WO2006010138A1 - Heteromultimeres de chimiokine bioactifs - Google Patents

Heteromultimeres de chimiokine bioactifs Download PDF

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WO2006010138A1
WO2006010138A1 PCT/US2005/024707 US2005024707W WO2006010138A1 WO 2006010138 A1 WO2006010138 A1 WO 2006010138A1 US 2005024707 W US2005024707 W US 2005024707W WO 2006010138 A1 WO2006010138 A1 WO 2006010138A1
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chemokine
monomer
heteromultimer
monomers
cxc
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WO2006010138A9 (fr
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Kevin H. Mayo
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Regents Of The University Of Minnesota
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5421—IL-8
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • Chemokines are a family of related proteins that regulate leukocyte migration. Chemokines are single polypeptides 70-100 amino acids in length sharing varying (20-95%) similarity. They are 8-12 kilodalton (kD), basic, heparin binding proteins that contain conserved cysteines forming essential disulfide bonds, and are divided into two major subfamilies, CC and CXC, based on the positions of the invariant cysteines near the amino terminus of the protein. If the first two cysteines are adjacent to each other they are classified in the CC family (beta). The CXC family (also referred to as alpha) has a single amino acid between the initial two cysteines.
  • CXC members are located in a gene cluster at human chromosome 4ql2-21, whereas most CC members are in a cluster at human chromosome 17ql 1-32.
  • the CXC chemokines predominantly target endothelial cells, neutrophils and subsets of T cells, whereas the CC chemokines target a variety of cell types, including T cells, macrophages, eosinophils, and basophils (Mackay, C.R., Nat. Immunol. 2, 95-99 (2001)).
  • C and CX 3 C families have also been described.
  • PF4 and IL8 are members of the CXC- chemokine family of small (8-10 kDa) proteins, a sub-family of chemokines within the cytokine super-family (Miller et al., Critical Reviews in Immunology 12, 17-46 (1992)).
  • CXC-chemokines are generally chemotactic for migratory immune cells, and thus are involved in the regulation of inflammatory processes and wound healing. In addition, they demonstrate biological activities in hematopoiesis, cell proliferation, angiogenesis and glycosaminoglycan binding.
  • CXC-chemokines which are known to self-associate as dimers and tetramers, exhibit high sequence and three-dimensional structural homology at both the tertiary (monomer) and quaternary (dimer and tetramer) levels.
  • Each folded CXC-chemokine monomer has an aperidoic, 2-disulfide bond-stabilized N-terminal segment, followed by a three-stranded antiparallel ⁇ -sheet domain, and a C-terminal ⁇ -helix that is folded onto the generally amphipathic ⁇ -sheet (Clore et ah, FASEB J. 9, 57-62 (1995)).
  • Dimers are formed by intermolecularly extending the monomer ⁇ -sheet into a six-stranded antiparallel ⁇ -sheet, originally termed AB-type dimer. Tetramers are formed by ⁇ -sheet sandwiching of two AB-type dimmers (St Charles et ah, J. Biol. Chem. 264, 2092-9 (1989)). Native PF4 dimers associate asymmetrically into tetramers, whereas in an N- terminal PF4 chimera (PF4M2), dimers associate symmetrically into tetramers with little change in biological activity (Mayo et ah, Biochemistry 34(36), 11399-409 (1995)). IL8 forms AB-type dimers and is not known to tetramerize (Clore, et ah, J. MoI. Biol. 217, 611-20 (1991)).
  • PF4 and IL8 mediate different biological activities. Although first recognized to bind heparin and act as an anticoagulant, PF4 is also known as an anti-angiogenic agent. On the other hand, IL8 binds heparin more weakly and can promote angiogenesis. The angiogenic functional difference may be due, at least in part, to N-terminal sequence differences. PF4 lacks the IL8 N-terminal tripeptide Glu-Leu-Arg (ELR) motif, which is known to mediate IL8 receptor binding and subsequent signal transduction. In addition, PF4 tetramer formation may also contribute to these functional variations.
  • ELR Glu-Leu-Arg
  • PF4 functions optimally as a tetramer in vitro (Mayo et al, Biochem J., 312(Pt 2), 357-65 ( 1995) and Mikhailov et al., J. Biol. Chem. 274, 25317-25329 (1999)), and therefore presumably as a tetramer in vivo due both to the presence of heparan- sulfate normally found on the surface of cells and because it is released from ⁇ - granules of platelets in relatively large quantities upon tissue injury.
  • the present invention provides heteromultimers (e.g., heterodimers and heterotetramers, and preferably heterodimers) including differing chemokine monomers, and methods of using these heteromultimers.
  • the chemokine monomers include at least one CXC chemokine monomer.
  • NMR spectroscopy and computational modeling demonstrate that when present together in solution, CXC chemokines PF4 and IL8 interact with each other and exchange subunits to form AB-type heterodimers.
  • the findings described herein indicate that hetero-association between/among other structurally homologous members of the chemokine family (e.g., CC chemokines), and perhaps among members of various cytokine sub-families, may be a general occurrence.
  • the heterodimers thus produced are also shown to have biological activity that is significantly different from homogenous tetramers prepared from single chemokines.
  • the present invention includes an isolated heteromultimer that includes at least two different analogous or homologous chemokine monomers.
  • the chemokine monomers are covalently linked.
  • the heteromultimer may in further aspects be a tetramer or a dimer.
  • the chemokine monomers include one or more CXC chemokine monomers, and in a yet further aspect the CXC chemokine monomer is selected from the group consisting of PF4, IL-8, GRO- ⁇ , GRO- ⁇ , GRO- ⁇ , MIP-2 ⁇ , MIP- 2 ⁇ , ENA-78, GCP-2, NAP-2, IP-10, MIG, I-TAC, SDF-I, and BCA-I.
  • the heterodimer may be an AB-type heterodimer comprising an IL-8 monomer and a PF4 monomer.
  • a chemokine heterodimer including an IL-8 and a PF4 monomer further includes a linker molecule.
  • the linker molecule connects the C-terminus of the IL8 monomer to the N- terminus of the PF4 monomer.
  • a preferred linker for use in making this connection has an amino acid sequence of GGSSSSSSG (SEQ ID NO:3).
  • the linker molecule connects the N-terminus of the IL8 monomer to the C-terminus of the PF4 monomer.
  • a preferred linker molecule for use in this role has an amino acid sequence of GGSSSSSSSSG (SEQ ID NO:4).
  • a chemokine heterodimer including a CXC chemokine monomer includes a CXC chemokine monomer that is angiostatic.
  • the angiostatic CXC chemokine monomer may be a PF4 monomer.
  • the CXC chemokine monomer is an angiogenic CXC chemokine monomer.
  • the angiogenic CXC chemokine monomer may be an IL- 8 monomer.
  • the chemokine heterodimer of the invention may also include a monomer that is a CC chemokine monomer.
  • the CC chemokine monomer may be selected from the group consisting of RANTES, MIP-Ia, MIP-I ⁇ , MIP-3 ⁇ , MCP-I, MCP-2, MCP-3, MCP-4, Eotaxin-1, Eotaxin-2, Eotaxin-3, TARC, MDC, ELC, SLC, I- 309, MEC, CTACK, TECK, HCC-I, HCC-2, and Regakine-1.
  • a heterodimer including two different analogous or homologous chemokine monomers that are covalently attached by a linker molecule is provided.
  • the chemokine monomers include one or more CXC chemokine monomers, while in a further aspect the linked chemokine monomers include one or more CC chemokine monomers.
  • the present invention also provides a method of modulating chemotaxis or growth of a chemokine receptor-bearing cell that includes contacting the cell with an isolated heteromultimer that includes two different analogous or homologous chemokine monomers.
  • the chemokine monomers are covalently linked.
  • the heteromultimer may be in the form of a dimer or a tetramer, or simply a dimer.
  • the heteromultimer includes a linker molecule.
  • the chemokine monomers may include one or more CXC chemokine monomers.
  • Modulating the chemotaxis or growth of a chemokine-receptor bearing cell may be used to affect cell-mediated immunity.
  • Cells affected in this fashion may include, for example, neutrophils, T-lymphocytes, or natural killer cells.
  • modulating the chemotaxis or growth of a chemokine- receptor bearing cell affects angiogenesis.
  • Cells affected in this fashion may include endothelial cells.
  • CXC chemokine monomers used in the method may be selected from the group consisting of PF4, IL-8, GRO- ⁇ , GRO- ⁇ , GRO- ⁇ , MIP-2 ⁇ , MIP-2 ⁇ , ENA-78, GCP-2, NAP-2, IP-IO, MIG, I-TAC, SDF-I, and BCA-I.
  • the heteromultimer is an AB- type heterodimer that includes an IL-8 monomer and a PF4 monomer.
  • the monomer may be an angiostatic CXC chemokine monomer.
  • the angiostatic CXC chemokine monomer is a PF4 monomer.
  • the CXC chemokine monomer may be an angiogenic CXC chemokine monomer.
  • the angiogenic CXC chemokine monomer may be an IL-8 monomer.
  • the method may also include use of chemokine heterodimers that include one or more CC chemokine monomers.
  • the CC chemokine monomer is selected from the group consisting of RANTES, MIP-Ia, MlP-l ⁇ , MIP-3 ⁇ , MCP-I, MCP-2, MCP-3, MCP-4, Eotaxin-1, Eotaxin-2, Eotaxin-3, TARC, MDC, ELC, SLC, 1-309, MEC, CTACK, TECK, HCC-I, HCC-2, and Regakine-1.
  • modulation of the chemotaxis of a chemokine-receptor bearing cell affects inflammation.
  • the chemokine receptor-bearing cells are selected from the group consisting of macrophages, monocytes, neutrophils, T-lymphocytes, eosinophils, and basophils.
  • the present invention also provides a method of treating a subject with a disorder responsive to chemokines including administering an isolated heteromultimer that includes at least two different analogous or homologous chemokine monomers to the subject.
  • the chemokine monomers are covalently linked.
  • the heteromultimer may be in the form of a dimer or tetramer, or specifically a dimer.
  • the heteromultimer includes a linker molecule.
  • Tthe cheraokine monomers in aspects of the method may include one or more CXC chemokine monomers.
  • the disorder may be tumor-associated angiogenesis, fibroproliferative disorders, blood clotting disorders, or wounds.
  • the CXC chemokine monomer is selected from the group consisting of PF4, IL-8, GRO- ⁇ , GRO- ⁇ , GRO- ⁇ , MIP-2 ⁇ , MIP-2 ⁇ , ENA-78, GCP-2, NAP-2, IP-IO, MIG, I-TAC, SDF-I , and BCA-I.
  • the CXC chemokine monomer may be an angiostatic CXC chemokine monomer.
  • the angiostatic CXC chemokine monomer may be a PF4 monomer.
  • the CXC chemokine monomer is an angiogenic CXC chemokine.
  • the angiogenic CXC chemokine may be an IL-8 monomer.
  • the chemokine monomers may include one or more CC chemokine monomers.
  • the CC chemokine monomer may be selected from the group consisting of RANTES, MIP-I ⁇ , MIP-I ⁇ , MIP-3 ⁇ , MCP-I, MCP-2, MCP-3, MCP-4, Eotaxin-1, Eotaxin-2, Eotaxin-3, TARC, MDC, ELC, SLC, 1-309, MEC, CTACK, TECK, HCC-I , HCC-2, and Regakine-1.
  • the disorder may be selected from the group consisting of rheumatoid arthritis, inflammation, respiratory diseases, allergy, and IgE-mediated allergic reactions.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) or is synthetically derived.
  • a naturally-occurring polypeptide present in a living animal is not isolated, but the same polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such a polypeptide could be part of a composition, and still be isolated in the composition, and not be a part of its natural environment.
  • Figures IA and IB are graphs showing the effect of IL8 binding on PF4 and PF4M2.
  • 1 H- 15 N HSQC spectra of I5 N-labeled PF4 (IA) and PF4M2 (IB) are shown in the absence (blue cross-peaks) and presence (red cross-peaks) of unlabeled IL8 (1 :1 molar ratio).
  • Figure 2 illustrates expanded regions of 1 H- 15 N HSQC spectra from Figure IA.
  • the addition of IL8 to 15 N-PF4 causes some sets of multiple resonances for a given residue to coalesce, vary their intensity ratios or merely shift. This is exemplified for residues H23 and A43, H35 and G48, 142 and 163, respectively.
  • Figures 3A and 3B illustrate the 1 H and 15 N chemical shift differences, ⁇ , between pure PF4 (3A) and PF4M2 (3B) and PF4 (or PF4M2) to which BL8 has been added at a molar ratio of 1 : 1 , averaged according to
  • hatched horizontal bars represent regions of contact between monomers in the dimer (xxxx) or tetramer (/////).
  • Black horizontal bars and oval shapes represent ⁇ - strand and ⁇ -helical elements of secondary structure, respectively.
  • FIGS 4A and 4B graph the relative 1 H and 15 N chemical shift changes.
  • Relative 1 H and 15 N chemical shift changes, ⁇ , in PF4 (4A) and PF4M2 (4B) induced by addition of IL8 at different PF4 (or PF4M2) to IL8 ratios are illustrated.
  • Each point results from subtraction of the chemical shift of a residue in pure PF4 (or PF4M2) from that of a PF4 subunit in the PF4/IL8 complex and then dividing by the chemical shift change between pure PF4 (or PF4M2) and PF4 (or PF4M2) after addition of IL8 at the ratio 1:2.
  • the arithmetic averages of relative chemical shifts for 1 H and 15 N are shown. Error bars represent the standard deviation of the chemical shifts calculated for different residues.
  • Figure 5 graphs the diffusion coefficients for PF4 and PF4M2 (solid and open squares) and for PF4 and PF4M2 upon addition of IL8 (solid and open circles) vs. the total protein concentration.
  • Figures 6A-6D graph the C ⁇ RMS deviations of PF4, IL8 homodimers, PF4/IL8 heterodimer (6A) and PF4M2, IL8 homodimers, PF4M2/IL8 heterodimer (6B) from initial structures as a function of the simulation time. Fluctuations from the average residue position during the last 800 picosecond (ps) of the simulation for the corresponding homo- and heterodimers are shown in panels 6C and 6D.
  • Figure 7 shows a single chain IL8/PF4 heterodimer designed using GG(S) 6 GG peptide as linker, linking the C-terminus of IL8 with the N-terminus of PF4.
  • Figure 8 shows a single chain IL8/PF4 heterodimer designed using GG(S) 8 GG peptide as linker, linking the N-terminus of IL8 with the C-terminus of PF4.
  • Figures 9A and 9B are graphs showing the effects of various monomers, dimers, tetramers, and heterodimers on the proliferation of endothelial cells (EC). Effects on proliferation of endothelial cells (EC) due to the presence of PF4 and IL8 alone and in combination at different molar ratios are shown in Figure 9A. Using the concentrations used in this assay and the known K D values for homo- and hetero-aggregate formation, concentrations of all possible monomer, dimer and tetramer species were calculated as illustrated in Figure 9B.
  • Figures lOA-lOC show the effect of heterodimerization on IL8 biological function.
  • Figure 1OA shows the metabolic response of CD34+ cells to the infusion of IL8 alone at a concentration of 100 nanogram (ng)/milliliter (mL) and to the infusion of IL8 at the same concentration preceded by the infusion of PF4 at the concentrations of 100 ng/mL (PF4:IL8 molar ratio 1:1) and 10 ⁇ g/mL (PF4:IL8 molar ratio 100:1).
  • the zero time point corresponds to the addition of IL8 to the CD34+ cells.
  • the metabolic response was assessed by measurement of extracellular proton excretion (acidification rate).
  • Figure 1OB shows the chemotactic response from Baf3/hCXCR2 cells to IL8 alone, PF4 alone, and PF4 and IL8 added together at different ratios.
  • the dashed line indicates the level of chemotactic activity at 1 ng/mL of IL8, if the presence of PF4 had no effect on activity.
  • Figure 1OC using the concentrations used in this assay and the known K D values for homo- and hetero-aggregate formation, concentrations of all possible monomer, dimer and tetramer species were calculated as illustrated. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE
  • a heteromultimer that includes at least two different chemokine monomers.
  • a heteromultimer is an aggregate of a plurality of different chemokine monomers.
  • the heteromultimer is a heterodimer or a heterotetramer, and more preferably it is a heterodimer.
  • a heterodimer refers to a dimer composed of two subunits in which the two subunits are different.
  • the differing subunits in heteromultimers of the present invention are chemokine monomers.
  • the heterodimers at AB-type dimers.
  • the heterodimers may be a part of a larger structure such as a tetramer.
  • Monomers associating to form a heteromultimer may be retained together in a variety of fashions.
  • monomers forming the heterodimer may associate through affinity of the two monomers, or they may be covalently linked.
  • the monomers are covalently linked, and more preferably, covalently linked using a linker molecule.
  • discussion herein regarding heterodimers is also intended to cover heteromultimeric chemokines such as tetramers and other multimers, as defined above.
  • a chemokine monomer is a polypeptide categorized as belonging to the chemokine family of proteins.
  • the chemokine family of proteins includes a variety of single polypeptides of about 70-100 amino acids in length sharing a varying degree of structural similarity.
  • Chemokines act by inducing chemotaxis in effected cells. Chemokines are generally 8-12 kilodalton (kD), basic, heparin binding proteins that contain conserved cysteine amino acids that form important disulfide bonds. The number and spacing of the first two cysteines is used to characterize chemokines into subfamilies.
  • Alpha chemokines referred to herein as CXC-chemokines, are chemokines in which a single amino acid (X) is present between the initial two cysteines (C), forming the CXC sequence.
  • Beta chemokines referred to herein as CC-chemokines, are chemokines in which the first two cysteines are adjacent to one another, forming the short CC sequence.
  • Chemokines act through chemokine receptors, a subfamily of 7- transmembrane, G-protein-coupled receptors. Chemokine receptors are found on a variety of cells, such as on hematopoietic cells, neurons, astrocytes, epithelial cells, and endothelial cells. The presence of chemokine receptors on these different types of cells highlights the various roles for chemokine receptors, such as leukocyte chemotaxis and stimulation of endothelial cell proliferation. Table 1 provides a list of a large number of known CXC and CC chemokines, as well as the receptors and cell types that they effect.
  • chemokine names are based on their cysteine subclass roots, followed by "L” for "ligand”. The numbers correspond generally to the same number used in the corresponding gene nomenclature. Because most chemokine receptors are restricted to a single chemokine subclass, the nomenclature system of chemokine receptors is rooted by the chemokine subclass specificity, followed by "R” for "receptor” and the number. For example, according to this nomenclature system, IL-8 is now called CXCL8.
  • Table 1 further provides abbreviations that can be utilized to obtain further information on these structures from the RCSB protein data bank (PDB) maintained online by the Research Collaboratory for Structural Bioinformatics.
  • At least one of the chemokine monomers is a monomer selected from the CXC subfamily of chemokines, i.e., a CXC- chemokine.
  • CXC chemokines are distinguished by the CXC amino acid sequence motif.
  • CXC chemokines have four highly conserved amino acid residues, and are characteristically heparin binding proteins.
  • CXC chemokine monomers include platelet factor 4 (PF4), interleukin-8 (IL-8), growth-related proteins (GRO- ⁇ , GRO- ⁇ , GRO- ⁇ ), macrophage inflammatory protein-2 ⁇ (M ⁇ P-2 ⁇ ), macrophage inflammatory ⁇ rotein-2 ⁇ (MIP-2 ⁇ ), epithelial neutrophil activating protein-78 (ENA-78), granulocyte chemotactic protein-2 (GCP-2), neutrophil activating protein-2 (NAP-2), interferon- ⁇ -inducible protein (IP-10), monokine induced by interferon- ⁇ (MIG), IFN-inducible T-cell alpha-chemoattractant (I-TAC), stromal cell-derived factor-1 (SDF-I), and B-cell attracting chemokine-1 (BCA- 1).
  • PF4 platelet factor 4
  • IL-8 interleukin-8
  • growth-related proteins GRO- ⁇ , GRO- ⁇ , GRO- ⁇
  • M ⁇ P-2 ⁇ macrophag
  • CXC chemokines can affect a variety biological functions, including angiogenesis and cell-mediated immunity.
  • CXC chemokines have thus been further categorized as angiogenic CXC chemokines and angiostatic CXC chemokines. In addition to their differing activities, these two groups may be structurally distinguished.
  • Angiogenic CXC chemokines contain a three amino acid sequence, Glu-Leu-Arg, referred to as the ELR motif, which precedes the first cysteine amino acid of the primary structure of these cytokines.
  • Angiogenic refers to the ability of a compound to stimulate angiogenesis.
  • Angiogenic CXC chemokines include IL-8, ENA-78, GRO- ⁇ , GRO- ⁇ , GRO- ⁇ , GCP-2, platelet basic protein (PBP), connective tissue activating protein-III (CTAP-III), beta-thromboglobulin ( ⁇ -TG), and NAP-2.
  • the chemokine heterodimer of the invention includes an angiogenic CXC chemokine monomer.
  • the angiogenic chemokine monomer may be interleukin-8 (IL-8).
  • Interleukin 8 also referred to as neutrophil activation protein, may in one aspect of the invention be the peptide including the amino acid sequence SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLD PKENWVQRVVEKFLKRAENS (SEQ ID NO: 1).
  • IL-8 cDNA encodes a 99- amino acid precursor protein with a signal sequence, which is cleaved to yield mainly 77- or 72-residue mature protein (Matsushima et al., J. Exp. Med. 169, 1485-1490 (1988)).
  • IL-8 is further processed at the NH 2 terminus yielding different truncation analogs (77-, 72-, 71-, 70-, 69-amino acid forms).
  • the truncation is caused by proteases that are released from IL-8-secreting cells or by accessory cells, and the occurrence of the NH 2 -terminal forms depends on the producer cells and culture conditions.
  • the two major forms are 77- and 72- amino acid proteins with a minor 69-amino acid protein.
  • fibroblasts and endothelial cells predominantly produce the 77-amino acid form, whereas leukocytes mainly secrete 72- or 69-amino acid forms.
  • IL-8 in a concentrated solution and crystallized state, occurs as a homodimer consisting of two identical subunits.
  • the monomer contains a disordered NH2 terminus, followed by a loop region, three antiparallel -strands, and an -helix extending from amino acids 57 to the COOH terminus.
  • concentrations greater than 100 ⁇ M EL-8 exists as a dimer stabilized by six hydrogen bonds between the first -strands of the partner molecules (residues 23- 29) and by other side-chain interactions.
  • the dimer consists of two antiparallel - • helices lying on top of a six-stranded antiparallel -sheet.
  • IL-8 occurs in its monomeric form.
  • IL-8 can be produced by leukocytic cells (monocytes, T cells, neutrophils, and natural killer cells) and nonleukocytic somatic cells (endothelial cells, fibroblasts, and epithelial cells). IL-8 production is not constitutive but inducible by proinflammatory cytokines such as IL-I and tumor necrosis factor TNF- ⁇ .
  • IL-8 production can be induced by bacteria (e.g., Helicobacter pylori, Pseudomonas aeruginosa), bacterial products [e.g., lipopolysaccharide (LPS)], viruses (e.g., adenovirus, respiratory syncytial virus, cytomegalovirus, rhinovirus), and viral products.
  • bacteria e.g., Helicobacter pylori, Pseudomonas aeruginosa
  • bacterial products e.g., lipopolysaccharide (LPS)
  • viruses e.g., adenovirus, respiratory syncytial virus, cytomegalovirus, rhinovirus
  • viral products e.g., IL-8/CXCL8 as a key mediator in neutrophil-mediated acute inflammation due to its potent actions on neutrophils.
  • IL-8/CXCL8 has a wide range of actions on various types of cells, including lymphocytes, monocytes, endothelial cells, and fibroblasts, besides neutrophils.
  • lymphocytes including lymphocytes, monocytes, endothelial cells, and fibroblasts, besides neutrophils.
  • IL- 8/CXCL8 has crucial roles in various pathological conditions such as chronic inflammation and cancer. See Mukaida N., Am. J. Physiol. Lung Cell MoI. Physiol. 284, L566-L577 (2003) for further details on the structure, mechanism, and role of IL-8.
  • Chemokine heterodimers of the invention may also include angiostatic CXC chemokines.
  • Angiostatic CXC chemokines are CXC chemokines that lack the ELR motif, and are inhibitors of angiogenesis.
  • Angiostatic chemokines include platelet factor 4 (PF-4), intefereron- ⁇ -inducible protein (IP-10), and monokine induced by interferon- ⁇ (MIG).
  • Angiostatic CXC chemokines are typically induced by interferon.
  • the chemokine heterodimer of the invention includes an angiostatic CXC chemokine monomer.
  • the angiostatic CXC chemokine monomer may be platelet factor 4.
  • Platelet factor 4 may in one aspect of the invention be the peptide including the amino acid sequence EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPH
  • Platelet factor 4 is generally found as a tetrameric molecule composed of four PF4 monomers, each of 70 amino acids in length (Deuel et al., PNAS USA 74, 2256- 2258 (1977)). PF4 is generally found in platelets and megakaryocytes, and has potent antiheparin activity and plays a role in heparin-induced thrombocytopenia. Platelet factor 4 is further distinguished by the existence of three major clusters of basic amino acids; one near the N-terminus, one in the middle, and one at the C-terminus.
  • PF4 also contains two disulfide bridges between CyslO and Cys 36, and Cys 12 and Cys 52.
  • the structure of human PF4 has been revealed by x-ray crystallography, and includes a single a-helix that spans from residue 61 to 70 in each monomer. Further information on PF4 is provided in a review by Bikfalvi (Bikfalvi, A, Semin. Thromb. Hemost. 30, 379-385 (2004).
  • At least one of the chemokine monomers is a monomer selected from the CC subfamily of chemokines, i.e., a CC-chemokine.
  • CC chemokines are distinguished by the CC amino acid sequence motif.
  • CC chemokines include Regulation on Activation Normal T cell Expressed (RANTES), the macrophage inhibitory protein (MIP) family, including MIP- Ia, MIP-I ⁇ , MIP-3 ⁇ , the monocyte chemoattractant protein (MCP) family, including MCP-I, MCP-2, MCP-3, MCP-4, Eotaxin-1, Eotaxin-2, Eotaxin-3, TARC, MDC, ELC, SLC, 1-309, MEC, CTACK, TECK, HCC-I, HCC-2, and Regakine-1.
  • MIP macrophage inhibitory protein
  • MCP monocyte chemoattractant protein
  • MCP-I monocyte chemoattractant protein
  • MCP-2 monocyte chemoattractant protein
  • MCP-4 monocyte chemoattractant protein
  • Eotaxin-1 Eotaxin-2
  • Eotaxin-3 Eotaxin-3
  • TARC MDC
  • ELC ELC
  • SLC SLC
  • chemokine monomers are demonstrated herein to associate to form chemokine heterodimers.
  • native PF4 and the PF4M2 N-terminal chimera were both used in NMR studies with BL8, because they show significant differences in their quaternary structures at dimer interfaces, as well as in their dimer and tetramer dissociation constants.
  • PF4M2 yields better-defined NMR spectra, primarily due to its forming symmetric tetramers and exhibiting less chemical exchange broadening.
  • chemokine heterodimers are demonstrated to form as a result of the conformation and affinity of the chemokine monomers.
  • chemokine monomers in a chemokine heteromultimer may be linked by a linker molecule.
  • linker molecules are preferred as the allow preparation of chemokine heterodimers at any desired concentration, and substantially increase the stability of the chemokine heterodimer.
  • a linker molecule may be an organic molecule of an appropriate size to covalently bond the two chemokine monomers forming the heterodimer. Examples of suitable linkers include organic polymers such as poly(alkylene oxides), polynucleotides, and oligosaccharides.
  • the linking molecule is a peptide.
  • Organic molecules suitable for use as linking molecules should be capable of directed binding to polypeptides, and preferably have relatively low strain energies and a narrow range of mobility when linked to the chemokine heterodimer monomers.
  • poly(alkylene oxides) may use N-ethyl malemide to provide a reactive binding site capable of binding to thiols.
  • Organic molecules suitable for use as linking molecules can be determined, for example, by using molecular modeling studies such as those described in Example 7 herein.
  • the linker molecule is a peptide
  • the peptide may be included in the heterodimer by using recombinant DNA technology, as known by those skilled in the art, to provide a fusion peptide in which the two monomers are expressed with the linker connecting the N-terminal of one chemokjne monomer to the C- terminal of the other chemokine monomer.
  • DNA fragments encoding chemokine monomers can be obtained using PCR amplification and/or restriction enzyme digestion.
  • the linker peptide with an appropriate length, determined, for example, by molecular modeling, is generated, preferably using conventional synthetic oligonucleotide synthesis.
  • the different fragments are then cloned together in frame by sequential digestion and ligation steps according to procedures known to those skilled in the art.
  • the DNA encoding the linked chemokine heterodimer is then included in to an appropriate expression organism such as a bacterial or yeast using a suitable vector.
  • Preferred linker peptides for use in this recombinant method include peptides with the sequences GG(S) n GG, where n is the number of serine peptides included.
  • suitable linker peptides include linker peptides wherein n varies from 2 to 10 serine peptides.
  • the linker peptide may include at least 2 serine peptides, more preferably at least 4, and even more preferably at least 6.
  • the linker peptide may include 10 or fewer serine peptide, more preferably 8 or fewer peptides, and even more preferably 7 or fewer peptides.
  • chemokine heterodimers covalently linked using linker molecules include IL8 and PF4.
  • the linker molecule connects the C-terminus of the IL8 monomer to the N-terminus of the PF4 monomer using a peptide linker.
  • a preferred linker for use in this embodiment has an amino acid sequence of GGSSSSSSGG (SEQ ID NO:3).
  • the linker molecule connects the N-terminus of the IL8 monomer to the C-terminus of the PF4 monomer.
  • a preferred linker is a linker with the amino acid sequence of GGSSSSSSSSGG (SEQ ID NO:4).
  • Chemokine monomers of the invention are peptides.
  • polypeptide refers broadly to a polymer of two or more amino acids joined together by peptide bonds.
  • polypeptide also includes molecules that contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that are joined together.
  • peptide, oligopeptide, and protein are all included within the definition of polypeptide and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.
  • Substitutes for an amino acid in the polypeptides of the invention are preferably conservative substitutions, which are selected from other members of the class to which the amino acid belongs.
  • conservative substitutions which are selected from other members of the class to which the amino acid belongs.
  • an amino acid belonging to a grouping of amino acids having a particular size or characteristic can generally be substituted for another amino acid without substantially altering the structure of a polypeptide.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.
  • Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Examples of preferred conservative substitutions include Lys for Arg and vice versa to maintain a positive charge; GIu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free -OH is maintained; and GIn for Asn to maintain a free NH 2 .
  • amino acids and derivatives thereof that can be used include 3- hydroxyproline, 4-hydroxyproline, homocysteine, 2-aminoadipic acid, 2- aminopimelic acid, ⁇ -carboxyglutamic acid, ⁇ -carboxyaspartic acid, ornithine, homoarginine, N-methyl lysine, dimethyl lysine, trimethyl lysine, 2,3- diaminopropionic acid, 2,4-diaminobutyric acid, homoarginine, sarcosine, hydroxylysine, substituted phenylalanines, norleucine, norvaline, 2- aminooctanoic acid, 2-aminoheptanoic acid, statine, ⁇ -valine, naphthylalanines, substituted phenylalanines, tetrahydroisoquinoline-3-carboxylic acid, and halogenated tyrosines.
  • chemokine monomers are readily available, and known by those skilled in the art.
  • polypeptide sequences for a variety of chemokines are available at Genbank, or at the protein data bank, as listed in Table 1.
  • Chemokine heterodimers of the present invention in linked and unlinked forms, can tolerate a certain degree of variation in its amino acid sequence without significant disruption of its bioactivity.
  • the terms used for various chemokine monomers of the present invention such as IL8 and PF4, as used herein, thus presume a certain allowed variation in structure, unless specifically restricted to a particular sequence.
  • Preferred variants of chemokine monomers or any of their constituent peptides include those sequences that are at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical, and most preferably at least 99% identical to a particular chemokine monomer or its constituent peptides.
  • Such variants contain one or more amino acid deletions, insertions, and/or substitutions relative to the original chemokine or its subunits, and may further include chemical and/or enzymatic modifications and/or derivatizations. Percent identity can be determined by a BLAST analysis.
  • Percent identity between two polypeptide sequences is generally determined by aligning the residues of the two amino acid sequences to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • two amino acid sequences are compared using the Blastp program, version 2.2.10, of the BLAST 2 search algorithm, as described by Tatusova et al. (FEMS Microbiol. Lett., 174, 247-250 (1999)), and available on the world wide web at the National Center for Biotechnology Information website, under BLAST in the Molecular Database section.
  • identity In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as "identity.”
  • Chemokine polypeptides of the invention can be produced on a small or large scale through use of numerous expression systems that include, but are not limited to, cells or microorganisms that are transformed with a recombinant vector into which a polynucleotide of the invention has been inserted. Such recombinant vectors and methods for their use are described below. These vectors can be used to transform a variety of organisms. Examples of such organisms include bacteria (for example, E. coli or B.
  • subtilis subtilis
  • yeast for example, Saccharomyces and Pichia
  • insects for example, Spodoptera
  • plants or mammalian cells (for example, COS, CHO, BHK, 293, VERO, HeLa, HEK, MDCK, Wl 38, and NIH 3T3 cells).
  • mammalian cells for example, COS, CHO, BHK, 293, VERO, HeLa, HEK, MDCK, Wl 38, and NIH 3T3 cells.
  • Also useful as host cells are primary or secondary cells obtained directly from a mammal that are transfected with a vector.
  • recombinant PF4, PF4M2 and BL8 used in the present invention were expressed in E. coli and purified as described by Yang et al., Biochemical Journal 304(Pt 2), 371-6 (1994)).
  • Synthetic methods may also be used to produce polypeptides and polypeptide subunits of the invention. Such methods are known and have been reported (Merrifield, Science, 85:2149 (1963), Olson et al., Peptides, 9, 301, 307 (1988)).
  • the solid phase peptide synthetic method is an established and widely used method, which is described in the following references: Stewart et al., Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc, 85 2149 (1963); Meienhofer in "Hormonal Proteins and Peptides," ed.; CH. Li, Vol. 2 (Academic Press, 1973), pp.
  • Polypeptides can be readily purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration • using, for example, Sephadex G-75; ligand affinity chromatography, and the like.
  • Polypeptides can also be readily purified through binding of a fusion polypeptide to separation media, followed by cleavage of the fusion polypeptide to release a purified polypeptide.
  • PF4 tetramers/dimers and IL8 dimers can readily exchange subunits to form heterodimers through their ⁇ -sheet interfaces, and that heterodimer formation modulates the biological functions of PF4 and IL8.
  • the combined presence of IL8 and PF4 in solution potentiates the anti-proliferative activity of PF4 against endothelial cells, attenuates IL8- mediated signaling in hematopoietic progenitor cells, and enhances chemotactic response to IL8 from Baf3 (bone marrow-derived pro-B-cells)/hCXCR2 cells.
  • PF4, PF4M2, and IL8 are highly homologous proteins, demonstrating up to 60% sequence similarity and exhibiting essentially the same three- dimensional overall folds in their monomer states. Differences among these CXC chemokines become most significant at the level of their quaternary structures. IL8 forms AB-type dimers, whereas PF4 and PF4M2 can also form asymmetric and symmetric tetramers, respectively. Heterodimer formation also explains most of the observed chemical shift changes in heteronuclear single quantum coherence (HSQC) spectra of PF4 and PF4M2 dimers.
  • HSQC heteronuclear single quantum coherence
  • Shift changes from residues within ⁇ -strands 1 and 2 and the C-terminal part of the helix are primarily explained by the presence of different intersubunit contacts between/among residues upon heterodimer formation. Other chemical shift changes may be accounted for by minor structural perturbations and/or absence of intersubunit dimer-dimer contacts from homotetramers.
  • CXC-chemokines e.g. growth-related protein (GRO)- ⁇ , macrophage inflammatory protein (MIP)-2 ⁇ (or GRO- ⁇ ), MIP-2 ⁇ (or GRO- ⁇ ), and neutrophil activating protein (NAP)-2) also show up to 60% amino acid sequence similarity and are all structurally homologous to PF4 and IL8 (Clore et al., FASEB J. 9(1), 57-62 (1995)). For example, superposition of folded structures of PF4 and IL8 monomers gives a backbone RMSD value of 1.8 A.
  • GRO growth-related protein
  • MIP macrophage inflammatory protein
  • NAP neutrophil activating protein
  • Chemokine heterodimers of the invention thus include only chemokine polypeptides that are analogous or homologous with respect to one another in terms of structure and sequence.
  • polypeptides may be referred to as heterologous, analogous, or homologous.
  • heterologous polypeptides are polypeptides that are distinctly different in structure.
  • U.S. Patent 6,730,296 provides chemokines linked to heterologous polypeptides, in which examples of heterologous proteins include chemokines and the Fc portion of an immunoglobulin, which are heterologous in terms of sequence and structure.
  • Heterologous polypeptides, as defined herein have an amino acid sequence similarity of less than 50%.
  • Analogous polypeptides are polypeptides with related folds but unrelated sequences.
  • analogous polypeptides may result that independently developed the same fold.
  • Folds are tertiary structure in a polypeptide that form a recognizable structural region, while related folds are tertiary structures that are recognized by those skilled in the art to be very similar in terms of geometry.
  • homologous polypeptides are two proteins with related folds and related sequences.
  • Related sequences as defined herein, are sequences with an amino acid sequence that has a 50% or more similarity.
  • CXC-chemokines may help resolve a number of discrepancies and unexplained observations in the literature.
  • the angiogenesis inhibitory effect from PF4 appears to be stronger and less variable in vivo than it is in vitro. This suggests that the full potency of PF4 as an antiangiogenic agent may not be realized fully unless a complement of CXC-chemokines, like IL8, is present in solution together with PF4. This is possible in vivo, but not necessarily in vitro where the chemokine composition in culture media is essentially predetermined.
  • CXC- chemokines that are constantly being expressed by cells and released into the serum in varying quantities. Depending on mass action, their monomer subunits could readily exchange with each other, forming heterodimers and thereby modulating various chemokine activities, as observed here for PF4 and IL8.
  • chemokines such as PF4 and IL8 are substantially different in vivo.
  • PF4 is present at micromolar to nanomolar concentrations
  • IL8 has been reported to be present at nanomolar to picomolar levels (Burrows et al., Biochemistry 33(43), 12741-12745 (1994), Clark-Lewis et al., J. Leukoc. Biol. 57(5), 703-11 (1995)).
  • Chemokines present at these levels, particularly at such divergent concentrations, will exhibit negligible heterodimer formation.
  • relative association binding constants which dictate the extent of heterodimerization, can be different in vivo from those values determined herein, because solution conditions vary considerably, i.e. temperature, pH, salt and the presence of numerous other biomolecules in the serum milieu.
  • IL8 alone, the influx of this CXC-chemokine would have to be on the micromolar scale to have a significant effect on PF4-mediated biology.
  • IL8 is normally present in serum at nanomolar to picomolar levels, insufficient quantities of a differing chemokine are present to effect formation of significant amounts of PF4 heteroaggregates in serum.
  • PF4/EL8 heterodimers also suggests a possible effect of PF4 on hematopoiesis by the interaction of PF4 with IL8 and/or other CXC- chemokines, and thus attenuating (or enhancing) the capacity to induce signaling.
  • Formation of PF4/EL8 heterodimers clearly enhances the ability PF4 to act as an antiproliferative agent against endothelial cells. This observation alone has therapeutic implications for the use of PF4 in the clinic as an antiangiogenic and/or anti-tumor agent.
  • PF4 by itself exhibits limited effectiveness as an anti-cancer agent in the clinic; however, if PF4 and IL8, for example, were administered as a conjugate or fusion product, the effects should be synergistic, as demonstrated by the present in vitro results.
  • An additional aspect of the invention provides methods of using chemokine heterodimers to module the biological activity of cells.
  • the data demonstrate that the formation of PF4/IL8 heterodimers is bio-functionally correlated, by examining the concentration-dependent influence of PF4 on IL8- induced migration of Baf 3 cells and IL8-induced signaling in CD34+ hematopoietic progenitor cells, as well as the influence of IL8 on anti ⁇ proliferative effects of PF4 against endothelial cells (EC).
  • Chemokines derive their name from their ability to direct cell migration, i.e., chemotaxis. Accordingly, one of the biological activities affected by chemokine heterodimers is chemotaxis.
  • chemokines have additional effects on cells.
  • chemokines also function to regulate activities such as angiogenesis and hematopoiesis, which are both examples of cell growth.
  • another biological activity of cells affected by chemokine heterodimers is cell growth.
  • chemokine heterodimers may indirectly affect a wide variety of cells
  • chemokine heterodimers have direct effects primarily on cells bearing chemokine receptors.
  • Chemokine receptors are a subfamily of 7-transmembrane G- protein coupled receptors, and are named according to the chemokine subfamily (e.g., CXC, CC) followed by a number.
  • Chemokines receptors share a 25-80% amino acid sequence identity when compared with one another.
  • One chemokine is often capable of binding multiple chemokine receptor types, and a single receptor may be receptive to binding by more than one type of chemokine. Again, this is illustrated by Table 1.
  • the present invention thus provides a method of modulating the chemotaxis or growth of a chemokine-receptor bearing cell.
  • the method includes contacting the cell with an isolated heterodimer that includes two different chemokine monomers, i.e., a chemokine heterodimer.
  • the chemokine monomers forming the chemokine heterodimer may be held together by conformation and affinity. In a further aspect, they may be held together using a linker molecule.
  • the chemokine heterodimer may be any of the chemokine heterodimers as described herein.
  • the chemokine heterodimer may include a CXC chemokine monomer and/or a CC chemokine monomer.
  • CXC chemokine monomers may have an effect on cell-mediated immunity.
  • Cell-mediated immunity refers to an immune response that involves immune cells other than ⁇ -lymphoctes involved in the production of humoral antibody.
  • cells affected by CXC chemokine monomers in mediation of cellular immunity include neutrophils, T- lymphocytes, and natural killer cells.
  • CXC chemokines may also have an effect on angiogenesis.
  • Angiogenesis as defined herein, is the formation of new blood vessels or the enlargement of existing blood vessels to enhance circulation.
  • CXC chemokines are categorized as either angiogenic if they contain the ELR motif, or angiostatic if they do not contain the ELR motif.
  • Angiogenic CXC chemokines stimulate angiogenesis, whereas angiostatic CXC chemokines inhibit angiogenesis.
  • Angiogenesis involves a variety of biological activities related to the formation of blood vessels. One of the more important of these activities is the proliferation of endothelial cells. Endothelial cells are known to bear chemokine receptors.
  • cells affected by CXC monomers in modulating angiogenesis may include endothelial cells.
  • chemokine heterodimers including a CC chemokine are used to modulate the chemotaxis of cells. Stimulating chemotaxis can be important in combating infection by recruiting host defense cells in an inflammatory response. While not intending to be bound by theory, chemokines mediate cell motility at multiple stages. For example, injured renal cells produce inflammatory mediators that enhance the expression of adhesion molecules on endothelial cells of capillaries adjacent to the inflamed lesion.
  • chemokines mediate the rolling process of leukocytes along the endothelial surface, thereby allowing contact of leukocytes with chemokines.
  • chemokines are tethered on endothelial plasma membranes using heparin sulfate proteoglycans following secretion by activated endothelial cells.
  • Chemokines bind to their respective receptors on the rolling leukocyte, which activates leukocyte-expressed ⁇ 2 -integrins, resulting in firm adhesion of the leukocyte to the endothelial surface as a prerequisite for leukocyte transmigration.
  • Other chemokine receptors appear to differentially influence spreading, diapedesis, and subsequent migration into the tissue space.
  • Chemokines also upregulate the secretion and activity of matrix metalloproteinases by infiltrating leukocytes, thus facilitating leukocyte transmigration through the basement membrane and extracellular matrix.
  • CC chemokines may effect the chemotaxis of a variety of cells, including macrophages, monocytes, neutrophils, T-lymphocytes, eosinophils, and basophils.
  • the invention provides methods of treating a subject with a disorder by administering a chemokine heterodimer.
  • the disorder is a disorder known to be responsive to chemokines.
  • a disorder, as defined herein, is an undesired disturbance of structure or function within a subject. The disturbance may result from exogenous factors such as disease, poison, or trauma, or it may result from developmental factors such as a genetic or embryological failure.
  • a "subject” is an organism, including, for example, an animal. This includes, for example, humans, nonhuman primates, sheep, horses, cattle, pigs, dogs, cats, rats, mice, birds, reptiles, fish, insects, arachnids, protists (e.g., protozoa), and prokaryotic bacteria. Subject also includes model organisms, including, for example, animal models, used to study tumor progression, growth, or metastasis, or to study wound healing. Preferably, the subject is a human or other mammal.
  • the chemokine monomers forming the chemokine heterodimer used in methods of treating a subject with a disorder may be held together by conformation and affinity. In a further aspect, they may be held together using a linker molecule.
  • the chemokine heterodimer may be any of the chemokine heterodimers as described herein.
  • the chemokine heterodimer may include a CXC chemokine monomer and/or a CC chemokine monomer.
  • the amount of chemokine heterodimer of the present invention that is delivered to the subject will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the subject has undergone. Ultimately, the attending physician will decide the amount of chemokine heterodimer with which to treat each individual patient. Initially, the attending physician will administer low doses of polypeptide of the present invention and observe the patient's response. Larger doses of polypeptide of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
  • compositions used to practice the method of the present invention should contain about 0.01 ng to about 100 mg (preferably about 0.1 ⁇ g to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg) of polypeptide of the present invention per kg body weight
  • chemokine heterodimers may have a variety of effects, based on the nature of the chemokine monomers included.
  • CXC chemokines may be used to treat tumor-associated angiogenesis, fibroproliferative disorders, blood clotting disorders, and wounds.
  • the choice of chemokine for a particular disorder depends on whether or not cell growth and chemotaxis are intended to be stimulated or inhibited.
  • CXC chemokines with the ELR motif are generally angiogenic
  • CXC chemokines without the ELR motif are generally angiostatic. Any consistently observed increase in angiogenesis in response to the presence of a particular composition is evidence of angiogenic activity.
  • CXC chemokines with significant angiogenic activity will often be preferred.
  • “Significant angiogenic activity” is defined herein as the establishment of angiogenesis in a previously silent system or model, or a consistently observed marked increase in angiogenesis.
  • the stimulation of angiogenesis will generally be useful in the context of wound and sore healing.
  • Other uses contemplated include, for example, in the treatment of vascular grafts and transplants and, particularly, in the treatment of skin, gastric and duodenal ulcers.
  • the invention provides methods for promoting wound-healing, which methods generally comprise contacting a wound or ulcer site of an animal with a biologically effective amount of a heterodimer including one or more CXC chemokines.
  • the present invention particularly contemplates the use of the heterodimers including CXC chemokines in the treatment of chronic wounds and ulcers. It is contemplated that treatment with CXC chemokines would be conducted for several days, with the dosage being reduced over time.
  • Biologically effective amounts will be those amounts that promote at least some wound-healing, with amounts that result in significant improvement of the healing process being preferred.
  • Angiostatic chemokines may be used to treat disorders in which undesired proliferation is occurring, such as fibroproliferative disorders, myocardial ischemia and reperfusion, and tumor-associated angiogenesis.
  • the angiostatic members of the CXC chemokine family include PF4, MIG, and IP- 10, all of which are induced by interferon and have been showin to inhibit vascularization.
  • angiostatic chemokines such as PF4 have been shown to bind to glycosaminoglycans, and inhibit endothelial cell migration, proliferation, and in vivo angiogenesis in response to fibroblast growth factor.
  • PF4 has been shown to inhibit fibroblast growth factor binding to its receptor.
  • PF4 has also been shown to prevent cell cycle progression by preventing cell entry into the S phase.
  • Angiogenesis has been recognized as playing an important role in the pathogenesis of chronic inflammatory and fibroproliferative disorders, as well as being necessary to supply tumor cells with sufficient blood supply for tumor proliferation.
  • macrophages isolated from rheumatoid synovium produce pro-angiogenic factors, and psoriasis is well known to be angiogenesis- dependent.
  • the angiogenic phenotype in these disorders appears to often be the result of a combined defect in the overexpression of angiogenic chemokines in addition to a deficiency in the production of angiostatic chemokines.
  • These disorders can be treated using angiostatic chemokines of the present invention that remedy the deficiency of angiostatic chemokines. See, for example, Belpario et al., J. Leuk. Biol. 68, 1 (2000).
  • Chemokines may also be used to stimulate cell-mediated immunity.
  • chemokine heterodimers of the invention may be used to treat many of the conditions in which cell mediated immunity plays an important role.
  • chemokine heterodimers may be used to direct immune cells to viral- infected cells.
  • chemokine heterodimers may be used to direct immune cells to tumor cells that have been recognized as foreign by the immune system. Accordingly, chemokine heterodimers of the invention may be used as an immunostimulant.
  • the chemokine heterodimers may induce a combined cell-mediated immunity and angiostatic effect.
  • Such an effect may be particularly useful for treating cancer, as the angiostatic effect serves to starve the proliferating cancer cells, while the cell-mediated immunity recruits an immune response to dispose of the cancer cells themselves.
  • the role of chemokines in cancer is addressed in more detail by Strieter et al (Strieter et al., Ann. N.Y. Acad. Sci. 1028, 1-10 (2004).
  • CXC chemokines may be used to treat a variety of disorders relating to cell growth. Chemokines may also be used to treat disorders in which chemotaxis, i.e. chemokine-stimulated cell migration, is involved. For example, CC chemokines may be used rheumatoid arthritis, inflammation, respiratory diseases, allergy, and IgE-mediated allergic reactions. CC chemokines such as RANTES are often upregulated after an allergic challenge, and play an important role in neutrophil recruitment and eosinophil chemotaxis and degranulation. Similar effects are observed in many inflammatory disorders.
  • Treatment using chemokine heterodimers in these types of situations would generally utilize chemokine heterodimers that antagonize the usual receptor for the CC chemokine incorporated through inclusion of an additional chemokine monomer that interferes with receptor activation by the chemokine heterodimer, or serves to reduce or inhibit receptor activation by CC chemokines present in vivo that are playing a part in the inflammatory disorder.
  • CXC and CC chemokines have been described as useful for treating disorders relating to cell growth and chemotaxis, respectively.
  • CXC and CC chemokines are not clearly divided in terms of their effects on various receptors and cell types.
  • the effects of cell growth and cell migration may be intertwined in the response of cells to chemokines in many situations.
  • heterodimers including CXC chemokines of the present invention may be used to treat disorders relating to chemotaxis, while CC chemokines may correspondingly be used to treat disorders relating to cell growth. This would be particularly true of chemokine heterodimers including both CC and CXC chemokines.
  • the invention also provides pharmaceutical compositions that can be used for the administration of chemokine heterodimers of the invention to a subject in need thereof.
  • a pharmaceutical composition can contain an IL8/PF4 heterodimer, or a linked version thereof, and a pharmaceutically acceptable carrier.
  • compositions of the invention may be prepared in many forms that include tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow-release formulations, such as shaped polymeric gels.
  • An oral dosage form may be formulated such that the polypeptide or antibody is released into the intestine after passing through the stomach. Such formulations are described in U.S. Patent No. 6,306,434 and in the references contained therein.
  • Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non ⁇ aqueous vehicles (which may include edible oils), or preservatives.
  • the chemokine heterodimers can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampules, prefilled syringes, small volume infusion containers or multi-dose containers with an added preservative.
  • the pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical compositions suitable for rectal administration can be prepared as unit dose suppositories. Suitable carriers include saline solution and other materials commonly used in the art.
  • chemokine heterodimers can be conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • chemokine heterodimers may take the form of a dry powder composition, for example, a powder mix of a modulator and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.
  • chemokine heterodimers may be administered via a liquid spray, such as via a plastic bottle atomizer.
  • Chemokine heterodimers can be formulated for transdermal administration. Chemokine heterodimers can also be formulated as an aqueous solution, suspension or dispersion, an aqueous gel, a water-in-oil emulsion, or an oil-in-water emulsion. A transdermal formulation may also be prepared by encapsulation of a chemokine heterodimer within a polymer, such as those described in U.S. Pat. No. 6,365,146. The dosage form may be applied directly to the skin as a lotion, cream, salve, or through use of a patch. Examples of patches that may be used for transdermal administration are described in U.S. Pat. Nos. 5,560,922 and 5,788,983.
  • chemokine heterodimers For use in promoting wound healing, although systemic administration is possible, local or directed administration of chemokine heterodimers is preferred.
  • the chemokines may be added to the wound site, e.g., in the form of a cream, ointment, gel or lyophilized powder; formulated in an ingestible composition to reach an ulcer in the stomach or duodenum; or may be incorporated into a wound dressing that is applied to the wound site.
  • chemokine heterodimer required for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient. Ultimately the attendant health care provider may determine proper dosage.
  • a pharmaceutical composition may be formulated as a single unit dosage form.
  • 2D 1 H- 15 N-HSQC (heteronuclear single-quantum coherence) spectra of PF4 and PF4M2 were recorded to monitor chemical shift changes upon addition of increasing concentrations of IL8.
  • the chemical shifts are quoted in parts per million (ppm) downfield from sodium 4,4-dimethyl-4-silapentane sulfonate (DSS).
  • HSQC spectra were acquired at 40 0 C on a Varian Unity Inova 600 MHz spectrometer equipped with a H/C/N triple-resonance probe and x/y/z triple-axis pulse field gradient unit.
  • the solvent deuterium signal was used as a field- frequency lock.
  • Carrier frequencies for 15 N and 1 H were positioned at 116.5 parts per million (ppm) and 5.2 ppm, respectively.
  • a gradient sensitivity-enhanced version of 2D 1 H- 15 N HSQC was applied with 400 (tl) x 2048 (t2) complex data points and spectral widths of 2500 Hz in tl ( 13 N) and 9000 Hz in t2 ( 1 H) dimensions.
  • Raw data were converted and processed by using NMRPipe (Delaglio et al., J. Biomol. NMR 6, 277-293 (1995)) and were analyzed by using NMRview (Johnson et al., J. Biomol. NMR 4, 603-614 (1994)).
  • the diffusion coefficient, D of the molecule of interest, was estimated from the diffusion attenuation of spin-echo by using:
  • ⁇ (g 2 ) A(0)exp[- ⁇ 2 ⁇ Vz)( ⁇ - ⁇ /3)] (Eq. 1) where ⁇ is the gyromagnetic ratio for protons, ⁇ is the duration of the pulsed field gradient, and ⁇ is the spacing between the front edges of pulsed field gradient.
  • non- isotopically-enriched IL8 was titrated into a solution of uniformly 15 N-enriched PF4, and chemical shift changes in 15 N-PF4 were monitored by recording 1 H- 15 N HSQC spectra upon addition of IL8. Spectra were collected at 40 °C in an aqueous solution of 20 mM NaCl adjusted to a pH value of 5.0.
  • Figure IA shows two HSQC spectra, one with 15 N-PF4 alone (blue cross-peaks) and the other with 15 N-PF4 and IL8 at a molar ratio of 1:1 (red cross-peaks).
  • PF4 tetramers predominate (Mayo, Biochemistry, 30(4), 925-34 (1991)).
  • PF4 resonance assignments cannot be definitively made, but can be tentatively made for some residues by analogy with 1 H- 15 N HSQC spectra of homologous PF4M2 as presented in Figure IB (blue cross-peaks).
  • PF4M2 is a PF4 chimera in which the N-terminus of PF4 is replaced with the N- terminus of IL8.
  • PF4M2 is a chimera of native PF4 in which the first 11 N-terminal residues of native PF4 are substituted by the first 8 N-terminal residues of IL8.
  • PF4M2 functions biologically like native PF4 (Mayo et al., Biochemistry, 34(36), 11399-409 (1995)).
  • PF4M2 forms symmetric tetramers, greatly simplifying its NMR spectra and allowing nearly complete ! H/ 15 N resonance assignments to be made and the structure to be determined.
  • the main quaternary structural difference between PF4 and PF4M2 arises from a difference in electrostatic repulsion between N-terminal segments and other parts of the molecule, essentially resulting in formation of asymmetric and symmetric tetramers for PF4 and PF4M2, respectively.
  • the C-terminal helix in PF4M2 (residues 58 to 70) is shifted slightly relative to the ⁇ -sheet domain, leading to an increased intersubunit distance between helices in two adjacent monomers.
  • N-terminal residues in PF4M2 and IL8 are the same, mutual orientation of PF4M2 and IL8 monomers in heteroaggregates is close to the orientation of two PF4M2 monomers in its homoaggregate. In contrast, mutual orientation of the two monomers in PF4/IL8 heteroaggregates differs more from that in the native PF4/PF4 homoaggregate. Therefore, chemical shifts of C-terminal residues are observed to be more perturbed in the native PF4/IL8 heteroaggregate. The same is true for residues in the region around H23. Because most N-terminal residues in native PF4 could not be assigned, little information about chemical shift changes at the N-terminus of native PF4 could be derived. However, based on PF4M2 chemical shift data, N-terminal residues are indeed affected by addition of IL8.
  • Equation 2 which gives the total concentration of protein, can therefore be rewritten to include the concentration of heterodimers, C HD ⁇
  • K HD (hq. 8)
  • Figure 5 illustrates diffusion coefficients, D, for PF4 and PF4M2, pure and mixed with IL8, vs. total protein concentration.
  • D diffusion coefficients
  • IL8 forms only heterodimers with native PF4 as it does with PF4M2
  • native PF4/IL8 heterodimers must be less stable than PF4M2/EL8 heterodimers, and the heterodimer KD value for PF4/IL8 heterodimers must have a value larger than that for PF4M2/IL8 heterodimers.
  • Fractional populations are calculated for protein molar concentrations of 0.43 mM (PF4 or PF4M2) and 0.86 mM (IL8), when present pure in solution (left side of table) or in combination with each other (right side of table).
  • the PDB entries were IRHP, IPFN and 1IL8. Crystallographic water molecules were deleted. Hydrogen atoms were added to the crystal structure using the HBUILD module of CHARMM (Brooks et al., J. Comput. Chem. 4, 187-217 (1983)). The ionization state of the system was set at pH 5.0, the pH value used in experimental studies. At this pH value, the total charge on monomers was +9e (PF4), +1 Ie (PF4M2) and +6e (IL8).
  • Molecular dynamics (MD) simulations were performed for homodimers of IL8, PF4, PF4M2 and heterodimers of PF4/IL8 and PF4M2/IL8.
  • two types of dimers were constructed: AB-type and ⁇ -sheet sandwiched type.
  • PF4/IL8 and PF4M2/IL8 AB-type heterodimers were formed by manually replacing one of the monomer subunits from an AB-type dimer of native PF4 or PF4M2 with a monomer subunit from IL8, following superposition of PF4 or PF4M2 and IL8 homodimers.
  • PF4/IL8 and PF4M2/IL8 ⁇ -sheet sandwiched heterodimers two adjacent and overlapping monomers from each AB-type dimer in the PF4 tetramer were first removed, leaving the ⁇ -sheet sandwiched homodimer. Then, an IL8 monomer was superimposed onto one of the sandwiched monomer subunits, and that PF4 subunit was deleted. All initial structures were built using the Insight-II program (Biosym Technologies Inc., San Diego, CA).
  • heterodimers are AB-type dimers arises primarily from two pieces of evidence: correlation of chemical shift changes with structure, and dimer stability derived from molecular dynamics simulations.
  • PF4 and PF4M2 in the presence of IL8, most residues that give rise to the largest chemical shift changes lie at the interface between monomer subunits ( Figure 3).
  • IL8-induced chemical shift changes with PF4 and PF4M2 nevertheless would be expected for perturbations in both AB- type and ⁇ -sandwich-type interactions.
  • the IL8 monomer that participates in the other half of the heterodimer is not known to form dimers other than the AB-type, and PF4 and PF4M2 AB-type dimers are thermodynamically more stable than their ⁇ -sandwich-type dimers.
  • EL8 in the presence of PF4 and PF4M2, induces formation of AB-type heterodimers.
  • Heterodimers were formed by manually replacing one of the monomer subunits from an AB-type dimer of native PF4 or PF4M2 and a monomer subunit from a ⁇ -sheet sandwich dimer of PF4 with a monomer subunit from the IL8 dimer, and aligning the two subunits to maintain the same general inter-subunit orientation as that found in the respective homodimer.
  • These initial heterodimeric structures were then energy- minimized at 300 K and subjected to a 1 nanosecond (ns) molecular dynamics simulation in explicit solvent using CHARMM (Berman et al., Nucleic Acids Research, 28, 235-242 (2000)).
  • CHARMM Brunauer-et al., Nucleic Acids Research, 28, 235-242 (2000).
  • a covalently-linked, single-chain (sc) heterodimer of IL8 and PF4 was designed and constructed, with the expectation that a covalently-linked heterodimer would not be able to dissociate like endogenous IL8 and PF4 homo- and heterodimers. This could provide a more stable heterodimer in which the full activity of the heterodimer could be more readily preserved.
  • Linker biochemistry was used is based on that from Huston and Landar (Huston et al., Methods EnzymoL, 203, 46-88 (1991); Landar et al., J. MoI. Biol. 299, 169-79 (2000), to link different homodimeric structures, in particular IL8 (Leong et al., Protein ScL, 6, 609-17 (1997)).
  • This approach has not been used previously to create heterodimers as discussed herein.
  • peptide fragments [ENSGG(S) n AKELRCQC] were constructed (Leong et al., Protein Sci. 6, 609- 617 (1997).
  • These peptides consist of three amino acids from the IL8 C- terminus, the linker, and eight amino acids from the IL8 N-terminus.
  • the following procedure was used to construct the sc IL8/PF4 heterodimer.
  • 20 conformations for peptides of various lengths were generated.
  • the generated peptides contained no unfavorable contacts with the rest of the protein.
  • Each of these conformations was then refined by using restrained molecular dynamics and energy minimization. The conformations were then evaluated using Ramachandran (backbone dihedral angle) analysis.
  • the criterion for choosing the most appropriate linker sequence was that designed peptide should have no unusual backbone angles, relatively low strain energies, and a narrow range of mobility for the functionally important ELR sequence in IL8. Based on these criteria, the peptide chosen was G 2 S 6 G.
  • the IL8 amino acid sequence at the N-terminus was first connected to the PF4 sequence at the C-terminus, and then the PF4 sequence at the N-terminus was connected to the IL8 sequence at the C-terminus. Because the C-terminal helix of PF4 is shorter than the C-terminal helix of IL8, one cannot use a linker of the same length that had been used for scIL8 homodimer. Therefore, the procedure to generate a linker was repeated, as described below. To generate the linker and refine the structure, the Homology module in the program Insight II was used.
  • Linker peptides consisting of GG(S) 4 GG, GG(S) 5 GG, GG(S) 6 GG, GG(S) 7 GG, and GG(S) 8 GG were tested. For each peptide, 10 conformations were generated and refined through the sequence of Steepest-descent and Conjugate minimization, dynamics, and Conjugate minimization again. Several steps were used to allow energy convergence. Molecular dynamics steps were used to avoid local energy minima. In Figure 7, the family of generated linkers for GG(S) 6 G is exemplified. For each linker peptide, one conformation with the minimum energy was chosen out of 10. The energies for these conformations were compared, and the lowest energy structure was chosen. In Figure 8, the IL8/PF4 heterodimer connected using the GG(S) 8 G linker is shown.
  • heterodimer DNA is shuttled into bacterial (pGEX-6P; GST fusion vector from Amersham Biosciences), eukaryotic (pSecTag2 and pcDNA4 His/Max; HIS fusion vectors for mammalian expression from Invitrogen), and yeast (pPICZ ⁇ ; HIS fusion vector for secreted expression in yeast from Invitrogen) specific expression vectors for protein production and isolation by using HPLC.
  • the bacterial expression system produces the construct used in the 3 H-thymidine incorporation system using endothelial cells, as was used previously.
  • the sc IL8/PF4 heterodimer displays essentially the same activity as noted in Nesmelova et al., J. Biol.
  • HUVEC Human umbilical vein derived endothelial cells
  • EC were seeded at 5000 cells/well in flat-bottomed fibronectin coated tissue culture plates and grown for 3 days in the absence or presence of regulators, in culture medium. During the last 6 hours of the assay, the culture was pulsed with 0.3 microcuries ( ⁇ Ci) [methyl- 3 H]- thymidine/well.
  • Extracellular Proton Excretion in CD34+ Cells Extracellular proton excretion (extracellular acidification rate) was measured using Cytosensor microphysiometer (Molecular Devices) (McConnell et al., Science 257, 1906-12 (1992)).
  • Human CD34+ cells isolated from bone marrow of healthy volunteers were suspended in agarose (Agarose Entrapment Reagent; Molecular Devices, Sunnyvale, CA) at a density of 10 5 cells/l ⁇ L and placed into the center of Capsule Cups (Molecular Devices), which were then loaded into a microphysiometer.
  • PF4 was introduced to separate populations of cells at concentrations of 0 nanograms per milliliter (ng/mL), 100 ng/mL, and 10 micrograms per milliliter ( ⁇ g/mL) for 8 minutes.
  • IL-8 was introduced at a concentration of 100 ng/mL either alone or with 100 ng/mL or 10 ⁇ g/mL PF4 for 20 minutes.
  • Cells were continuously perfused with a low-buffered medium at a rate 100 microliters per minute ( ⁇ L/min). The buffer flow was periodically halted, and extracellular acidification rates were measured every 2 minutes and expressed as percent change with regard to baseline acidification rate.
  • 2 x 10 5 Baf3/hCXCR2 cells were plated in 100 microliters ( ⁇ L) of Gey' s balanced salt solution supplemented with 1 milligrams per milliliter (mg/mL) of BSA into a the upper chamber of a 96- well chemotaxis plate (NeuroProbe, Cabin John, MD) equipped with a 5 ⁇ m pore size polycarbonate filter. Varying concentrations of chemoattractants (IL8 alone or together with PF4) were added to the lower chamber in aliquots of 75 uL.
  • chemoattractants IL8 alone or together with PF4
  • PF4 and IL8 heterodimerization modulates biological function
  • PF4 is a known antiangiogenic agent, acting via inhibition of endothelial cell proliferation (Gupta et al., J. Cell Biol. 127(4), 1121-1127 (1994)).
  • PF4 normally inhibits proliferation of cultured EC by about 40-50%.
  • IL8 depending on its concentration, can be a stimulator of angiogenesis (Koch et al., Science 258, 1798-1801 (1992)).
  • endothelial cell proliferation was tested in the presence of PF4 or IL8 alone and in combination.
  • the modulation of the biological activity of IL8 associated with the presence of PF4 was assessed through the measurement of the metabolic response from CD34+ cells to IL8 and the IL8-induced migration of Baf3/hCXCR2 cells. In both assays the activity of IL8 alone and in combination with PF4 was tested.
  • IL8 stimulates a rise in intracellular calcium in a myeloid progenitor cell line, a signal typically associated with an increase in the extracellular acidification rate.
  • Figure 1OA illustrates that infusion of IL8 to CD34+ human progenitor cells isolated from bone marrow results in an increase in the acidification rate.
  • infusion of PF4 alone does not cause any increase in the acidification response of CD34+ cells at the concentrations tested (100 ng/mL and 10 ⁇ g/mL).
  • PF4 and EL8 are coinfused, the CD34+ cells response to IL8 is completely negated at the PF4:IL8 ratios investigated.

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Abstract

L'invention concerne des hétéromultimères de chimiokine notamment différents monomères de chimiokine homologues ou analogues. L'association de monomères de chimiokine différents afin de former des hétéromultimères, notamment des hétérodimères, est mise en évidence par l'analyse à résonance magnétique nucléaire et le modelage moléculaire. Les hétéromultimères de chimiokine peuvent, en outre, être stabilisés par liaison covalente de ceux-ci au moyen de brins lieurs peptidiques. Les hétéromultimères de chimiokine peuvent être utilisés afin de modifier les cellules supportant le récepteur de chimiokine et dans le traitement de troubles sensibles aux chimiokines. La formation d'hétéromultimères permet d'améliorer diverses activités biologiques des chimiokines.
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WO2008021263A2 (fr) * 2006-08-11 2008-02-21 Medarex, Inc. Procédés d'identification d'anticorps qui se lient aux formes oligomériques de chimiokines et leurs utilisations
US8207228B2 (en) 2004-10-04 2012-06-26 Regents Of The University Of Minnesota Calixarene-based peptide conformation mimetics, methods of use, and methods of making

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Cited By (11)

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US8207228B2 (en) 2004-10-04 2012-06-26 Regents Of The University Of Minnesota Calixarene-based peptide conformation mimetics, methods of use, and methods of making
US8642656B2 (en) 2004-10-04 2014-02-04 Regents Of The University Of Minnesota Calixarene-based peptide conformation mimetics, methods of use, and methods of making
US8716343B2 (en) 2004-10-04 2014-05-06 Regents Of The University Of Minnesota Calixarene-based peptide conformation mimetics, methods of use, and methods of making
WO2007105224A1 (fr) * 2006-03-16 2007-09-20 Protagonists Ltd. Combinaison de cytokines et du récepteur de cytokines destinée à modifier le fonctionnement du système immunitaire
AU2007226155B2 (en) * 2006-03-16 2014-04-03 Protagonists Ltd. Combination of cytokine and cytokine receptor for altering immune system functioning
US8703911B2 (en) 2006-03-16 2014-04-22 Symthera Canada Ltd. Cytokine receptor peptides, compositions thereof and methods thereof
US9416158B2 (en) 2006-03-16 2016-08-16 Symthera Canada Ltd. Cytokine receptor peptides, compositions thereof and methods thereof
US9931376B2 (en) 2006-03-16 2018-04-03 Symthera Canada Ltd. Cytokine receptor peptides, compositions thereof and methods thereof
US11207381B2 (en) 2006-03-16 2021-12-28 Symythera Canada Ltd. Cytokine receptor peptides, compositions thereof and methods thereof
WO2008021263A2 (fr) * 2006-08-11 2008-02-21 Medarex, Inc. Procédés d'identification d'anticorps qui se lient aux formes oligomériques de chimiokines et leurs utilisations
WO2008021263A3 (fr) * 2006-08-11 2008-04-24 Medarex Inc Procédés d'identification d'anticorps qui se lient aux formes oligomériques de chimiokines et leurs utilisations

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