used as reagents in methods of screening compounds that modulate FPRL1 receptor activity. Such compounds may be useful in the treatment of disorders characterized by insufficient or excessive production of FPRL1 receptor or FPRL1 receptor ligand, or production of forms of FPRL1 receptor or FPRL1 receptor ligand that may have aberrant activity in comparison with wild type molecules. In general, such compounds can be used to modulate biological functions involving the FPRL1 receptor / FPRL1 receptor ligand.

The invention provides methods (screening analysis) for identifying modalities, i.e., candidate or test compounds or agents (eg, peptides, peptidomimetics, small molecules, or other drugs), foods, combinations thereof, etc., affecting the FPRL1 receptor or the FPRL1 receptor ligand. This can be a stimulating or inhibiting effect. The invention also includes the compounds identified in such screening analyzes.

It is desirable to test compounds that increase or decrease FPRL1 receptor activity in response to, or independent of, a ligand. A compound can modulate the activity of the FPRL1 receptor by increasing or decreasing the activity of the FPRL1 receptor itself (agonists and antagonists).

Test compounds can be obtained using any of the numerous approaches to combinatorial library methods, including: biological libraries; spatially addressable parallel phase or solid phase libraries; synthetic library methods that require deconvolution; the "one account-one compound" library method; and synthetic library methods using selection by affinity chromatography. The biological library approach is limited to peptides, while the other four include libraries of peptide, non-peptide, or small molecule oligomeric compounds (Lam, 1997).

A "small molecule" refers to a composition having a molecular weight of less than about 5 kD and more preferably less than about 4 kD, and most preferably less than 0.6 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algae extracts, are known in the art and can be screened with any of the assays of the invention. Examples of methods for the synthesis of molecular libraries have been described (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 can be presented in solution (Houghten et al., 1992) or on beads (Lam et al., 1991), on chips (Fodor et al., 1993), bacteria, spores (Ladner et al., Patent No. 5,223,409, 1993), plasmids (Cull et al., 1992) or on phages (Cwirla et al., 1990; Devlin et al., 1990; Felici et al., 1991; Ladner et al. ., US Patent No. 5,223,409, 1993; Scott u Smith, 1990).

Many assays are available for screening candidate or test compounds that bind to or modulate FPRL1 receptor activity. A cell-free assay comprises, for example, contacting the FPRL1 receptor or a biologically active fragment with a SHAAGtido compound that binds to the FPRL1 receptor to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the FPRL1 receptor, wherein determining the ability of the test compound to interact with the FPRL1 receptor comprises determining the ability of the FPRL1 receptor to preferentially bind to, or modulate, activity of the test compound. Cell-based assays include, for example, calcium flux assays, binding assays, and cell migration assays discussed in the examples.

Immobilization of either a molecule containing a SHAAGtido sequence or one of its accompanying molecules (such as FPRL1) can facilitate the separation of complexed forms from non-complexed forms of one or both proteins, as well as adaptation to high performance analysis. The binding of a test compound to a SHAAGtido molecule or an FPRL1 receptor molecule, or the interaction of a SHAAGtido molecule with an FPRL1 receptor molecule in the presence and absence of a candidate compound, can be completed in any suitable container for contain the reactants, such as microtiter plates, test tubes, and microcentrifuge tubes. A fusion protein can be provided that adds a domain that allows one or both proteins to bind to the matrix. For example, GST (glutathione S-transferase) -SHAAGtido fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione-sepharose beads (SIGMA Chemical, St. Louis, MO) or glutathione derived microtiter plates. which are then combined with either the test compound or the test compound and either the non-adsorbed FPRL1 receptor or the SHAAGtido molecule, and the mixture is incubated under conditions that lead to complex formation (eg, under conditions physiological salt and pH). After incubation, the beads or wells of the microtiter plates are washed to remove any of the unbound components, the matrix is immobilized in the case of the beads, the complex is determined directly or indirectly. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of SHAAGtido determined using conventional techniques.

Other techniques for immobilizing proteins on matrices can also be used in screening analyzes. See, for example, the United States Patent Application co-pending with Serial Number 09 / 721,902. A SHAAGtido molecule or an FPRL1 receptor molecule can be immobilized using biotin-avidin or biotin-streptavidin systems. Biotinylation can be completed by using many reagents, such as biotin-NHS (N-hydroxy-succinimide; PIERCE Chemicals, Rockford, IL), and immobilizing them in the wells of 96-well plates coated with streptavidin (PIERCE Chemical). Alternatively, antibodies or antibody fragments reactive with SHAAGtido molecules or FPRL1 receptor molecules but which do not interfere with SHAAGtido binding to FPRL1 receptor molecule, can be derivatized into the wells of the plate and entrapped the receptor molecule FPRL1 or SHAAGtido in the wells by conjugation with the antibodies. Methods for detecting such complexes, in addition to those described for GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with FPRL1 receptor molecules or SHAAGtido molecules, as well as ligated enzyme analysis that detects activity enzyme associated with FPRL1 receptor molecules or SHAAGtido molecules.

To demonstrate that the compounds are FPRL1 receptor antagonists, it can be determined whether they inhibit the activity of a SHAAGtido on the receptor. Such compounds preferably have at least one of the following characteristics:

(one)

potentially inhibit the binding of a SHAAGtido or a molecule comprising a SHAAGtido sequence to the FPRL1 receptor;

(2)

significantly inhibit the Ca2 + response of a SHAAGtido or a molecule comprising a SHAAGtido that binds to FPRL1;

(3)

have a limited, non-specific Ca2 + response; or

(4)

inhibit chemotactic activity.

Conventional in vitro binding assays can be employed to demonstrate the affinity of the compounds for the FPRL1 receptor (thereby inhibiting the activity of a SHAAGtide through competitive interaction with the receptor). See the examples below. Preferably, the active compounds show an IC50 value of <10 µM, more preferably <5 µM, most preferably <1 µM.

Compounds that inhibit SHAAGtido activity affect intracellular Ca2 + concentration in SHAAGtido-stimulated cells. Binding of the ligand to the FPRL1 receptor results in G protein-induced phospholipase C activation, which leads to the conversion of phosphatidyl inositol phosphate to inositol phosphate and diacylglycerol. Inositol phosphate in turn binds to a receptor located at intracellular sites to release Ca2 + into the cytoplasm. In addition to increases in Ca2 + concentration due to release from intracellular stores, binding of inositol phosphate to its receptor leads to increased extracellular calcium flux through the membrane and into the cell. Other G protein signaling pathways may be involved.

Thus, the activation of the FPRL1 receptor by a SHAAGtido, and, subsequently, the inhibition of activation by the compounds of the invention can be determined by analyzing the increase in the levels of free intracellular Ca2 +. Typically, this can be accomplished through the use of calcium sensitive fluorescent probes such as quin-2, fura-2, and indo-1. The effect of active compounds on blocking the Ca2 + response depends on the amount of active compound and chemokine present. Generally, when 10 nM of chemokine are present, 10 µM of active compound should produce a 20 to 100% inhibition of the Ca2 + response.

To determine if the active compound produces a non-specific Ca2 + response, cells bearing multiple receptors, including the receptor targeted by the active compound, are incubated with the compound. The cells are then stimulated with a ligand for the target receptor and successively followed by ligand stimulation for the other receptors found in the sample cells. A comparable response of non-target receptors to the ligand in the presence or absence of compound indicates that the active compound is specific for the target receptor.

To determine chemotaxis, any format of cell migration analysis can be used, such as the ChemoTx® system (NeuroProbe, Rockville, MD) or any other suitable system or device (Bacon et al., 1988; Penfold et al., 1999). In summary, these analyzes of cell migration work as follows. After harvesting and preparing the cells carrying the active target chemokine receptor, the cells are mixed with the candidate antagonists. The mixture is placed in the upper chamber of the cell migration apparatus. To the lower chamber, a stimulating concentration of chemokine ligand is added. The migration analysis is then run, terminated, and cell migration is evaluated.

The authors of the invention have shown SHAAGtido activity in the FPRL1 receptor expressed in monocytes, neutrophils, Immature Dendritic Cells and Mature Dendritic Cells. Accordingly, such cells can be used in in vitro analysis methods. Populations of enriched or essentially purified cells can be used in in vitro chemotaxis assays. These cell populations can be prepared by a variety of methods known in the art depending on the specific cell type desired. Typically, essentially purified cell populations are prepared by culturing under specific conditions, for their physical characteristics such as behavior on a density gradient, by classification according to characteristic markers (eg, by fluorescence-activated cell sorting ( FACS) using antibodies (preferably monoclonal antibodies) to cell surface proteins, immunoprecipitation), or other methods.

Cells can be identified by histology (see, eg, Luna, 1968), by immunological staining and similar methods (see, eg, Harlow et al. 1998; Coligan et al., 1991). Methods for preparing essentially purified cell compositions for use in in vitro chemotaxis assays are briefly described below and in the Examples. However, the invention does not require the use of any particular purification method, provided the desired cells are obtained; many variations and alternative methods are known to those skilled in the art. Additionally, many other purification and detection methods are known in the art or can be readily developed, including methods for cells not specifically enumerated herein. In addition, cloned cell lines derived from immune system tissues can be used in the chemotaxis assays described herein, if desired. General immunological, purification, and cell culture methods are described in Coligan et al. (1991), including supplements up to 1999. Unless otherwise specified, cultured cells are incubated at 37 ° C in 5% CO2.

Suitable methods for purification of monocytes are found in Bender et al., 1996. (see also US Patent No. 5,994,126). Briefly, monocytes are isolated from PBMC by depleting T cells using antibodies immobilized against a pan T CD2 cell surface marker. Conveniently, a commercially available CD2 antibody source anchored to magnetic beads is used (Dynal; Lake Success, NY). PBMCs isolated from a buffy coat (typically 35 ml containing 400 x 106 PMBC) are resuspended by conventional Ficoll gradient centrifugation methods in MACS buffer (DPBS (HyClone; Logan, UT) with 1% BSA (Sigma) ) at 20 x 106 cells per ml. DPBS is Dulbecco's Phosphate Buffered Saline (CaCl2 (0.1g / l), KCl (0.2g / l), KH2PO4 (0.2g / l), MgCl2-6H2O (0.1g / l ), NaCl (8.0 g / l), Na2HPO4 (2.16 g / l)). An appropriate amount of immobilized CD2 + magnetic beads (typically 10 µl per 106 cells) is added to the cells. The mixture is incubated for 15 minutes at 4 ° C with gentle rotation. Magnetically labeled T cells are separated from unlabelled cells on a magnetic cell sorter (Dynal) according to the manufacturer's protocols. Unlabelled cells mainly contain monocytes and B cells.

The B cells of the previous preparation are separated taking advantage of the differential adhesion properties. In summary, PBMCs with depleted T cells are allowed to adhere to the plastic of a T-175 tissue culture flask (100 x 106 cells / flask; Costar; Acton, MA) for 3 hours at 37 ° C. Non-adherent cells (comprising mostly B cells) are aspirated. To completely remove non-adherent cells, the flasks are rinsed 3 more times with DPBS. The resulting cells are highly enriched (that is,> 90%) in monocytes.

Monocytes can also be isolated by positive selection for CD14 antigen. In summary, PBMCs isolated from peripheral blood, such as the buffy coat, by standard Ficoll gradient centrifugation methods are resuspended in MACS buffer at 1 × 10 6 cells / ml. Antibodies immobilized against CD14 surface antigen, such as CD14 + magnetic microbeads (Milteyni) (1 µl beads per 1 × 10 6 cells) are added and the mixture is incubated at 4 ° C for 15 minutes. Monocytes are separated from the other cell populations by passing the mixture through a positive selection column on a magnetic cell sorter (Miltenyi Biotech; Auburn, CA) according to the manufacturers protocol. Monocytes that are retained in the column are eluted with MACS buffer once the column is separated from the MACS apparatus. The cells are then pelleted by centrifugation and resuspended in RMPI plus 10% FCS medium at 106 cells per ml. Monocytes isolated by this method are cultured in essentially the same way as isolates by the CD2 + depletion method.

Suitable methods for purifying dendritic cells, including separating mature and immature populations, are known in the art. Essentially purified dendritic cells (including mature and immature cell subpopulations) can be prepared by selective in vitro culture conditions.

Dendritic cells are widely distributed in all tissues that have contact with potential pathogens (eg, skin, gastrointestinal and respiratory tracts, and T-cell rich areas of secondary lymphoid tissues). In the skin and upper respiratory tract they form a network of widely branched cells (called Langerhans cells in the skin). After capturing the antigen, dendritic cells from peripheral tissues such as the skin and intestine circulate it through the draining lymph vessels to the T-cell areas of the lymph nodes where they present the internalized antigen. Immature dendritic cells work by absorbing and processing antigens. During subsequent migration to the draining lymph node, DCs mature. Mature dendritic cells function as a key CPA to initiate immune responses by inducing the proliferation of pathogen and adjuvant-specific cytotoxic T cells.

Essentially pure populations of dendritic cells can be produced by in vitro culture, below). Furthermore, there are marked changes in the expression of chemokine receptors during dendritic cell maturation that can be used to identify cell phase (Campbell et al. 1998; Chan et al. 1999; Dieu et al. 1998; Kellermann et al. . 1999). For example, immature dendritic cells predominantly express CCR1, CCR5, and CXCR4. After maturation, these receptors, with the exception of CXCR4, are down-regulated.

In culture, immature forms of dendritic cells undergo maturation that is thought to be analogous to events during migration of dendritic cells from the point of contact with the antigen to secondary lymphoid tissues. Human or macaque dendritic cells of different stages of development can be generated in culture from CD14 + blood progenitors using specific cytokines. A separate lineage of dendritic cells can be differentiated from spinal cord or bone marrow CD34 + precursor cells. In one embodiment of the invention, dendritic cell subpopulations are generated for in vitro analysis for the identification of chemotactic compositions (that is, to assess the potency and selectivity of chemotaxin against defined CD subtypes). Illustrative dendritic cell subpopulations are: (1) cells derived from peripheral blood monocytes; (2) cells derived from mature peripheral blood monocytes, and (3) cells derived from CD34 + precursors. Subpopulations are isolated or produced by a variety of methods known in the art. For example, mature and immature dendritic cells of PBMC are produced according to Bender et al. supra.

In summary, PBMC T cells are depleted using antibodies immobilized against the CD2 cell surface marker (present in all T cells). Commercially available CD2 + dynabeads (Dynal) can be used according to the manufacturer's protocol. The mixture with depleted T cells is separated into adherent vs. non-adherent fractions by incubating the cells on tissue culture quality plastic for 3 hours at 37 ° C. Non-adherent cells are gently separated, and adherent cells (generally CD14 + monocytes) are placed in culture medium (eg RMPI + 10% FCS) with a supplement of 1000 U / mL each of GM-CSF and IL-4 (R&D Systems, Minneapolis, MN) ("Day 1"). Between days 3-7 the cells begin to show a veiled morphology, and are replenished with cytokines on days 2, 4, and 6, at which time the cells can be harvested as immature dendritic cells. In one embodiment, cells from this in vitro phase are isolated and used in the analysis. Typically approximately 10 × 10 6 dendritic cells are obtained from 400 x 10 6 PBMC.

On day 7 the immature dendritic cells show the typical morphology of dendritic cells, with an elongated cell body and many processes. Cell size increases significantly compared to precursor monocytes. Immature dendritic cells can be phenotypically characterized by controlling their expression of cell surface markers.

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

In the MCM method, 6-day immature dendritic cells are harvested by centrifugation and resuspended at 106 cells / ml in maturing medium (eg, diluted MCM (up to 1: 1 with RPMI containing 10% FCS) GM-CSF (1000 U / ml) and IL-4 (1000 U / ml) are added The cells are cultured for three more days, with a new 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, immature dendritic cells are harvested from Day 6 and resuspended in a conventional culture medium (RPMI plus 10% FCS) with a supplement of 20 µg / ml poly (I: C ) (Sigma), 1000 U / ml GM-CSF and IL-4. Cells are cultured another three days without additional cytokines. Day 9 cells are used as mature dendritic cells.

The mature dendritic cells generated by these two different methods show phenotypic and functional properties different from those of immature dendritic cells or precursor monocytes. The mature dendritic cells of each preparation are perfectly characterized by FACS to ensure that the desirable cell types have been obtained.

Remarkably, the generated mature dendritic cells express a significantly higher level of MHC class II on the cell surface than immature cells. The expression of CD80, CD83 and CD86 is also upregulated. Chemokine receptor expression also changes dramatically during the maturation process. For example, CCR1, CCR5 are down-regulated in mature cells, while CCR7 is up-regulated and appears on the cell surface within hours of MCM addition. Functionally, mature dendritic cells are no longer capable of efficiently absorbing the antigen, but they acquire the ability to stimulate proliferation of T cells and B cells not pretreated. Mature dendritic cells also change their migratory behaviors; they no longer respond to ligands for CCR1, CCR2, and CCR5, such as MIP-1α, RANTES, and MIP-1β. Instead, they respond to ligands for CCR7 SLC and ELC.

The MCM is prepared as described by Romani et al. 1996, with minor modifications. Briefly, petri dishes (100mm, Falcon) are coated with 5ml of human Ig (10mg / mL) for 30 min at 37 ° C and washed with PBS 2-3 times immediately prior to use. 50 × 106 PBMC in 8 ml are layered on the Ig coated plates for 1-2 hours. Non-adherent cells are washed and discarded. Adherent cells are incubated in fresh fresh medium (RPMI + 10% normal human serum) at 37 ° C, and the resulting medium (MCM) is collected after 24 hours. TNF-α concentration in the MCM is determined by the standard ELISA method (eg, using an ELISA kit for TNF-α (R&D Systems, Minneapolis, MN)). The final TNF-α level in the MCM is adjusted to 50 U / ml by mixing an appropriate amount of MCM with RPMI / 10% fetal calf serum.

Suitable methods for purification of neutrophils are known in the art. According to a 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. Twenty-five ml of WB plus 25 ml of dextran results in approximately 35 ml of supernatant after 30 minutes of sedimentation. The supernatant is layered over 12-15 ml Ficoll and centrifuged at 400 x g for 30-40 minutes at 1820 ° C. The plasma / platelet layer containing mononuclear cells and Ficoll-Paque is separated by aspiration. Neutrophils are in the white layer above the red blood cell layer (RBC). (In some preparations, the neutrophil and erythrocyte layers are mixed. In these cases, the RBCs are separated by hypotonic lysis: 12.5 ml of cold 0.2% NaCl is added to the neutrophil / RBC pellet while undergoing vortex. 12.5 ml of cold 1.6% NaCl are immediately added while vortexing. Cells are centrifuged at 60-100 xg for 10 m and recovered. If necessary, the lysis step is repeated). The resulting neutrophils are> 95% pure (with eosinophils as the remaining primary cells).

Prognostic Analysis

The diagnostic methods described herein can further be used to identify subjects who have or are at risk of developing a disease or disorder associated with the aberrant expression or activity of the FPRL1 receptor or FPRL1 ligand. For example, the assays described can be used to identify a subject who has or is at risk of developing a disorder such as a neurodegenerative disorder. Typically, a test sample is obtained from a subject and the FPRL1 receptor or FPRL-1 ligand is detected or its activity is analyzed. For example, a test sample can be a biological fluid (eg, serum), a sample of cells, or a tissue.

Prognostic analyzes can be used to determine whether a modality (eg, an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, food, etc.) can be administered to a subject to treat a disease or disorder. associated with aberrant expression or activity of the FPRL1 receptor or FPRL1 ligand. Such methods can be used to determine if a subject can be effectively treated with an agent for a disorder. Also described herein are methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of the FPRL1 receptor or FPRL1 ligand. In such an analysis, a test sample is obtained and an SHAAGtide or nucleic acid is detected (eg, where the presence of SHAAGtide or nucleic acid is diagnostic for a subject to whom the agent can be administered to treat an associated disorder with aberrant expression or activity of the FPRL1 receptor or FPRL1 ligand).

The following examples are provided to illustrate the invention and are not intended to be limiting.

EXAMPLES

Example 1: CKβ8-1 (25-116), like other variants of CKβ8, stimulates intracellular calcium flux in cells expressing CCR1.

Recombinant human chemokines, leukotactin, three known CKβ8 variants CKβ8 (1-99), CKβ8 (2599), CKβ8-1 (1-116), and a novel NH2-terminal truncated form of CKβ8-1, CKβ8-1 ( 25-116) were obtained from R&D Systems (Minneapolis, MN). CKβ8-1 (25-116) was compared with the other three variants to determine the ability to achieve intracellular calcium mobilization in HEK239 cells transfected with stable human CCR1. Human HEK293-CCR1 cells were prepared using Fugena 6 (Roche, IN) following the manufacturer's protocol. HEK-293 cell lines were maintained in DMEM with 10% FBS with a supplement of 800 µg / ml G-418.

Stable expression of the human chemokine CCR1 receptor in HEK293 cells was obtained as follows: Whole cDNA encoding CCR1 was cloned by polymerase chain reaction (PCR) from genomic DNA isolated from human peripheral blood cells. The PCR product was cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) using standard molecular cloning procedures and completely sequenced to confirm its identity.

Two micrograms of the CCR1 / pcDNA3.1 construct were used to transfect HEK293 cells as follows. FuGENE: DNA complex was prepared by mixing 6.0 µl of FuGENE 6 reagent (Roche Molecular Biochemicals, CA) and 2 µg of CCR1 / pcDNA3.1 in 100 µl of serum free medium (Hyclone, CO). After incubating for 30 minutes at room temperature, the complex was added to a 60 mm culture plate containing 0.5 -1 X 106 cells in 10 ml of DMEM medium with a supplement of 10% FBS (Hyclone, CO). After mixing, the cells were returned to the incubator to cultivate at 37 ° C for two days. Within 48 hours of transfection, Genetinin (G418) (Mediatech, Hemdon, VA) was added to a final concentration of 800 µg / ml. Cells were then plated into 96-well plates at a concentration of 20,000 cells / well. After 2-3 weeks under selection with G418, stable geneticin resistant CCR1 expressing cells were evaluated for their ability to mobilize calcium in response to MIP-1α at a concentration of 1-500 nM.

Ca2 + mobilization responses were performed using the intracellular radiometric fluorescent dye, Indo-1. Cells were loaded with Indo-1 / AM (3 µM; Molecular Probes, Eugene, OR) in culture medium (45 min, 20 ° C, 107 cells / ml). After loading the dye, the cells were washed once with 10 ml of PBS) and resuspended at 106 cells / ml in HBSS containing 1% FBS. Cytosolic [Ca2 +] release was determined using excitation at 350 nm using a Photon Technology International fluorometer (excitation at 350 nm, dual emission adjusted at 400 and 490 nm).

With HEKCCR1-293 transfectants, CKβB8-1 (25-116) and the other CKβ8 variants induced rapid calcium flux at 100 nM. The two truncated variants CKβ8 (25-99) and CKβ8-1 (25-116) induced a high calcium response, while the signals generated by variants CKβ8 (25-99) and CKβ8-1 (1-116) were lower. . None of these chemokines induced a signal with untransfected parental HEK293 cells, demonstrating that the activity is due to CCR1 and not to an endogenous receptor. The maximal receptor stimulations obtained with 100nM CKβ8 (25-99) and CKβ8-1 (25-116) were equivalent to those obtained with the same concentration of the CCR1 agonist, leukotactin.

Example 2: CCL23 variant CKβ8-1 (25-116) exhibits a unique activity profile in human monocytes and neutrophils that is not a CCR1-linked event.

Recombinant human chemokines, leukotactin, MIP-1α, three known CKβ8 variants CKβ38 (1-99), CKβ8 (25-99), CKβ8-1 (1-116) and a novel NH2-terminal truncated form of CKβ8- 1, CKβ8-1 (25-116) were obtained from R&D Systems (Minneapolis, MN). Human monocytes were generated from buffy coats (Stanford Blood Center, Palo Alto, CA) following a standard protocol. Briefly, PBMC were isolated by standard density gradient centrifugation (Ficoll-Paque-Plus, Pharmacia). Monocytes were purified using magnetic positive selection with CD14 Microbeads (Miltenyi, Auburn, CA). Human neutrophils were isolated from fresh peripheral blood from healthy individuals by gradient centrifugation on Ficoll-Hypaque (Hyclone, CA).

The activity of CCL23 variants was tested on freshly prepared human monocytes and neutrophils using the calcium flux assay described in Example 1. Although all chemokines somewhat stimulated calcium release in monocytes, CKβ8 (1-99) and CKβ8-1 (1-116) showed little activity, even at 250nM. CKβ8 (25-99) showed slightly higher calcium stimulation. However, CKβ8-1 (25116) showed a unique calcium flux with an extended calcium release. The maximum receptor stimulation obtained with 100 nM CKβ8-1 (25-116) was at least twice as high as that obtained with the same leukotactin concentration.

In neutrophils, 100 nM leukotactin induced calcium flux but neither MIP-1α nor CKβ8 (1-99), CKβ8 (25-99) nor CKβ8 (1116) induced calcium flux. However, CKβ8-1 (25-116) induced a unique calcium release. The magnitude was much greater than that observed for the same amount of leukotactin stimulation.

Example 3: Cross desensitization test carried out on HEK293-CCR1 transfectants, monocytes and neutrophils.

In cross-desensitization assays, cells were successively stimulated with leukotactin and then chemokines CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116), and CKβ8-1 (25-116) . In HEK293-CCR1 transfectants (prepared in Example 1), leukotactin induced similar patterns of receptor desensitization for all variants. When the cells were pretreated with 100 nM leukotactin, the calcium flow response to all ligands was completely inhibited.

Similar receptor cross-desensitization assays were performed using both monocytes and neutrophils (prepared as in Example 2). In monocytes, leukotactin completely desensitized the variants of CCL23, CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116). In contrast, pre-stimulation with leukotactin did not sensitize CKβ8-1 (25-116) activity on monocytes. In neutrophils, CKβ8 (1-99), CKβ8 (25-99), and CKβ81 (1-116) were inactive and pre-stimulation with leukotactin had no effect. However, pre-stimulation with leukotactin did not desensitize stimulation with CKβ8-1 (25-116).

Example 4: CCL23 variants compete with I125-MIP-1α for binding to cells expressing CCR1.

The binding characteristics of the CCL23 variants were compared in cells expressing human CCR1. The ability of MIP-1α and CCL23 variants CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116), and CKβ8-1 (25-116) to compete with the I125-binding. MIP-1α was investigated in HEK293: CCR1 cells (prepared as described in Example 1). Cells were incubated with I125-labeled MIP-1α (final concentration ∼ 0.05 nM) in the presence of unlabeled chemokine (3 hrs at 4 ° C: 25mM HEPES, 140mM NaCl, 1mM CaCl2, 5mM MgCl2 and 0.2% BSA, adjusted to pH 7.1). Reaction mixtures were aspirated onto PEI-treated GF / B fiberglass filters using a cell harvester (Packard). Filters were washed twice (25mM HEPES, 500mM NaCl, 1mM CaCl2, 5mM MgCl2, adjusted to pH 7.1). Scintillation agent (MicroScint-10; 35 µl) was added to dry filters and the filters were counted in a Packard Topcount scintillation counter. Data was analyzed and plotted using the Prism program (GraphPad Software, San Diego, CA).

Competition curves were observed with increasing concentrations of MIP-1α or CCL23 variants. MIP-1α produced an IC50 of 0.54 nM. The CCL23 variants produced IC50 values of 64 nM, 1.34 nM, 206 nM, and 112 nM, respectively. CKβ8-1 (1-116) showed 3-4 times less potency than its transfectant CKβ8 (1-99) for the displacement of bound I125-MIP-1α from CCR1, coinciding with its relatively weak affinity for CCR1. Also, as expected, the truncation of CKβ8 (1-99), CKβ8 (25-99), showed a 40-fold increase in IC50. However, the IC50 for CKβ8-1 (25-116), the truncated variant on the same amino acid as CKβ8-1 (1-116), only increases once.

Simian binding competition assays were carried out on monocytes and neutrophils. These cells were prepared as in Example 2. Competition for binding between MIP-1α in neutrophils could not be studied, since MIP-1α does not bind neutrophils. In monocytes, MIP-1α has an IC50 of 0.27 nM, and CCL23 IC50 variants of 10 nM, 0.25 nM, 55 nM, and 5 nM, respectively. In general, CKβ8 (1-99) and CKβ8 (2599) showed an IC50 similar to that observed in HEK293-CCR1 cells. However, CKβ8-1 (1-116) and CKβ81 (25-116) showed superior MIP-1α displacement activities in monocytes, especially CKβ8-1 (25116) which was 10-fold higher. Competition data for binding of I125-MIP-1α (IC50) is shown in Table

6. The IC50 for each interaction was obtained from nonlinear least squares curve fitting.

Table 6. I125I-MIP-1α Union Competition Data for HEK293-CCR1 and Monocyte Transfectants

HEK293-CCR1

Monocytes

MIP1α

0.54 nM 0.27 nM

CKβ8 (1-99)

64 nM 10.3 nM

CKβ8 (25-99)

1.34 nM 0.25 nM

CKp8-1 (1-116)

206 nM 55 nM

CKβ8-1 (25-116)

112 nM 5.1 nM

Example 5 Variant CKβ8-1 (25-116) induces migration of human monocytes and neutrophils with a

novel immigration property.

5 Migration analyzes were carried out on monocytes and neutrophils. Human monocytes and neutrophils (prepared as in Example 2) were harvested and resuspended in chemotaxis medium (CM). The CM consisted of Hank's buffered saline (Gibco, MA) containing CaCl2 (1mM) and MgSO4 (1mM) with 0.1% BSA added (Sigma, St. Louis, MO). Analyzes were performed on ChemoTx® 96-well microplates (Neuroprobe,

10 MA). Leukotactin, MIP-1α and chemokines (CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116) or CKβ8-1 (25116), prepared as in Example 1) were added to the wells lower (final volume 29 µL), and 20 µL of cell suspension (5 × 10 6 cells / mL for monocytes; 2.5 X 10 6 cells / mL for neutrophils) were added to the polycarbonate filters (pore size 5 µm for monocytes; 3 µm pore size for neutrophils). After incubating for 90 min (37 ° C, 100% humidity, 5% CO2), the cells separated from the upper surface of the

15 filter scraping. Cells migrating to the lower chamber were quantified using the Quant Cell Proliferation Analysis Kit (Molecular Probes, OR).

In monocytes, CKβ8-1 (25-99), CKβ8-1 (1-99), and CKβ8-1 (1-116) showed moderate activity at all concentrations up to 100 nM. CKβ8-1 (25-116) showed a spectacularly higher activity than the others

Three variants at 100 nM, although their activity at 1 and 10 nM was very similar to that of the other variants.

The same test was performed on human neutrophils. Human neutrophils generally lacked response to CCR1 ligands. Coinciding with the calcium flux results, none of the known CCR1 ligands including leukotactin and M1P-1α were active. However, CKβ8-1 (25-116) induced a robust response.

25 to 100 nM. This magnitude is comparable to many other potent CXCR1 and CXCR1 ligands including IL-8 and GRO-α.

Example 6 CKβ8-1 (25-116) is capable of inducing intracellular calcium flux and chemotaxis in cells expressing formylated peptide receptor type 1 (FPRL1).

30 The functional activities of CKβ8-1 (25-116) were investigated in human FPRL1-L1.2 transfectants and in native L1.2 cells. Stable expression of the formylated peptide type 1 receptor (FPRL1) in L1.2 cells was obtained as follows. Complete cDNA encoding FPRL1 was cloned, using polymerase chain reaction (PCR), from genomic DNA isolated from undifferentiated HL-60 cells. The product of the reaction in

The polymerase chain was cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) using standard molecular cloning procedures and completely sequenced to confirm its identity. 20 µg of the FPRL1 / pcDNA3.1 construct was linearized by digestion with Bsm 1 (New England Biolabs, Beverly, MA) and used to transfect the L1.2 murine B cell line as follows. 25 million cells were washed twice and resuspended in 0.8 ml PBS. Cells were incubated for 10 min at room temperature with DNA

40 of the linearized FPRL / pcDNA3.1 construct and transferred to a 0.4 cm cuvette, and a single pulse of electroporation was applied at 250 V, 960 µF. Electroporated cells were incubated for 10 min at room temperature and transferred to a 37 ° C culture in RPMI with a 10% FCS supplement. Geneticin (G418) was added to a final concentration of 800 µg / ml 48 h after transfection and the cells were plated in 96-well plates at 25,000 cells / well. After 2-3 weeks under drug selection,

45 evaluated stable geneticin resistant FPLR1 expressing cells to determine their ability to mobilize Ca ++ in response to SHAAGtido or CKbeta 8-1 at concentrations of 1 to 1000 nM.

A calcium flux assay was performed with these transfectants using the method described in Example 1. Of the CCL23 variants, only CKβ8-1 (25-116) stimulated calcium release in cells expressing FPRL1.

Synthetic peptides Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm) and Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) ("W 1 and 2 peptides") ( obtained from Phoenix Pharmaceuticals (Belmont, CA)), known unnatural ligands for FPRL1, produced a robust calcium flux. No CKβ8-1 (25-116) induced calcium release was observed in parental cells or in cells transfected with other chemokine receptors. When a dose response analysis of CKβ8-1-induced calcium flux (25-116) was performed, an EC50 of 10-20 nM was observed in these cells. CKβ8-1 (1-116) showed no activity on cells expressing FPRL1, even at 200 nM.

The ability of CKβ8-1 (25-116) to achieve migration of cells expressing FPRL1 was examined. The test conditions were as in Example 5. Although the parental L1.2 cells did not migrate in the analysis,

5 cells expressing FPRL1 migrated in a bell-shaped dose-dependent manner in response to concentrations of CKβ8-1 (25-116) ranging from 1 nM to 1 µM. Semi-maximum cell migration was observed at 30 nM. The magnitude of the maximum response was higher than that observed with the synthetic peptides WKYMVm and WKYMVM. Overall, compared to other chemokines, CKβ8-1 (25-116) showed a wider bell-shaped curve in FPRL1-mediated migration. Therefore, in addition to its activity on CCR1, CKβ8-1 (25-116) also functions through the FPRL1 receptor expressed in monocytes and neutrophils.

Example 7 CKβ8-1 (25-116) is able to displace binding of I125-labeled WKYMVm in monocytes

human and cells expressing FPRL1

Binding of CKβ8-1 (25-116) to FPRL1 was determined by measuring the ability of CKβ8-1 (25-116) to displace I125-labeled WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH2 I125-labeled, Perkin Elmer Life Science (Boston, MA)) of human FPRL1-L1.2 transfectants and human monocytes. Cells were incubated with 0.01 nM WKYMVm-I125 in the presence of increasing concentrations of unlabelled WKYMVm or CKβ8-1 (25-116) for three hours at 4 ° C. The IC50 for each interaction was obtained from the fit of the nonlinear least squares curve of the data using the Prism program (GraphPad Software).

For FPRL1-L1.2 transfectants and monocytes, competition curves were observed with increasing concentrations of WKYMVm or CKβ8-1 (25-116) (Table 7). Such curves were not observed for other variants of CCL23.

Table 7 Competition curves for I125-labeled WKYMVm observed with WKYMVm and CKβ8-1 (25-116).

IC50

Human monocytes

L1.2 EPRL1 cells

WKYMVM

1.5 nM 80 nM

CKβ8-1 (25-116)

31 nM 196 nM

Example 8 Chemokine or SHAAGtido-Induced Calcium Mobilization by Immature Dendritic Cells, Mature Dendritic Cells, or Neutrophils.

Recombinant human CKβ8-1 (25-116) chemokine was obtained from R&D Systems (Minneapolis, MN). The SHAAGtid peptides SEQ ID NO: 1 and SEQ ID NO: 6 and a control protein (the reverse sequence of SEQ ID NO: 1) were synthesized and purified by HPLC using routine techniques such as those described by Sambrook et al., 1989, and Ausubel et al., 1999.

35 Human monocytes were generated either from buffy coats (Stanford Blood Center, Palo Alto, CA) or from fresh blood from healthy individuals following a conventional protocol. Briefly, PBMC were isolated by Ficoll-Paque gradient centrifugation (Ficoll-Paque-Plus, Pharmacia). Monocytes were purified by positive magnetic selection with CD14 Microbeads (Miltenyi). Human neutrophils were isolated from fresh blood by dextran sedimentation and Ficoll-Paque gradient centrifugation. All cells were washed and resuspended (1 × 107 / ml) in RPMI medium with 10% FBS.

Immature CDs were obtained by culturing CD14 + monocytes in the presence of GM-CSF and IL4. Briefly, 106 cells / ml were grown in a T-175 flask in RPMI / 10% FCS. Human GM-CSF and IL4 were added on day 0, 2, 4 and 6 at a final concentration of 1000 u / ml and 500 u / ml, respectively. Cells were harvested on day 7 in

45 forms of immature DC and were characterized to determine the expression of the surface protein by FACS analysis. CD maturation was carried out by culturing 6 days immature CDs in macrophage conditioned medium (MCM). Briefly, CDs were harvested by centrifugation and resuspended in MCM at 10 6 cells / ml. To the medium was added a supplement of 1000 u / ml of GM-CSF and 500 u / ml of IL4. After an additional three days of culture, the cells were harvested as mature CDs and characterized by flow cytometry to determine the expression of the surface protein.

MCM was prepared as follows: PBMCs isolated from buffy coat were incubated at 37 ° C in a plastic flask previously coated with 10 mg / ml human IgG (Sigma, St Louis, MO) for 30 minutes. After 30 minutes, the non-adherent cells were separated and the adherent cells were washed three times with DPBS,

Then they were grown in RPMI / 10% human serum. The conditioned medium was collected after 24 hours. TNF-α concentration, which is critical for CD maturation, was determined using a TNF-α ELISA kit (R&D Systems, MN). The final TNF-α level was adjusted to 50 u / ml by mixing with RPMI / 10% human serum, and stored at 80 until use. 2. 3

Ca2 + mobilization responses were performed using an intracellular radiometric fluorescent dye, Indo-1. Cells were loaded with lndo-1 / AM (3 µM; Molecular Probes (Eugene, OR)) in culture medium (45 min, 20 ° C, 107 cells / ml). After loading the dye, the cells were washed once (10 ml PBS) and

5 resuspended at 106 cells / ml in HBSS containing 1% FBS. Cytosolic [Ca2 +] release was determined using excitation at 350 nm using a Photon Technology International fluorometer (excitation at 350 nm, dual emission adjusted at 400 and 490 nm).

The SHAAGtides of SEQ ID NO: 1 and SEQ ID NO: 6, as well as CKβ8-1 (25-116), produced robust calcium flux

10 in human monocytes and neutrophils and were partially active on immature Dendritic Cells and mature Dendritic Cells. No significant calcium flux was observed with the control peptide. Since immature Dendritic Cells express high levels of CCR1, CKβ8-1 (25-116) induces Ca2 + release in these cells.

Example 9 Chemokine-induced calcium mobilization, SHAAGtido, and truncated variants of SHAAGtido by stable expressed FPRL1 cells.

Stable expression of human FPRL1 in L1.2 cells was obtained as in Example 6. Calcium flux assays were carried out on the transfectants using the method described in Example 1. Chemokines

20 (CKβ8 (1-99), CKβ8 (25-99), CKβ8-1 (1-116) and CKβ8-1 (25-116), were prepared as in Example 1. Peptides W 1 and 2 were obtained as in Example 6. In addition, the following SHAAGtido sequences and truncated variants (prepared as in Example 8) were tested:

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 peak calcium flux response was determined. Table 8 demonstrates that CKβ8 (25-116) (SEQ ID NO: 16) and certain SHAAGtides induced calcium mobilization in FPRL1 transfectants. However, CKβ8 (1-116), which does not contain a free SHAAGtido N-terminus, did not cause significant mobilization. The data also indicates that the N-terminus of SHAAGtido is important for its activity in FPRL1 transfectants. Those SHAAGtids that have a truncated N-end have a

Greatly reduced calcium mobilization. SHAAGtides that had a truncated or substituted C-terminus did not show the same loss of activity that was observed after truncation of the N-terminus.

Table 8 - Induction of Calcium Flow in FPRL1-L1.2 cells. (IC50 - where IC50 value is not listed, the sequence showed low activity or showed no significant activity.)

CI 50

SEQ ID NO: 1

150 nM

SEQ ID NO: 2

> 50 µM

SEQ ID NO: 3

68 nM

SEQ ID NO: 4

-

SEQ ID NO: 5

7.4 nM

SEQ ID NO: 6

38 nM

SEQ ID NO: 7

-

SEQ ID NO: 8

45 nM

CI 50

SEQ ID NO: 9

-

SEQ ID NO: 10

-

SEQ ID NO: 13 -CKβ8 (1-99)

-

SEQ ID NO: 14 -CKβ8 (25-99)

-

SEQ ID NO: 15 CKβ8 (1-116)

-

SEQ ID NO: 16 CKβ8 (25-116)

11 nM

peptide W 1

0.7 nM

peptide W 2

<0.1 µM

Example 10 Chemotactic activity of chemokines, SHAAGtido and truncated variants of SHAAGtido on FPRL1-L1.2 cells.

The chemotactic activity of the chemokines (CKβ8 (1-99) and CKβ8 (25-99), the SHAAGtides (SEQ ID NO: 1 and SEQ ID NO: 2) and the W 1 and 2 peptides (obtained as in the Example 6) in FPRL1-L1.2 cells was determined in migration analysis Chemokines were prepared as in Example 1. The SHAAGtido sequences SEQ ID NO: 1 and SEQ ID NO: 2 were prepared as in Example 8. FPRL1-L1.2 cells were prepared as in Example 6.

Migration analyzes were carried out on ChemoTx® 96-well microplates (Neuroprobe) using the protocol described in Example 5. Both SEQ ID NO: 1 and CKβ8-1 (25-116) migrated the transfectants, indicating that they are functional for this receptor.

Example 11 Chemotactic activity of chemokines, SHAAGtido and truncated variants of SHAAGtido in 15 monocytes and human neutrophils.

The chemotactic activity on monocytes and human neutrophils of chemokines: CKβ8 (1-99), CKβ8 (25-99), CKβ8 (1-116) and CKβ8 (25-116); W 1 and 2 peptides and SHAAGtid sequences SEQ ID NO: 1 and SEQ ID NO: 2 were determined in migration analyzes. The above peptides were prepared as in the previous examples.

Migration analyzes were performed on CHEMOTX® 96-well microplates (Neuroprobe) using the protocol described in Example 5. Both SHAAGtido SEQ ID NO: 1 and CKb8-1 (25-116) caused migration of both neutrophils like monocytes.

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Claims (19)

one.

An isolated protein or polypeptide that modulates FPRL1 receptor activity that comprises a sequence at its N-terminus, the sequence having an identity of at least 90% with SEQ ID No. 1 throughout its length, where the protein or the polypeptide is not one of SEQ ID Nos. 15 and 16.

2.

The isolated protein or polypeptide of claim 1, wherein said N-terminal sequence has an identity of at least 95% with SEQ ID No. 1.

3.

The isolated protein or polypeptide of claim 1, wherein the N-terminal sequence has 100% sequence identity with SEQ ID No. 1.

Four.

The isolated protein or polypeptide of claim 1, wherein said N-terminal sequence is SEQ ID No. 1 or SEQ ID No. 6.

5.

An isolated protein or polypeptide comprising a sequence corresponding to SEQ ID NO. 3 at the N-terminus, where the polypeptide is not one of SEQ ID No. 15 and 16.

6.

A kit comprising a pharmaceutical composition comprising the protein or polypeptide of any one of claims 1 to 5, a pharmaceutically acceptable carrier, and a syringe.

7.

An isolated nucleic acid encoding the protein or polypeptide of claim 1.

8.

The isolated nucleic acid of claim 7 comprising a nucleic acid sequence having an identity of at least 90% with SEQ ID No. 20.

9.

The isolated nucleic acid of claim 7, comprising a nucleic acid sequence having at least 95% identity with SEQ ID No. 20.

10.

The isolated nucleic acid of claim 7, comprising a nucleic acid sequence having at least 99% identity with SEQ ID No. 20.

eleven.

An isolated nucleic acid having a sequence complementary to the nucleic acid of claim 10.

12.

The isolated nucleic acid of claim 10, comprising SEQ ID No. 20 or SEQ ID No. 25.

13.

A fusion protein comprising the protein or polypeptide of claim 1 fused to a protein

or polypeptide other than the protein or polypeptide of SEQ ID No. 1.

14.

A method of identifying a modulator of the binding of a protein or polypeptide comprising an N-terminal sequence, where the N-terminal sequence has at least 90% identity with SEQ ID No. 1, to an FPRL1 receptor, which includes:

contacting a cell expressing an FPRL1 receptor with a candidate compound in the presence of a labeled protein or polypeptide comprising an N-terminal sequence, where the Nterminal sequence is at least 90% identical with SEQ ID No. 1; and comparing the level of binding of said protein or polypeptide to the FPRL1 receptor in the presence of the candidate compound with the level of binding of said protein or polypeptide to the FPRL1 receptor in the absence of the candidate compound, where decreased binding indicates that the compound candidate is a binding inhibitor, and an increase in binding indicates that the candidate compound is a binding enhancer.

fifteen.

The method of claim 14, wherein the candidate compound is an antibody, a peptide, a nucleic acid.

or a small molecule. 16. The method of claim 14, wherein the cell is a neutrophil, monocyte, T lymphocyte, or dendritic cell. Figure 1 FPRL1 Referred Ligands

Ligands

Calcium Flow (CE50) Migration (CE50) Union (CE50)

Endogenous Ligands: Lipoxin A4 Serum Amyloid A-Amyloid (1-42) Prion Peptide PrP (106-126) LL-371 D2D3 (88-274) 2 Encoded by Viruses and Bacteria: T21 / DP107 (gp41 HIV) gp120 ( 414-434) HIV H. pylori peptide, Hp (2-20) 4 fMLP Non-Natural Ligands: WKYMVm WKYMVM MMK-1

? > 0.2 μM> 2 μM> 2 μM> 5 μM? > 0.5 μM 5-10 μM 10 μM> 5 μM 0.1-1 nM 1-10 nM little nM ? > 0.2 μM 10 μM 25-50 μM 5-10 μM 0.1 nM (over 293-FPRL1) 3> 0.5 μM 5-10 μM 0.1-1 nM 1-10 nM little nM 2-10 nM for H3-LPX 250 nM for I125-SSA 83 nM for I125-D2D3 (88-274) 10 nM for I125-Peptide W> 10 nM for I125-Peptide W

Notes: 1. Bacterial peptide derived from human cathelicidin, LL-37. 2. Urokinase type plasminogen activator (uPA), D2D3 (88-274). 3. In neutrophils, neither uPA nor D2D3 (88-274) induce calcium flow. 4. Helicobacter pylori peptide, Hp (2-20).