Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/195—Chemokines, e.g. RANTES
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/52—Assays involving cytokines

Definitions

• the invention is directed to the use of RANTES and its analogs in the prevention and treatment of infections caused by paramyxovirus. More specifically, the invention concerns both treatment and diagnosis of paramyxoviral infection, especially in children.
• the present invention identifies an endogenous agent useful in combating this infection. As the agent is endogenous, not only does the endogenous agent provide a treatment means for this condition, it also provides a method to screen for susceptibility.
• the present invention is based on the discovery that the RANTES protein is this endogenous agent.
• RANTES is a 68 amino acid protein which is a "C-C" chemokine; other members of this family include MCP-1 and MCP-3.
• C-C chemokines are generally active on a variety of leukocytes including monocytes, lymphocytes, eosinophils, basophils, NK cells and dendritic cells. There is an extensive literature describing this secreted protein as well as its analogs. For example, Struyf, S., et al., Eur. J. Immunol. (1998) 28: 1262-1271 isolated a natural form of human RANTES lacking two N-terminal residues
• RANTES RANTES (3-68)
• RANTES (3-68) inhibited RANTES (1-68) induced chemotaxis as well as chemotaxis induced by related cytokines.
• Another RANTES antagonist, the RANTES protein extended at the amino terminus by a single methionine residue was reported by Proudfoot, A.E.I., et al, J. Biol. Chem. (1996) 271 :2599-2593. Eisner, J., et al, J. Biol. Chem.
• AOP-RANTES amino oxypentane - RANTES
• HAV human deficiency virus type I
• AOP-RANTES down regulates the CCR5 receptor but is less effective than RANTES itself in down modulation of CCRl.
• the ability of both RANTES and AOP-RANTES to inhibit HIV infection is also reported by Vila-Coro, A.J., et al, J. Immunol. (1999) 163:3037-3044.
• U.S. patents 5,705,360; 5,739,103; 5,854,412; and 5,459,128 relate to
• N-terminal deletions of C-C chemokines including RANTES. These N-terminal deleted chemokines are described as inhibiting endogenous chemokine binding and inhibiting activation of a responsive chemokine receptor.
• PCT publication WO94/07512 assigned to the University of Texas System claims a method for treatment of allergic or chronic inflammatory disease which comprises administering the RANTES protein.
• PCT publication WO97/19696 to Lusso, P., et al describes the use of RANTES and other C-C chemokines as having HIV suppressive activity; this publication claims a method of treating retrovirus infection by administering RANTES or related chemokines.
• PCT publication WO98/13495 assigned to British Biotech Pharmaceuticals describes mutants of human RANTES which are said to retain the ability to inhibit HIV infection, but have reduced pro-inflammatory properties. These mutants are disaggregated and are modified forms of human RANTES where substitution mutations at Glu-26 and Glu-66 are apparently preferred.
• N-terminally modified RANTES analogs such as N-nonyl RANTES and AOP-RANTES and their use in treatment of allergic conditions and HIV infection.
• PCT publication WO99/20759 to Genetics Institute is directed to N-terminally modified forms of a multiplicity of chemokines other than RANTES.
• Recombinant viral vectors containing a RANTES-encoding insert are described and claimed as a method to inhibit binding of an immunodeficiency virus to cells in WO99/27122 assigned to TransGene S.A. WO99/28474 assigned to the U.S. government
• DHHS is directed to RANTES (3-68) and methods to inhibit HIV infection using this protein.
• RANTES mutants which are antagonists of the interaction between HIV virus and a chemokine receptor where there is a mutation in the N-terminal region, in the N loop region, in the 40's loop region or in all three regions.
• WO99/37815 assigned to Akzo Nobel N. V. describes an isothermal transcription based amplification assay for detection or quantitation of chemokine RNA which is useful for the detection of levels of RANTES and related chemokines.
• RANTES chemokine has a significant effect on the respiratory infections caused by paramyxovirus.
• RANTES acts downstream of viral entry and signals through the specific CCRl and/or CCR5 chemokine receptors as to interrupt the death pathway of macrophages which have been infected by the virus.
• RANTES not only inhibits apoptosis of the infected macrophage, but also clears the macrophages of infection.
• the invention is directed to methods of prophylactic and therapeutic treatment of paramyxovirus infection using RANTES protein or a recombinant system for RANTES expression.
• the invention also relates to a method to detect individuals who are susceptible to paramyxovirus infection by assessing the levels of expression of RANTES or its relevant receptors (CCRl and CCR5) in such individuals or detecting defects in the genes for RANTES or its receptors. Knowledge of the interaction of RANTES with CCRl and CCR5 permits these receptors to be used to identify alternatives to RANTES as medicaments in the treatment of paramyxovirus infection.
• the invention is directed to a method to treat paramyxovirus infection which method comprises administering to a subject in need of such treatment an effective amount of RANTES protein or an effective amount of an expression system for said protein, including "naked DNA.”
• the invention is directed to a method of identifying individuals susceptible to paramyxovirus infection which method comprises assessing the level of expression of RANTES protein in said individuals, whereby an individual having an abnormally low level of such expression is identified as susceptible. Genetic testing can also be used to determine mutations in the genes encoding for RANTES and its receptors.
• the RANTES locus in humans has also recently been identified by Nickel, et al, J. Immunol. (2000) 164:1612-1616.
• the invention is directed to methods to identify medicaments which are useful in treating paramyxovirus infection by testing candidate medicaments for their ability to agonize CCRl and/or CCR5 receptors.
• the invention is directed to medicaments so identified and to methods to treat paramyxovirus infection using these medicaments.
• Figure 1 is a cartoon which compares a model of the interaction of RANTES with CCR3 as related to HIV infection with the interaction of RANTES with CCRl or CCR5 as related to paramyxovirus infection.
• Figure 2 shows the wild type RANTES genomic locus, a targeting construct to disrupt the first exon, and the resulting "null" locus.
• Figure 3 is a series of graphs comparing macrophage, PMN's, and lymphocytes at various times after infection in RANTES (+/+) and (-/-) mice.
• Figure 4 is a graph showing the survival of RANTES (+/+) compared to (-/-) mice after viral infection.
• Figure 5 is a series of graphs showing the effect of administering RANTES on viral load and cell death in infected mice.
• the present invention centers on the discovery that the RANTES protein is able to prevent macrophage cell death and to enhance clearance of virus in infected subjects thus permitting the treatment of paramyxovirus infection using this protein or an expression system therefor. Because it is clear that RANTES protein is an endogenous defense with respect to paramyxovirus infection, it is also possible to screen populations for susceptibility to paramyxovirus infection by determining their status relative to endogenous production of effective RANTES protein. Thus, the present invention is directed to methods of treatment of paramyxovirus infection and methods to determine susceptibility to this infection. The latter is important as individuals identified as susceptible can be subjected to precautionary conditions and can be monitored for early treatment.
• treat or “treatment” refers both to therapeutic and prophylactic administration of the active ingredient.
• susceptible individuals known to be in situations where they are exposed to paramyxovirus can be administered the
• RANTES protein or appropriate DNA or an individual already infected can be therapeutically administered these materials.
• RANTES protein is meant either the naturally occurring known human 68 amino acid protein or an analog thereof which retains the ability to activate the CCRl and/or CCR5 receptor.
• RANTES protein there are a number of analogs of the RANTES protein known in the art which behave as antagonists to the native RANTES protein. These analogs are believed to be ineffective in the method of the invention; rather analogs which retain the ability to activate the CCRl and/or CCR5 receptor are included in the definition of "RANTES protein” herein.
• useful analogs are allelic variants which retain CCR5 activation activity and synthetically prepared modifications of the 68-mer.
• RANTES variants could be screened in a macrophage cell culture system or a transfected cell line that expresses the chemokine receptors CCRl or CCR5. Receptor activation could be detected by calcium flux, chemotaxis, or apoptosis-inhibition assays. RANTES variants could be generated by targeted or random mutagenesis designed to generate compounds with receptor-activating properties.
• the same assay systems are used to screen for small molecules other than peptides that selectively activate the RANTES signaling pathway for blocking apoptosis due to viral infection and for clearing the infection.
• a chemokine receptor antagonist has been identified with a similar approach (Liang, et al, J. Biol. Chem. (2000) 270:19000-19008). After candidates are identified, their therapeutic action may be verified in a mouse model of viral bronchitis.
• a candidate medicament can be screened for its agonist ability with respect to the critical CCRl and/or CCR5 receptor in a manner similar to that used above to confirm the agonist activity of analogs of the RANTES protein.
• medicaments are small molecules, although peptides could also be employed.
• small molecules is meant the typical type of organic molecule generally found in pharmaceuticals and, in most cases, amenable to oral administration as indigestible in the digestive tract. Such small molecules are typically non-polymers, unlike peptides or polysaccharides.
• the RANTES peptide or small molecule agonist may be used perse, or an expression system for the RANTES protein may be administered.
• expression system is meant either a nucleotide sequence encoding the RANTES protein under control of additional sequences to effect its expression administered in a suitable vector or naked DNA - i.e., a nucleotide sequence encoding the RANTES protein administered in such a manner that delivery into the hosts' cells is able to effect expression.
• RANTES protein As paramyxoviral infection is a respiratory infection, localized administration of the protein or the expression system for the encoding DNA is preferred.
• the dosage administered is designed to reproduce therapeutic levels of RANTES protein in the airway macrophages; the levels will be roughly equivalent to physiologic levels of 1 ng/ml in culture.
• a preferred mode of administration is through an aerosol designed to obtain the appropriate levels in airway macrophage.
• Formulations for aerosols are well-known in the art and exemplary formulations may be found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA, the contents of which are incorporated by reference as they describe such formulations.
• an aerosol formulation using standard excipients, and containing RANTES, RANTES variant, or small molecule RANTES agonist is administered through an inhaler.
• Typical protocols would employ 1 to 2 inhalations at a single dosage and the dosage would be adjusted to achieve maximal efficacy to prevent infection or to treat it after it has developed. Dosage would be determined by measurements of RANTES levels in cell culture and mouse model systems and by efficacy trials in these same model systems.
• nucleotide sequence encoding said RANTES or RANTES variant protein may also be introduced into the subject.
• vectors for administration of nucleic acids include viral vectors such as adenovirus, retrovirus, Sendai virus, and the like.
• viral vector carriers typically designed specifically for this purpose, are known in the art, and one study using a RANTES vector (Braciak, T.A., et al, J. Immunol (1996) 157:5076- 5084) and one using RANTES -encoding DNA vaccine (Youssef, S., et al, J. Clin. Invest. (2000) 106:361-367) have been reported .
• RANTES As shown hereinbelow, the effect of RANTES on paramyxoviral infection is different in mechanism from the known ability of RANTES protein or its antagonists to inhibit HIV infection. This is illustrated schematically in Figure 1.
• RANTES In inhibiting HIV, as shown on the left, in addition to simply blocking the interaction of HIV with the CCR5 receptor, RANTES interacts with the CCR3 receptor on cytotoxic T lymphocytes to stimulate the production of FasL and induces consequent Fas-dependent killing of infected cells.
• paramyxovirus stimulates the production of RANTES by infected host cells, such as airway epithelial cells and macrophage, which RANTES then binds to CCRl and/or CCR5 on infected macrophage.
• the invention includes methods to detect individuals who are susceptible to paramyxoviral infection.
• the ability of the individual to express the nucleotide sequence encoding the RANTES protein is determined by assessing levels of RANTES protein in, for example, airway epithelial cell and macrophage samples using standard detection techniques, for example, by ELISA or other immunoassay of bronchoalveolar or nasal lavage or serum obtained at the time of infection.
• the levels of RANTES encoding mRNA can be determined using, for example, kinetic PCR or the isothermal transcription based assay described in WO99/37815 noted above, and incorporated herein by reference using epithelial cells and macrophages obtained by airway lavage or using circulating leukocytes.
• the individual may be subjected to standard methods of genetic testing to determine the presence of mutations in the RANTES locus as identified recently (Nickel, et ⁇ ., J. Immunol (2000) 164:1612-1616).
• mice containing a RANTES null locus were generated using a replacement vector derived from a 6.6 kb Apa 1-Xba I fragment isolated from a lambda FixII library prepared from mouse strain 129/Sv genomic DNA (Stratagene), as shown in Figure 2. Exons are shown as open boxes. A334 Bgl II fragment spanning the transcription and translation start sites in exon 1 (-267 to 67) was replaced with a hygromycin-resistance cassette (Hygro from B. Jones, Princeton, NJ) in opposite direction to the native gene. An upstream diphtheria toxin A cassette (DTA) was also added for negative selection. Linearized vector was transfected into El 4 strain 129 embryonic stem cells, and after selection in hygromycin, targeted clones were injected into C57BL/6J blastocysts which were implanted to generate chimeric mice.
• DTA diphtheria toxin A cassette
• Chimeric males were mated with C57BL/6J female mice and transmission of RANTES null allele was verified by Southern blot analysis of tail DNA.
• RANTES deficiency was initially verified by Northern blot analysis and ELISA of peritoneal exudative cells stimulated with LPS.
• RANTES (+/-) mice from the third backcross into the C57BL/6J strain were used to generate (+/+) and (-/-) mice that were characterized by PCR of genomic tail DNA using primers (5'-GGGAACTTCCTGACTAGGGG-3'; 5'-CTGGACTGGAGGGCAGTTAG-3'; and 5'-AGTGAGGATGATGGTGAGGG-3') corresponding to RANTES sequence in the null, wild-type, and both null and wild-type alleles, respectively.
• mice were maintained under pathogen-free conditions in the University biohazard barrier facility in micro-isolator cages for study at 7-9 weeks of age. Sentinel mice (specific pathogen-free ICN strain) and experimental control mice were handled identically to inoculated mice and exhibited no serologic or histologic evidence of exposure to
• Sendai virus (SdV, strain 52) and culture vehicle (allantoic fluid) were obtained from ATCC and stored at -70°C. After anesthesia, mice were inoculated intranasally with either 5,000 EID 50 (bronchitis) or 50,000 EID50 (broncho-pneumonia) (50% egg infectious dose, EID 50 ) of SdV or with UV-inactivated SdV or culture vehicle diluted in 30 ⁇ L PBS. The expression of RANTES was measured both by assessing lung tissue levels of RANTES mRNA and by immunostaining of lung tissue. Northern blot analysis used lung RNA isolated with RNA STAT-60 from TEL-TEST, Inc. (Friendswood, TX) and
• RANTES riboprobe was synthesized using the 0.5-kb RANTES cDNA fragment positionally cloned into the EcoRI and Xhol sites of pG ⁇ M3Zf-l (Promega). Plasmid DNA was linearized either with Apal or with BamEl and was transcribed in vitro with either T3 or T7 RNA polymerase to produce
• RANTES (+/+) and (-/-) mice were inoculated with 5,000 EID 50 SdV as described in Example 1 and BAL fluid was obtained by tracheal cannulation with 4 aliquots of 0.8 ml sterile PBS with 2% FBS. The BAL fluid was subjected to hopotonic lysis and centrifugation. The cell pellet was then used for determining total and differential cell counts as the mean of values from 3 blinded observers. The results are shown in Figure 3, where the open bars represent (+/+) and the dark bars represent (-/-) mice. As seen, macrophage levels in both cases reach a maximum about 8 days after infection and lymphocytes increase through day 12. However, the (-/-) mice showed a decreased number of immune cells in the airspace as well as an accumulation of immune cells in the sub- epithelium.
• lungs from post-infection days 3, 5, 8 and 12 were immunostained with anti-macrophage (Mac-3) monoclonal antibody and avidin-biotin horseradish peroxidase 3,3'-diaminobenzidine reporter and counterstained with hematoxylin.
• the immunostaining showed that macrophage accumulated in the sub-epithelium at day 5, in the epithelium at day 8, and in the airspace by day 12 post-infection in the (-/-) mice.
• RANTES (-/-) mice showed more virus in the epithelium than the (+/+) mice and were more lethargic, lost more weight, and had worse airway function.
• mice were inoculated at 50,000 EID 50 .
• Serial lung sections were tested for viability by the TUNEL cell death assay which employs TdT-mediated dUTP Nicked End Labeling using the ApoTag Plus Fluorescein In Situ Apoptosis Detection kit from Intergen (Purchase, NY).
• the results indicate enhanced survival of lung tissue cells in (+/+) mice compared to in (-/-) mice.
• lungs were subjected to Western blotting against anti-SdV antibodies and detection by enhanced chemiluminescence.
• Macrophage cultures were prepared from RANTES (+/+) and (-/-) mice and incubated with 5,000 EID 50 or 50,000 EID 5 o SdV for 4 days. The cultures were stained with anti-SdV antibody and CY3 reporter (red fluorescence) and counterstained with
• the top row of graphs in Figure 5 shows that the level of infection after 8 days is roughly the same in both (+/+) and (-/-) mice; however, the addition of RANTES to the medium clearly shows that infection was cleared after 8 days. The clearing appears to commence even after day 4.
• the bottom row of graphs indicates that both RANTES (+/+) derived macrophage and macrophage treated with RANTES (10 ⁇ g/ml) resists apoptosis while macrophage derived from (-/-) mice and not treated with RANTES undergo substantial cell death even after day 4 and increasingly through day 8.
• RANTES acts via chemokine receptor mediated signaling via CCR5 to impact virus induction of mitochondrial permeability.

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