EP1613736A2 - Inhibition of viral pathogenesis - Google Patents
Inhibition of viral pathogenesisInfo
- Publication number
- EP1613736A2 EP1613736A2 EP04775837A EP04775837A EP1613736A2 EP 1613736 A2 EP1613736 A2 EP 1613736A2 EP 04775837 A EP04775837 A EP 04775837A EP 04775837 A EP04775837 A EP 04775837A EP 1613736 A2 EP1613736 A2 EP 1613736A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- erb
- viras
- tyrosine kinase
- cell
- ligand
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
-
- 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
-
- 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
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
Definitions
- This invention relates to control of viral infection, and more particularly to control of poxvirus (e.g., smallpox) infection.
- poxvirus e.g., smallpox
- Example 2 describes poxvirus proteins with relatively high homo logy to the mammalian protein epiregulin (EPI), especially in their epidermal factor (EGF)-like domains. These poxvirus proteins are designated herein epiregulin-like growth factors (ELGF).
- EPI mammalian protein epiregulin
- ELGF epiregulin-like growth factors
- SPGF smallpox growth factor
- VEF vaccinia growth factor
- the invention is based in part on the observations that binding of the recombinant epidermal growth factor (EGF)-like domain of D4R (the SPGF from the Bangladesh strain of variola major) to cell-surface erb-B 1 receptors results in: (a) activation of the tyrosine kinase activity of such receptors in cells expressing them; (b) enhanced proliferation and/or survival of such cells; (c) rapid intemalization of erb-B 1 receptors; and (d) viral pathogenesis resulting from enhanced replication and attendant inflammation.
- EGF epidermal growth factor
- a SPGF (e.g., on the surface of a variola virion or shed from a variola virion) plays a central.role in variola virus entry into, and replication in, host cells and subsequent inflammation.
- the inventors have found that protein tyrosine kinase activity stimulated by binding of the SPGF to cells via the erb-B 1 receptor and intemalization of the erb-B 1 receptor subsequent to ligation by the SPGF are inhibited by erb-B protein tyrosine kinase inhibitors. This inhibition of erb-B 1 intemalization was apparently due to inhibition of the inducible association between c-Cbl and erb-B 1.
- CI-1033 a quinazoline protein tyrosine kinase inhibitor, inhibited clinical symptoms and decreased viral load in animals infected with vaccinia vims.
- This therapeutic effect of CI-1033 was enhanced by co-administration of a neutralizing vaccinia-specific monoclonal antibody (mAb) (anti-LlR iriAb).
- mAb neutralizing vaccinia-specific monoclonal antibody
- an erb- B tyrosine kinase inhibitor decreased transmission of variola vims from one cell to another.
- CI-1033 also enhanced T cell-mediated immunity to vaccinia vims in the infected animals and that this enhancement was further increased by co- administering the anti-LlR mAb to the infected animals.
- a mAb that binds to NGF the vaccinia ortholog of SPGF
- inhibitors of erb-B tyrosine protein kinase activity can be effective agents against poxvirus infection of animals (e.g., human subjects). These inhibitors appear to act by inhibiting , at least, viral replication in cells and viral egress from cells as well as by enhancement of immunity to the vims. The inhibitors may also reduce viral entry into cells.
- the invention features a method that includes: (a) identifying an animal as likely to have been, or as likely to be, exposed to a vims, the vims containing a gene encoding a viral erb-B ligand; and (b) treating the animal with a compound that inhibits the activity of an erb-B tyrosine kinase or inhibits the activation of an erb-B tyrosine kinase.
- the animal can be identified as having been infected, before, during or after step (b), with the vims.
- the viral erb- B ligand can be a poxvims erb-B ligand such as an orthopox vims (e.g., variola major, variola minor, monkeypox vims, or vaccinia) erb-B ligand.
- the erb-B ligand can be an epiregulin-like growth factor (ELGF) such as smallpox growth factor (SPGF) or vaccinia growth factor (NGF) and the erb-B tyrosine kinase can be erb-B 1, erb-B2, or erb-B4 tyrosine kinase.
- ELGF epiregulin-like growth factor
- SPGF smallpox growth factor
- NTF vaccinia growth factor
- the erb-B tyrosine kinase can be erb-B 1, erb-B2, or erb-B4 tyrosine kina
- the compound can be, for example, a non-agonist antibody that binds to the erb-B tyrosine kinase or a non-agonist erb-B ligand or a non-agonist fragment of an erb-B ligand.
- the compound can be a small molecule erb-B tyrosine kinase inhibitor such as, for example, a quinazoline-based compound (e.g., 4-anilinoquinazoline), a pyridopyrimidine-based compound, a quinoline-3-carbonitrile-based compound, or a pyrrolopyrimidine-based compound.
- the small molecule erb-B tyrosine kinase inhibitor can be an irreversible or a reversible inhibitor of the erb-B tyrosine kinase activity.
- a quinazoline-based compound can be, for example, CI- 1033, PD168393, PD160678, PD160879, PD174265, PD153035, ZD1839, GW572016, GW974,
- a pyridopyrimidine-based compound can be, e.g., PD69896, PD153717, or PD158780, a quinoline-3-carbonitrile-based compound can be, e.g., EKB-569, and a pyrrolopyrimidine-based compound can be, e.g., CGP59326A.
- the animal can be a human.
- the method can further comprise administering to the animal an antibody that has the ability to substantially neutralize one or more forms of the vims, e.g., an antibody that binds to the intracellular mature virion (LMV) form, the extracellular enveloped vims (EEN) form, and/or the cell-associated envelope vims form (CEN) of an orthopox vims (e.g., variola major, variola minor, vaccinia, or monkeypox vims).
- LMV intracellular mature virion
- EEN extracellular enveloped vims
- CEN cell-associated envelope vims form
- treatment with the compound can enhance an immune response to the vims in the animal.
- the immune response can be a T cell response, e.g., a CD8+ T cell response or a CD4+ T cell response, and the T cell response can be an interferon- ⁇ (IF ⁇ - ⁇ )- producing T cell response.
- the immune response can be an antibody-producing B cell response.
- the vims can be a poxvims expression vector and can further contain: (a) a heterologous nucleic acid sequence encoding an immunogen; and (b) a transcriptional regulatory element (TRE), the TRE being operably linked to the heterologous nucleic acid sequence.
- the poxvims expression vector can be a vaccinia vims expression vector, an attenuated vaccinia vims expression vector, a canarypox vims vector, or a fowlpox vims vector.
- the invention also features a method that includes: (a) identifying an animal susceptible to infection by a vims that comprises a gene encoding an erb-B ligand; and (b) treating the animal with (i) an antibody that substantially neutralizes one or more forn s of the vims or a vaccine that stimulates an immune response against the vims and (ii) a compound that inhibits an erb-B tyrosine kinase or inhibits the activation of an erb-B tyrosine kinase.
- the vims, the animal, the erb-B ligand, the compound, and the antibody that substantially neutralizes one or more forms of the vims can be any of those described above.
- the method can enhance any of the immune responses recited above and the vims can be any of the poxvims expression vectors described above.
- Another aspect of the invention is an in vitro method that includes: (a) providing an isolated compound that binds to an erb-B tyrosine kinase and inhibits the activity of an erb-B tyrosine kinase or activation of an erb-B tyrosine kinase; (b) contacting the compound with a cell that expresses the erb-B tyrosine kinase; and (c) before, simultaneous with, or after step (b), contacting the cell with a viral erb-B ligand or a functional fragment of the ligand.
- the compound can reduce activation of the erb-B tyrosine kinase on the cell by the ligand or fragment.
- the method can further include determining whether the compound reduces activation of the erb-B tyrosine kinase on the cell by the ligand or fragment.
- the viral erb-B ligand, the erb-B tyrosine kinase, and the compound can be any of those described above.
- the invention also provides a method of determining whether a compound is an antiviral compound.
- the method includes: (a) providing a compound that inhibits the activity of an erb- B tyrosine kinase or inhibits activation of an erb-B tyrosine kinase; (b) administering the compound to an animal susceptible to infection with a vims that contains a gene encoding an erb-B ligand; (c) before, during, or after step (b), exposing the animal to the vims; and (d) determining whether the compound reduces a symptom of viral infection in the animal.
- the vims can be a poxvims, e.g., an orthopoxviras such as variola major, variola minor, monkeypox vims, or vaccinia.
- the animal, the erb-B ligand, and the compound can be any of those described above.
- Yet another method featured by the invention involves: (a) identifying an animal susceptible to infection by a vims that comprises a gene encoding an erb-B ligand; and (b) treating the animal with an (i) antibody that binds to the ligand and (ii) an antibody that substantially neutralizes one or more forms of the vims or a vaccine that stimulates an immune response against the. vims.
- the animal can be identified as having been infected, before, during or after step (b), with the vims.
- the treatment can enhance an immune response to the vims and the immune response can be any of those listed above.
- the vims can be a poxvims expression vector and can further contain: (a) a heterologous nucleic acid sequence encoding an immunogen; and (b) a transcriptional regulatory element (TRE), the TRE being operably linked to the heterologous nucleic acid sequence.
- the poxvims expression vector can be a vaccinia vims expression vector, an attenuated vaccinia vims expression vector, a canarypox vims vector, or a fowlpox vims vector.
- the antibody that binds to the ligand can be the 13E8 monoclonal antibody (ATCC accession no. PTA-5040) or the 11D7 monoclonal antibody (ATCC accession no. PTA-5039).
- the vims, the animal, the erb-B ligand, the compound, and the antibody that substantially neutralizes one or more forms of the vims can be any of those described above.
- Polypeptide and “protein” are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification. Polypeptides for use in the invention include those with conservative substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.
- variant polypeptides with conservative substitutions will contain no more than 40 (e.g., no more than: 35; 30; 25; 20; 15; 13; 11; 10; nine; eight; seven; six; five; four; three; two; or one) conservative substitution(s). All that is required is that the variant polypeptide have at least 20% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; 99.8%; 99.9%; or 100% or more) of the activity of the wild-type polypeptide.
- activation of a cell means triggering enhanced proliferation of the cell or enhanced survival of the cell.
- Enhanced proliferation of the cell or enhanced survival of the cell means increased relative to: (a) a lower level of cell proliferation or cell survival; or (b) substantially no cell proliferation or cell survival.
- enhancing an immune response means increasing an immune response relative to: (a) a lower immune response; or (b) substantially no immune response.
- isolated compound refers to a compound (e.g., a protein) that either has no naturally-occurring counterpart or has been separated or purified from components which naturally accompany it, e.g., in tissues such as pancreas, liver, spleen, ovary, testis, muscle, joint tissue, neural tissue, gastrointestinal tissue or tumor tissue, or body fluids such as blood, serum, or urine.
- a naturally occurring biological compound is considered “isolated” when it is at least 70%, by dry weight, free from proteins and other naturally-occurring organic molecules with which it is naturally associated.
- a preparation of a compound for use in the invention is at least 80%, more preferably at least 90%, and most preferably at least 99%, by dry weight, that compound.
- the degree of isolation or purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC (high pressure liquid chromatography) analysis.
- Isolated compounds, and additional agents useful for the invention can be obtained, for example, by: (i) extraction from a natural source (e.g., from tissues or bodily fluids); (ii) where the compound is a protein, by expression of a recombinant nucleic acid encoding the protein; or (iii) by standard chemical synthetic methods known to those in the art.
- a natural source e.g., from tissues or bodily fluids
- a protein that is produced in a cellular system different from the source from which it naturally originates is "isolated," because it will necessarily be free of components which naturally accompany it.
- FIG. 1 is a photograph of a Coomassie blue-stained SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel of recombinant forms of the epidermal growth factor (EGF)-like domains of murine epiregulin (“Epi”), human EGF, and variola major Bangladesh strain smallpox growth factor (“SPGF”). These recombinant proteins are referred to herein as recEGF, recEPI, and recSPGF, respectively.
- the positions on the gel of molecular weight (MW) markers are indicated on the left side of the photograph, the values shown being MW x 10 "3 .
- Fig. 2 is a depiction of amino acid sequence alignments (for optimum homology) of human epiregulin (SEQ ID NO:6) and mouse epiregulin (SEQ ID NO:7) and the orthopox viral orthologs D1L (SEQ LD NO:l), CMP11R (SEQ ID NO:3), CI 1R (SEQ ID NO:4), and D3R (SEQ LD NO:5) from variola major strain India, camelpox, vaccinia, and monkeypox vimses, respectively.
- the regions corresponding to leader sequences and transmembrane domains are indicated and the most conserved segments of the EGF-like domains are boxed and labeled "EGF".
- Figs. 3A and 3B are depictions of the amino acid sequences of the SPGF of variola major strain India (D1L) (SEQ ID NO:l) and variola major strain Bangladesh (D4R) (SEQ TD NO:2), respectively. Positions at which the two sequences differ are shown in bold face and underlined.
- Fig. 4 is a line graph showing the percentages of human primary fibroblasts (SC-J) in the S -phase of the cell cycle after 18 hours of culture in the indicated concentrations of recEGF (“EGF”), recEPI (“EPI”), and recSPGF (“SPGF”).
- Fig. 5 is bar graph showing the proliferation of primary human keratinocytes (“N keratinocyte”) and primary human fibroblasts (“R2F fibroblast”) during culture in either standard tissue culture medium containing 10% fetal bovine serum (FBS) ("Complete”) or in tissue culture medium containing 2% FBS and the indicated concentrations of recSPGF (“SPGF”) or recEGF (“EGF”).
- FBS fetal bovine serum
- FIG. 6 is a pair of photographs of western blots of immunoprecipitates from MB453 cells ("453") or MB468 cells (“468").
- the immunoprecipitates were prepared by incubating the cells with biotinylated recSPGF, exposing the cells to the cross-linking agent BS 3 (in order to crosslink biotinylated recSPGF molecules bound to erb-B receptors on the surface of the cells), lysing the cells, and immunoprecipitating ("IP”) aliquots of the lysates with antibodies specific for the four erb-B receptors, i.e., erb-B 1 ("B 1 "), erb-B2 ("B2”), erb-B3 ("B3"), or erb-B4 ("B4").
- Fig. 7 is a line graph showing the binding to MB468 cells ("on 468") of the indicated amounts ("ng") of biotinylated recSPGF (detected with phycoerythrin (PE)-conjugated streptavidin) ("SPGF") in the absence (open circles) and presence ("+mAb”; closed circles) of a blocking antibody specific for erb-B 1.
- the data are presented as mean fluorescence intensities ("MFI"), which were determined by fluorescence flow cytometry (FFC). Binding of the recSPGF to MB453 cells ("on 453”), which do not express erb-Bl, was also tested ("on 453"; open triangles).
- Fig. 8 is a line graph showing the relative ability (expressed as % inhibition) of the indicated concentrations of recSPGF ("SPGF”), recEPI (“EPI”), and recEGF (“EGF”) to inhibit the binding of biotinylated recSPGF to MB468 cells.
- Figs. 10A-D are a series of fluorescence photomicrographs showing the cellular distribution of erb-Bl receptors (as detected with a mouse antibody specific for erb-Bl receptors and fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ig antibody) on HeLa cells that either were unstimulated ("(-)"; Fig. 1 A) or were stimulated for 10 minutes with either recSPGF ("SPGF”; Fig. 1 OB), recEPI (“EPI”: Fig, 10C), or recEGF (“EGF”; Fig. 10D).
- Fig. 11 A is a photograph of a western blot of whole cell lysates of HeLa cells that were either untreated (“(-)") or were stimulated with recSPGF ("SPGF”), recEPI (“EPI”), or recEGF ("EGF”).
- the western blot was stained with a mAb specific for phosphotyrosine residues ("WB: 4G10").
- WB: 4G10 The positions on the gel of molecular weight (MW) markers are shown on the left of the photograph, the values shown being MW x 10 "3 .
- IB is a photograph of a western blot of immunoprecipitates of cell lysates of HeLa cells that were either untreated ("(-)") or were stimulated with recSPGF ("SPGF”), recEPI ("EPI”), or recEGF ("EGF”).
- the immunoprecipitates were prepared with an antibody specific for erb-Bl ("IP: EGFR”).
- IP EGFR
- the western blot was stained with a mAb specific for phosphotyrosine residues ("WB: 4G10").
- Figs. 12A-12D are depictions of: the generic chemical structure of 4-anilinoquinazoline compounds ("Generic 4-anilinoquinazoline”; Figure ID) showing the numbering of relevant atoms; and three specific 4-anilinoquinazoline compounds, PD153035 (Fig. 12A), PD168393 (Fig. 12B), and CI-1033 (Fig. 12 C).
- Fig. 13 is a photograph of a western blot of whole cell lysates of HeLa cells that were untreated ("(-)") or were stimulated with recSPGF ("SPGF") either without pre-exposure to an erb-B protein tyrosine kinase inhibitor ("(+)") or with pre-exposure to one of three erb-B protein tyrosine kinase inhibitors, PD153035, PD158780, and PD168393.
- the western blot was stained with a mAb specific for phosphotyrosine residues.
- the positions on the gel of molecular weight (MW) markers are shown on the left of the photograph, the values shown being MW x 10 "3 .
- Fig. 14A is a line graph showing the percentages of human primary fibroblasts (SC-J) in the S-phase of the cell cycle after: no pretreatment ("SPGF alone") or pretreatment with either PD153035, CI-1033, or PS168393; and stimulation overnight with the indicated concentrations of recSPGF ("ng/ml").
- 14B is a photograph of a western blot of whole cell lysates of HeLa cells that were either untreated (“(-)") or were stimulated with recSPGF ("SPGF") without pre-exposure to an erb-B protein tyrosine kinase inhibitor ("(+)”) or with pre-exposure to the Src family protein tyrosine kinase inhibitor PP2 or the the erb-B protein tyrosine kinase inhibitors PD153035,
- PD168393 or CI-1033 The western blot was stained with a mAb specific for phosphotyrosine residues ("WB: P-Y (4G10)").
- WB: P-Y (4G10) The positions on the gel of molecular weight (MW) markers are shown on the left of the photograph, the values shown being MW x ION
- the arrows on the right side of the photograph indicate the positions of proteins phosphorylation of which was affected by erb-Bl protein tyrosine kinase inhibition.
- Fig. 14C is a bar graph showing the level of expression (in MFI determined by FFC) of erb-B 1 receptors on A431 cells after: pretreatment with no inhibitor or pretreatment with PP2, PD153035, PD168393 or CI-1033; and a 5 minute incubation with biotinylated recSPGF.
- Fig. 14D is a series of photographs of western blots of immunoprecipitates ("IP") of cell lysates of HeLa cells that were either unpretreated (“(+)”) or pretreated with PP2, PD153035, PD168393 or CI-1033 and then stimulated for 5 minutes with recSPGF. A control in which the cells were neither pretreated nor stimulated recSPGF (“(-)”) was also performed.
- the immunoprecipitates ("IP”) were prepared with either an antibody specific for erb-Bl ("EGFR”) or an antibody specific for c-Cbl (“c-Cbl”).
- the western blots were stained ("WB") with mAbs specific for erb-Bl ("EGFR”; upper panel) or c-Cbl (middle and lower panel).
- Fig. 15 A is a line graph showing the survival (in percentages of mice in the various treatment groups) over time of mice that were challenged intranasally with vaccinia vims and either untreated ("Control”; open circles), pretreated once with anti-LlR mAb ("anti-LlR”; closed circles), treated daily with CI-1033 ("CI-1033"; open triangles), or pretreated once with anti-LlR mAb and treated daily with CI-1033 ("CI-1033 + anti-LlR”; closed inverted triangles).
- Fig. 15B is a line graph showing the clinical score (derived as described in Example 1) of the mouse experimental groups described for Fig. 15 A. The same symbols used in Fig. 15 A are used to indicate the various experimental groups.
- Fig. 15 A is a line graph showing the survival (in percentages of mice in the various treatment groups) over time of mice that were challenged intranasally with vaccinia vims and either untreated ("Control”; open circles), pretreated once with anti-
- 15C is a bar graph showing the average weight of a whole lung from mice six days after a lethal challenge with vaccinia vims and either no treatment ("Control"), pretreatment with anti-LlR mAb ("anti-LlR”), daily treatment with CI-1033 (“CI-1033”), or one pretreatment with anti-LlR mAb and daily treatment with CI-1033 ("CI-1033 + anti-LlR”).
- Control no treatment
- anti-LlR anti-LlR mAb
- CI-1033 daily treatment with CI-1033
- CI-1033 + anti-LlR anti-LlR
- 15D is a bar graph showing the average viral titer (in "Log 10 PFU”; PFU, plaque forming units) in the lungs of mice eight days after infection with vaccinia vims and either one pretreatment with anti-LlR mAb ("anti-LlR”), daily treatment with CI-1033 ("CI-1033”), one pretreatment with the 13E8 mAb, one pretreatment with anti-LlR mAb and the 13E8 mAb, or one pretreatment with anti-LlR mAb and daily treatment with CI-1033 ("CI-1033 + anti-LlR”).
- mice were infected with vims but received no pretreatment or treatment, was from a separate experiment in which the mice were sacrificed on day 7 since all in the mice in the control group in the present experiment were dead on day 8.
- Fig. 16A is a photograph of an autoradiogram from an Rnase protection assay using RNA prepared from the lungs of mice: that were either uninfected ("0”); on the indicated days (“4", “6”, or “8") after infection with vaccinia vims. Infected animals were either untreated ("Control”), pretreated once with anti-LlR mAb ("anti-LlR”), treated daily with CI-1033 ("CI- 1033”), or pretreated once with anti-LlR mAb and treated daily with CI-1033 ("CI-1033 + anti- L1R”).
- RNA was hybridized with the cytokine-specific probes in the mCK-2b template set (BD Biosciences-Pharmingen, San Diego, CA) as indicated on the left side of the photograph. Protected fragments were resolved on a DNA sequencing gel, which was then exposed to x-ray film. Cytokine mRNA whose levels were increased by CI-1033 (alone or in combination with anti-LlR mAb) are indicated by arrows on the right side of the photograph. Fig.
- 16B is a series of two-dimensional FFC profiles showing the levels of intracellular interferon- ⁇ ("IFN- ⁇ (log)”) in, and levels of cell surface CD44 ("CD44 (log)”) on, mouse spleen CD8 + cells that had been stimulated in vitro with: normal MC57G cells ("MC57G”); or vaccinia vims-infected MC57G cells ("NV-infected MC57G”).
- IFN- ⁇ (log) intracellular interferon- ⁇
- CD44 (log) cell surface CD44
- mice that had been infected with vaccinia vims (5 days prior to sacrifice and assay) and were untreated ("Control"), pretreated once with anti-LlR mAb ("anti-LlR”), treated daily with CI-1033 (“CI-1033”), or pretreated once with anti-LlR mAb and treated daily with CI-1033 ("CI-1033 - anti-LlR”).
- the data shown are from a representative mouse from each experimental group of mice.
- Fig. 17 is a series of two-dimensional FFC profiles showing the levels of intracellular interferon- ⁇ ("LF ⁇ - ⁇ (log)”) in, and levels of cell surface CD44 ("CD44 (log)”) on, mouse spleen CD8 + cells that had been stimulated in vitro: with normal MC57G cells ("MC57G”); vaccinia vims-infected MC57G cells ("VN-infected MC57G”); or anti-CD3 mAb ("anti-CD3").
- LF ⁇ - ⁇ (log) intracellular interferon- ⁇
- CD44 cell surface CD44
- the data shown are from a representative mouse from each experimental group of mice.
- Fig. 18A is a series of photographs showing the immunohistochemical staining of variola strain Solaimen plaques obtained by culturing BSC-40 cell monolayers in either the absence ("-") or presence ("+") of the indicated concentrations of CI-1033.
- Fig. 18B is a bar graph showing the effect of CI-1033 on the number of plaques obtained by culturing BSC-40 cell monolayers in either the absence ("0") or presence of the indicated concentrations of CI-1033.
- Fig. 18C is a bar graph showing the number of comets obtained by culturing BSC-40 cell monolayers in either the absence ("0") or presence of the indicated concentrations of CI-1033.
- the experimental findings outlined above in the Summary section indicate that compounds that inhibit either erb-B 1 protein tyrosine kinase activity or the stimulation of erb-B 1 protein tyrosine kinase activity can be effective in treatment of, and/or prophylaxis against, orthopox vims-mediated pathogenesis (e.g., smallpox or vaccinia infection).
- the invention is not limited by any particular mechanism of action, it is believed that such compounds may act by inhibiting entry of vims particles into a host cell, intracellular viral replication, and/or expulsion of the vims from the cell, and by enhancing adaptive T cell immunity to the vimses.
- the genomes of orthopox vimses contain genes that encode immunosuppressive factors, it is very possible that the inhibition of viral replication and enhancement of adaptive immunity are functionally related.
- erbB receptors in the infection of cells by, for example, hepatitis B vims, Epstein-Barr vims, RNA tumor vimses (e.g., avian erythroblastosis vims), cytomegalovirus (CMV), and reoviras (see references in Example 7), in light of the present observations, suggest that inhibitors of erb-B protein tyrosine kinases are likely to be effective therapeutic and/or prophylactic agents against not only multiple poxvimses but also a wide variety of other vimses. Of particular interest are the smallpox (variola major and variola minor) and cowpox (vaccinia) vimses.
- Variola vims enters subjects through the respiratory tract; human- human transmission usually occurs as a result of coughing out of vims in oralpharyngeal secretions. The incubation period is 7-19 days, followed by fever, headache and backache. After 2-3 days, the fever falls and a rash appears on the face, trunk, and extremities and progresses to vesicles, pustules and scabs lasting for several weeks. Variola major infection is fatal in approximately 40% of unvaccinated human beings. Death is primarily due to internal bleeding (disseminated intravascular coagulation) and vascular collapse.
- Variola is one of the largest and most complex of double-stranded DNA vimses and is visible by light microscopy. Sequence data are available on variola major vimses and variola minor vims. Current sequence data for variola in GenBank include: (a) five nucleotide sequence entries and 626 amino acid sequence entries for variola major; and (b) three nucleotide sequence entries and 619 amino acid sequence entries for variola minor.
- D1L epidermal growth factor (EGF)-like membrane-bound protein with ⁇ 97% homology to corresponding molecules of other variola strains, 86-89% identity with the corresponding vaccinia protein, and 30% identity to human epiregulin.
- EGF epidermal growth factor
- ELGF epiregulin-like growth factors
- SPGF smallpox growth factors
- VEF vaccinia growth factors
- FIG. 3 A and B are shown the amino acid sequences of SPGF from two variola strains, i.e., D1L from variola major India (Fig. 3 A; SEQ ID NO:l) and D4R from variola major Bangladesh (Fig. 3B; SEQ LD NO:2). Note that these two SPGF differ at only two amino acid positions (indicated in bold and underlined in Figs. 3 A and B). Various aspects of the invention are described below.
- the invention includes methods of inhibiting activation of a cell that expresses a cell surface erb-B protein tyrosine kinase molecule.
- one or more isolated compounds that inhibit erbB protein tyrosine kinase activity or inhibit the activation of erb-B protein tyrosine kinase activity are delivered to the cell of interest.
- Delivery to the cell of the one or more compounds can occur prior to, simultaneous with, or after contacting of the cell with a viral erb-B ligand (e.g., an ELGF) or a functional (i.e., erb-B-binding and activating) fragment of an erb-B ligand.
- the method can include, prior to the delivery step, identifying a cell expressing an erb-B protein tyrosine kinase.
- the cell can be any cell expressing an erb-B protein tyrosine kinase molecule, e.g.: any kind of epithelial cell, for example, a pulmonary epithelial cell such as a lung epithelial cell, a gastrointestinal epithelial cell (e.g. a stomach or colonic epithelial cell), a skin epithelial cell (e.g., a keratinocyte), or an epithelial cell of the genitourinary system (e.g., a bladder or uterine epithelial cell); or a fibroblast.
- the cells can be normal cells or malignant cells.
- the erbB protein tyrosine kinase can be erb-Bl, erb-B2, or erb-B4 protein tyrosine kinase, a homodimer of any of these three proteins, or a heterodimer between any two of erb-B 1 , erb-B2, erb-B3, and erb-B4 protein tyrosine kinases.
- the viral erb-B ligand (EBL) can be from any vims whose genome contains a nucleic acid (DNA or RNA) encoding a protein that binds to an erb-B protein tyrosine kinase.
- the viral EBL can be, for example, from any of the vimses listed herein. It can be, e.g., from poxvimses.
- Poxvimses family Poxviridae of interest include but are not limited to Orthopoxviruses (e.g., variola, vaccinia, and monkeypox), Avipoxvimses (e.g., fowlpox and canarypox), Capripoxviruses (e.g., sheep poxvims), Leoporipoxviruses (e.g., myxoma and Shope vims), Parapoxvimses (e.g., orf vims and swinepox), Molluscipoxvimses (e.g., Molluscum contagiosum vims), and Yatapox vimses (e.g., Yata monkey tumor vims).
- Orthopoxviruses e.g., variola
- the poxvims ELGF can be, for example, a SPGF (e.g., the D1L protein from the variola major India strain or the D4R protein from the variola major Bangladesh strain) or a VGF (from vaccinia vims).
- SPGF e.g., the D1L protein from the variola major India strain or the D4R protein from the variola major Bangladesh strain
- VGF from vaccinia vims
- a functional fragment of a viral EBL is a fragment that is shorter than the full-length, wild- type, mature protein but has at least 20% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 97%; 98%; 99%; 99.5%; 99.7%; 99.8%; 99.9%, or 100% or more) of the ability of the full- length, wild-type, mature protein to bind to one or more of the erb-B receptors to which the full- length, wild-type protein binds.
- Methods of measuring the ability of ligands to bind to receptors are known in the art (see below for an example of such a method).
- Functional fragments can, for example, be or contain an EGF-like domain, e.g., amino acids 40 to 90 of SPGF D1L (SEQ ID NO:l) or D4R (SEQ LD NO:2).
- EGF-like domain e.g., amino acids 40 to 90 of SPGF D1L (SEQ ID NO:l) or D4R (SEQ LD NO:2).
- sequence alignment see Fig.2
- the viral EBL that contacts the cell-surface erb-B protein tyrosine kinase can be a component of a viral particle, and indeed binding of a viral EBL in this form to a cell-surface erb-B molecule can be at least one early step in the infection of the cell by the viral particle.
- the viral EBL (or a functional fragment thereof) can either be secreted by a cell infected with the vims or be shed from an extracellular viral particle.
- the viral EBL (or functional fragment thereof) can be a molecule that has been, for example, partially or completely purified from the vims or has been chemically or recombinantly produced.
- a compound that inhibits an erb-B protein tyrosine kinase can be an antibody that binds to an erb-B protein.
- Such an antibody can be a non-agonist antibody, i.e., an antibody that activates substantially no signal through the receptor to which it binds.
- Antibodies specific for the ectodomain of erb-Bl have been described that compete for ligand binding and block erb-Bl signaling; moreover these antibodies were shown to cause cell cycle arrest and/or cell death [Arteaga (2001) J. Clin Oncol. 19(18) (September 15 supplement): 32s-40s and references cited therein].
- Another class of relevant compounds includes antibodies that bind to a viral EBL, e.g., a mAb that binds to an SPGF or VGF such as the 3D4R-13E8 and 3D4R-11D7 mAbs (also referred to herein as the 13E8 and 11D7 mAbs, respectively).
- a compound useful for the method of the invention can be a non-agonist erb-B ligand or a non-agonist fragment of an erb-B ligand that binds to an erb-B protein.
- Methods for determining whether a compound that binds to a receptor of interest is an agonist or a non- agonist of that receptor are known in the art.
- Mammalian ligands of erb-B include, but are not limited to, EGF, epiregulin, transforming growth factor- ⁇ (TGF ⁇ ), amphiregulin, heparin binding EGF, betacellulin, and heregulin [Arteaga et al. (2001) J. Clin. Oncol. 19:32s-40s].
- Other appropriate compounds include non-agonist fragments of viral proteins, e.g., an ELGF such as SPGF or a VGF.
- Small molecule erb-B tyrosine kinase inhibitors Compounds of particular interest are small molecule inhibitors of erb-B protein tyrosine kinase activity. A large number of such compounds have been described. There are essentially three categories of such compounds. In category A are compounds that appear to act substantially by competing for the Mg-ATP binding site of the catalytic domain of erb-B protein tyrosine kinases [Al-Abeidi (2000) Oncogene 18:5690-5701]. In category B are compounds that apparently act substantially by competing for the tyrosine site on the erb-B protein tyrosine kinase [Posner et al. (1994) Mol. Pharmacol.
- Category A compounds include, but are not limited to, bicyclic quinazoline-based compounds (e.g., 4-anilinoquinazolines), pyridopyrimidine-based compounds, quinoline-3- carbonitrile-based compounds, pyrrolopyrimidine-based compounds, pyrazolopyrimidine-based compounds, as well as tri cyclic derivatives of quinazolines (such as imidazoquinazolines, pyrroloquinazolines, and pyrazoloquinazolines) and the other bicyclic compounds listed above [Fry et al.
- quinazoline-based compounds e.g., 4-anilinoquinazolines
- pyridopyrimidine-based compounds e.g., quinoline-3- carbonitrile-based compounds
- pyrrolopyrimidine-based compounds quinoline-3- carbonitrile-based compounds
- pyrrolopyrimidine-based compounds pyrazolopyrimidine-based compounds
- quinazoline-based compounds include: ZD1839/Iressa [Fry et al. (2003)]; GW572016 (N- ⁇ 3-Chloro-4-[3-fuorobenzyl)oxy]phenyl ⁇ -6-[5-( ⁇ [2- methylsulfonyl)ethyl]amino ⁇ methyl)-2-furyl]-4-quinazoline [Xia et al. (2002) Oncogene 21 :6255-6263; and Cockerill et al. (2001) Bioorg. Med. Chem. Lett.
- useful pyridopyrimidine-based compounds include PD69896, PD153717, and PD158780 [Fry et al. (1997) Biochem. Pharmacol. 54(8):877-887, incorporated herein by reference in its entirety].
- a useful quinoline-3-carbonitrile is EKB-569 [Fry et al. (2003)]
- a useful pyrrolopyrimidine is CGP59326A/PKI-166 [Lydon et al. (1998) Int. J. Cancer 76(1):154- 163, incorporated herein by reference in its entirety]
- useful pyrazolopyrimidines include, for example, compounds 9 and 11 in Traxler et al. [(1997) J. Med. Chem.
- PD 160879, and PD 174265 by virtue of an acrylamide group, act as irreversible inhibitors by alkylating, for example, the Cys 773 residue of erb-Bl.
- a comparison of the relative efficacy of a series of such acrylamide derivatives is described in Smaill et al [(1999) J. Med. Chem. 42(10): 803-1815, incorporated herein by reference in its entirety].
- substituting these compounds e.g., at the 7 position of quinazoline and pyridopyrimidine compounds
- alkylamine and alkoxyamine side chains serves to overcome aqueous solvent solubility problems [Smaill et al. (2000) J. Med. Chem.
- category A Also in category A are compound DAPH 1 (4,5-Dianilinophthalimide) and derivatives of it [Buchdunger et al. (1994) Proc. Natl. Acad. Sci. USA 91 :2334-238, incorporated herein by reference in its entirety] and the fungal products quercetin, genistein and lavendustin A [Levitzki et al. (1995) Science 267:1782-1788, incorporated herein by reference in its entirety].
- category B In category B are the compounds AG537 and AG538 [Posner et al. (1994) Mol. Pharmacol.
- flavone derivatives such as compounds 10a, 10c, and 10m in Cushman et al. [(1994) J. Med. Chem. 37:3353-3362, incorporated herein by reference in its entirety].
- flavone derivatives such as compounds 10a, 10c, and 10m in Cushman et al. [(1994) J. Med. Chem. 37:3353-3362, incorporated herein by reference in its entirety].
- category C are: thiazolidine-diones such as compounds 1, 2, and 3 in Geissler et al. [(1990) J. Biol. Chem. 265(36):22255-22261, incorporated herein by reference in its entirety]; 3- substituted 2,2'-diselenobis(lH-indoles) such as compounds 33 and 34 in Showalter et al. [(1997) J. Med. Chem.
- small molecule erb-B tyrosine kinase inhibitors refers to all the compounds described above under the title "Small molecule erb-B tyrosine kinase inhibitors". Both large molecule (e.g., antibodies) and small molecule compounds can be delivered to a cell singly or in combinations of 2 or more, e.g., three, four, five, six, seven, eight, nine, ten, 15, or 20. Such combinations can include combinations involving only one or more than one (e.g., two, three, or four) of the above-described classes of inhibitors.
- supplementary agents can be delivered to the cell or the environment of the cell, e.g., cultures or animals containing the cell, in order to enhance the effect of the above-described compounds.
- One such supplementary agent that can be used is an antibody that binds to one or more forms of the vims containing a gene that encodes the EBL of interest and substantially neutralizes the vims.
- substantially neutralizes means has the ability to reduce the titer of vims at least 5- fold, e.g., at least: 10-fold; 20-fold; 50-fold; 100-fold; 1,000-fold; 10,000-fold; 100,000-fold; 1,000,000-fold or more.
- Examples of such antibodies include neutralizing antibodies that bind to poxvims intracellular mature virions (IMN) (e.g., an antibody specific for the L1R vaccinia protein or for its variola orthologue), extracellular enveloped vims (EEV), or cell-associated enveloped vims (CEV).
- IFN poxvims intracellular mature virions
- EEV extracellular enveloped vims
- CEV cell-associated enveloped vims
- All the antibodies described above can be polyclonal antibodies or mAb and can be from any of a wide range of species, e.g., a human, a non-human primate (e.g., a monkey or a chimpanzee), a cow, a horse, a goat, a sheep, a pig, a cat, a dog, a rabbit, a guinea pig, a hamster, a gerbil, a rat, a mouse, or a chicken.
- a human e.g., a human, a non-human primate (e.g., a monkey or a chimpanzee), a cow, a horse, a goat, a sheep, a pig, a cat, a dog, a rabbit, a guinea pig, a hamster, a gerbil, a rat, a mouse, or a chicken.
- a non-human primate e.g.
- antibody refers not only to whole antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecules, but also to antigen-binding fragments, e.g., Fab, F(ab')2, Fv, and single chain Fv (scFv) fragments.
- An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived.
- chimeric antibodies e.g., humanized antibodies.
- Antibody fragments that contain the binding domain of the molecule can be generated by known techniques.
- F(ab') 2 fragments can be produced by pepsin digestion of antibody molecules; and Fab fragments can be generated by reducing the disulfide bridges of F(ab') 2 fragments or by treating antibody molecules with papain and a reducing agent.
- Fab fragments can be produced, for example, as described in U.S. Patent No. 4,642,334, which is incorporated herein by reference in its entirety.
- Chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using methods described in Robinson et al., International Patent Publication PCT/US86/02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240, 1041-43; Liu et al. (1987) J.
- Fully human antibodies can be produced by immunizing transgenic animals (e.g., mice) that contain gene segments encoding gene segments encoding all human immunoglobulin (i.e., variable, joining, diversity, and constant) regions (see, for example, U.S. Patent Nos. 5,545,806 and 5,569,825).
- transgenic animals e.g., mice
- gene segments encoding gene segments encoding all human immunoglobulin (i.e., variable, joining, diversity, and constant) regions see, for example, U.S. Patent Nos. 5,545,806 and 5,569,825).
- Applicants have deposited under the Budapest Treaty the 3D4R-11D7 and 3D4R-13E8 hybridomas with the American Type Culture Collection (ATCC), Rockville, MD 20852, U.S.A.
- the 3D4R-11D7 hybridoma was assigned the ATCC accession no.
- the methods of inhibiting activation of a cell can be in vitro or in vivo. In vitro application of the methods of the invention can be useful in basic scientific studies on viral infection and mechanisms of cellular resistance to viral infection.
- the compounds can be cultured with cells of interest (see above) and any of the forms of the viral EBL recited above. Measurements of, for example, viral titer, level of cell proliferation/survival, and/or protein phosphorylation can be made after various times of incubation using methods described herein and those known in the art. Moreover, such methods can be useful for screening test compounds for their ability to inhibit viral infection of cells. In such assays, a test compound binds to an erb-B receptor interest can be contacted with a cell that expresses the erb-B receptor by culturing the test compound with the cell.
- the cell Before, simultaneous with, or after the contacting, the cell can be contacted with a viral EBL or a functional fragment of an EBL. A determination is then made of whether the test compound reduces activation of the erb-B receptor by the ligand or the functional fragment.
- Activation of erb-B receptors can be measured by methods known in the art, e.g., by (a) detecting or measuring tyrosine phosphorylation of the erb-B receptor or downstream intracellular proteins that are phosphorylated directly by the activated erb-B receptor or indirectly by proteins activated in a cascade- fashion following activation of the erb- B receptor; (b) detecting or measuring intemalization of the erb-B receptor; or (c) detecting or measuring proliferation and/or survival of a cell.
- the in vitro methods of the invention in which the compound used is known to activate an erb-B receptor can also be "positive controls" in the above-described screening assays for compounds with the ability to inhibit activation of a cell by a viral EBL.
- the methods of the invention will preferably be in vivo. These applications can be useful in the therapy and prophylaxis of relevant viral diseases. They can also be useful for diminishing the side effects of vaccination with live vims (e.g., cowpox).
- an animal infected e.g., a human smallpox patient
- an animal that will be infected e.g., a human subject to be vaccinated with vaccinia vims
- an animal at risk of being infected e.g., a human subject at risk of being infected by a variola major vims in the course of an anticipated bioterrorist attack
- therapy of or prophylaxis from the clinical symptoms caused by the vims can be achieved.
- prophylaxis can mean complete prevention of the symptoms of a disease, a delay in onset of the symptoms of a disease, or a lessening in the severity of subsequently developed disease symptoms.
- therapy can mean a complete abolishment of the symptoms of a disease or a decrease in the severity of the symptoms of the disease. Modifications of the above-described in vivo methods of the invention can be used as screening assays for compounds that are effective prophylactic and/or therapeutic agents against a vims of interest.
- a test compound that inhibits the activity of an erb-B tyrosine kinase or inhibits the activation of an erb-B tyrosine kinase is administered to an animal that is susceptible to infection with a vims that contains a gene encoding an EBL. Before, during, or after the administration, the animal is exposed to the vims.
- a determination of the efficacy of the compound is then made by, for example, detecting or measuring a change in the titer of the vims in a body fluid (e.g., blood, urine, or mucous), an organ or tissue (e.g., lung, spleen, or liver tissue), or a lavage (e.g., a lung, intestinal, bladder, or vaginal lavage).
- a body fluid e.g., blood, urine, or mucous
- an organ or tissue e.g., lung, spleen, or liver tissue
- a lavage e.g., a lung, intestinal, bladder, or vaginal lavage.
- a reduction in one or more symptoms e.g., fever, rash, limb paralysis
- the methods of the invention can be applied to a wide range of species, e .g., humans, non-human primates, horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, mice, and birds such as chickens, turkeys and canaries.
- species e .g., humans, non-human primates, horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, mice, and birds such as chickens, turkeys and canaries.
- the isolated compound itself is administered to the subject.
- the compounds will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous (i.v.) infusion, or injected subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
- a pharmaceutically-acceptable carrier e.g., physiological saline
- intravenous (i.v.) infusion injected subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
- the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other dmgs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.0001-100.0 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art.
- Administrations can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-,100-, 150-, or more fold).
- Encapsulation of the compound in a suitable delivery vehicle e.g., polymeric microparticles or implantable devices
- a suitable delivery vehicle e.g., polymeric microparticles or implantable devices
- useful compounds are proteins
- they can be delivered to cells in vivo by administering expression vectors containing nucleic acids encoding such proteins, or cells (e.g., cells derived from the animal) transformed with such expression vectors, to the animal in which the cells reside. Such methods are well-known in the art.
- the invention features methods of enhancing immune responses.
- the immune responses enhanced can be CD8 + or CD4 + T cell responses.
- T cell responses enhanced can be cytokine/lymphokine-producing or cytotoxic T lymphocyte (CTL) responses.
- Cytokine/lymphokine-prod ⁇ cing responses are preferably Thl-type cytokine/lymphokine responses, e.g., interleukin (IL)-12- and IFN- ⁇ -producing responses.
- IL interleukin
- cytokines or lymphokines such as interleukin (IL)-2, IL-3, IL-4, IL-10, IL-13, IL-15, granulocyte- colony stimulating factor (G- CSF), macrophage colony stimulating factor (M-CSF), or granulocyte macrophage-colony stimulating factor (GM-CSF).
- the methods of the invention have been shown to enhance CD4 + T cell responses as well as CD8 + T cell responses (Examples 6 and 7), and (2) B cell (antibody-producing) responses generally require the activity of CD4 + helper T cells, the immune responses that can be enhanced by the methods of the invention include B cell responses.
- B cell responses can be in vivo and are essentially the same as the above- described in vivo methods of inhibiting activation of cells. In addition they can be applied to the same subjects recited above for the latter method.
- the methods can include the additional steps of, following one or more treatments, testing for an immune response in the animal and/or characterizing a response as, for example, a T cell response, a CD4+ T cell response, a CD8+ T cell response, or an antibody-producing B cell response by methods familiar to those in the art.
- a response as, for example, a T cell response, a CD4+ T cell response, a CD8+ T cell response, or an antibody-producing B cell response by methods familiar to those in the art.
- supplementary agents in addition to antibodies that substantially neutralize one or more forms of a vims of interest (see above), can be used.
- a method of enhancing an immune response of particular interest involves administering one or more of the above-described antibodies that bind to a viral EBL (e.g., mAbs 13E8 and 11D7) and one or more antibodies that substantially neutralize one or more forms of a vims of interest, e.g, the anti-LlR antibody.
- Additional supplementary agents useful for enhancing immune response include, for example, appropriate viral antigens.
- An antigen can be a component of the vims with which the subject is, will be, or at is risk of being infected and that is the source of the viral EBL whose viral replication-enhancing effects the administered compound inhibits, e.g., where the method of enhancing immunity is being used for enhancing the efficiency of vaccination with live vims (e.g., vaccination against smallpox with vaccinia vims).
- additional antigens can be administered.
- Such antigens can be, for example, killed vims, viral proteins or peptide fragments of viral proteins.
- the antigens can be administered alone or with adjuvant, e.g., cholera toxin (CT), E.
- CT cholera toxin
- MCT heat labile toxin
- MCT mutant CT
- MLT mutant E. coli heat labile toxin
- MCT E. coli heat labile toxin
- MCT and MLT contain point mutations that substantially diminish toxicity without substantially compromising adjuvant activity relative to that of the parent molecules.
- Other useful adjuvants include alum, Freund's complete and incomplete adjuvant, and RIBI.
- chemokines such as MlP-l ⁇ , MIP-3 ⁇ , RANT ⁇ S, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, interferon- (IFN-) ⁇ , ⁇ , or ⁇ , granulocyte- colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), or granulocyte macrophage-colony stimulating factor (GM-CSF).
- G-CSF granulocyte- colony stimulating factor
- M-CSF macrophage colony stimulating factor
- GM-CSF granulocyte macrophage-colony stimulating factor
- antibodies specific for chemokines, lymphokines, or cytokines that inhibit an immune response of interest can be used as supplementary agents.
- antibodies specific for IL-4 or IL-10 may be used as supplementary agents.
- antibodies specific for IL-12 or IFN- ⁇ may be employed as supplementary agents.
- a poxvims expression vector containing a heterologous nucleic acid sequence encoding an immunogen of interest is administered to an appropriate animal (e.g., a human, a non-human primate (e.g., a monkey or a chimpanzee), a cow, a horse, a goat, a sheep, a pig, a cat, a dog, a rabbit, a guinea pig, a hamster, a gerbil, a rat, a mouse, a chicken, or any other species recited herein) at a frequency determined to be effective by methods known in the art.
- an appropriate animal e.g., a human, a non-human primate (e.g., a monkey or a chimpanzee), a cow, a horse, a goat, a sheep, a pig, a cat, a dog, a rabbit, a guinea pig, a hamster, a ger
- the heterologous nucleic acid sequence is operably linked to a transcriptional regulatory element (TR ⁇ ).
- TR ⁇ s include promoters and enhancers and are known in the art.
- an expression control sequence e.g., a TRE
- Poxvims vectors of interest include vaccinia vectors, attenuated vaccinia vectors, canarypox vectors, and fowlpox vectors.
- Routes of administration will preferably be cutaneous (e.g., by scarification), intramuscular, subcutaneous, intravenous, or intraperitoneal, or mucosal (e.g., intranasal or intrarectal).
- cutaneous e.g., by scarification
- intramuscular e.g., subcutaneous, intravenous, or intraperitoneal
- mucosal e.g., intranasal or intrarectal.
- any of the above- described supplementary agents can also be administered to the animal.
- the frequencies of administration of the compounds and supplementary agents are also readily determinable by one ofskill in the art.
- the above-described genetic immunization strategy using poxvims vectors can be effective at enhancing both cellular (CD8+ and CD4+ T cell) or B cell-mediated responses to immunogens encoded by heterologous nucleic acid sequences in the poxviral vector used for immunization. They can be used for generating therapeutic, prophylactic, or non-therapeutic/non-prophylactic immune responses to a wide variety of immunogens.
- replication-competent poxvims vectors e.g., vaccinia vectors in humans
- non-replicating poxvims vectors e.g., canarypox vectors in humans
- a replication-competent poxvims vector containing an immunogen encoding sequence of interest is administered to a subject.
- Sufficient time is allowed for the vector to replicate sufficiently and produce sufficient immunogen to initiate a potent immune response in the subject.
- Methods well-known in the art can be used to establish an effective time period.
- One or more erb-B inhibitory compounds (with or without one or more supplementary agents) is then administered to the subject.
- This administration serves to both reduce the titer of the viral vector in the subject and to enhance the immune response to the immunogen.
- Immunogens of interest can be, for example, microbial (e.g., viral, bacterial, fungal, yeast, or protozoan) antigens, parasite (e.g., nematode)-derived antigens, tumor antigens, or proteins against which it is desired to make antibodies (polyclonal or monoclonal) for investigational, therapeutic, prophylactic, or diagnostic purposes.
- microbes from which immunogen-encoding nucleic acid sequences can be obtained include, without limitation, Mycobacteria tuberculosis, Salmonella enteriditis, Listeria monocytogenes, M. leprae, Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Borrelia burgdorferi, Actinobacillus pleuropneumoniae, Helicobacter pylori, Neisseria meningitidis, Yersinia enterocolitica, Bordetella pertussis, Porphyromonas gingivalis, mycoplasma, Histoplasma capsulatum, Cryptococcus neoformans, Chlamydia trachomatis, Candida albicans, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Entamoeba histolytica, Toxoplasma brucei, Toxo
- heat-shock proteins e.g., the Y. enterocolitica heat shock protein 60 [Konieczny et al. (2000) supra; Mertz et al. (2000) J. Immunol. 164(3): 1529- 1537] and M. tuberculosis heat- shock proteins hsp60 and hsp70; the Chlamydia trachomatis outer membrane protein [Ortiz et al. (2000) Infect. Immun. 68(3):1719-1723]; the B. burgdorferi outer surface protein [Chen et al. (1999) Arthritis Rheum. 42(9):1813-1823]; the L.
- tumors from which immunogen-encoding nucleic acids to be inserted into poxvims vectors can be obtained include neural tissue cancer, melanoma, breast cancer, lung cancer, gastrointestinal cancer, ovarian cancer, testicular cancer, lung cancer, prostate cancer, cervical cancer, bladder cancer, vaginal cancer, liver cancer, renal cancer, bone cancer, a hematological cell cancer, and vascular tissue cancer.
- PSMA prostate-specific membrane antigen
- MUC-1 mucin such as MUC-1
- the heterologous nucleic acid sequences in the poxvims vectors can encode full-length immature proteins, full-length mature proteins, segments of proteins containing immunogenic epitopes, or T cell epitope-containing peptide fragments. They can encode a single polypeptide sequence or multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, 11, 12, 15, 18, 20, 25, 30, or more) polypeptides.
- the heterologous nucleic acid sequence encodes more than one polypeptide
- the polypeptides can be from one or more than one (e.g., two, three, four, five, six, seven, eight, nine, or ten) microbial organism(s) and/or tumor(s).
- poxvims vector/erb-B inhibitor strategies are not limited to genetic immunization but can be applied generally to poxvims vector gene therapy methodologies in which, instead of an immunogen, the heterologous nucleic acid sequence encodes a therapeutic molecule such as, for example, a cytokine or growth factor (such as any of those listed herein), a drag or pro-drag, or an enzyme for which a subject of interest is deficient, e.g., adenosine deaminase.
- a therapeutic molecule such as, for example, a cytokine or growth factor (such as any of those listed herein), a drag or pro-drag, or an enzyme for which a subject of interest is deficient, e.g., adenosine deaminase.
- a therapeutic molecule such as, for example, a cytokine or growth factor (such as any of those listed herein), a drag or pro-drag, or an enzyme for which a subject of interest
- test regimen is therapeutic for, or prophylactic against, a particular disease.
- a test population displaying symptoms of the disease e.g., humans or experimental animals having smallpox
- a control population also displaying symptoms of the disease, is treated, with a placebo or a different regimen.
- Disappearance or a decrease of the disease symptoms in the test subjects would indicate that the test regimen is an effective therapeutic methodology.
- EXAMPLES Example 1. Materials and Methods Production of recombinant EGF-like domain of the SPGF D4R A polypeptide containing the EGF-like domain (amino acid residues 40-90 of SEQ ID NO:2) of the SPGF D4R was expressed in E. coli DL21 cells as inclusions and the resulting protein was compared with analogous EGF-like domains made from human EGF and mouse epiregulin (EPI).
- EGF-like domain of the SPGF D4R A polypeptide containing the EGF-like domain (amino acid residues 40-90 of SEQ ID NO:2) of the SPGF D4R was expressed in E. coli DL21 cells as inclusions and the resulting protein was compared with analogous EGF-like domains made from human EGF and mouse epiregulin (EPI).
- EPI mouse epiregulin
- these EGF-like domains are refe ⁇ ed to as recSPGF, recEGF, and recEPI, respectively.
- the inclusions were dissolved in 6 M guanidine-HCl solution and the polypeptides were purified by Ni2+-NTA column chromatography exploiting N-terminal tag sequences in the polypeptides that contain six histidine residues.
- the partially purified EGF-like domains were refolded in redox buffer (2mM reduced and lmM oxidized glutathione) and the polypeptides were further purified using RP-HPLC (reverse phase-high pressure liquid chromatography).
- RecSPGF (8 kDa) was compared to recEGF (6 kDA) and recEPI (5 kDa) by 15% sodium SDS-PAGE; proteins were stained with C ⁇ omassie blue (Fig. 1).
- Mitogenicity assays were performed by plating cells at low density in the relevant medium but without EGF and with a reduced amount of serum (and a reduced amount of BPE for N cells) such that their proliferation in response to a range of concentrations of added erb-B ligands could be assessed.
- 3000 R2F cells were plated in replicate 9 cm 2 culture wells in DMEM/F12 medium containing 1% calf serum. The next day, and on the 4th day after plating, the wells were fed with medium but supplemented with recEGF or recSPGF (at various concentrations) or with complete fibroblast medium.
- human foreskin fibroblasts SC-J
- FBS fetal bovine serum
- the cells were pretreated with tyrosine kinase inhibitors for 1 hour at 37°C and then stimulated with different amounts of recSPGF. Eighteen hours later, the cells were harvested and fixed in 80% ice-cold ethanol at 4°C for 1 hour.
- RecSPGF biotinylation and binding to epithelial cells analyzed by FFC RecSPGF was biotinylated using the ECL protein biotinylation module (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's protocol. 1 x 10 5 MB468 (or MB453) epithelial cells were used for each test sample. For the direct binding assay, one aliquot of cells was incubated with 1 ⁇ g anti-erb-Bl blocking antibody (mAb 528; Santa Cruz Biotechnology, Inc., Santa Craz, CA) at 4°C for 1 hour.
- mAb 528 Santa Cruz Biotechnology, Inc., Santa Craz, CA
- the inhibition of ligand binding is expressed as the percentage of (MFIe x p - MFI m j n /MFI ma ⁇ - MFI m i n ) where MFI exp is the MFI detected with an experimental sample, MFI min is the MFI detected with a negative control sample to which no biotinylated recSPGF was added, and MFI max is the MFI detected with a positive control sample to which biotinylated recSPGF was added but no inhibitor was added.
- HeLa cells (80%-90% confluent) in 10 cm culture dishes were stimulated with various erb-Bl ligands at a concentration of 50 ng/ml at 37°C for 10 minutes. The dishes were washed once with ice-cold PBS.
- the cells were then lysed directly in the dishes with 1 ml lysis buffer (25 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100, ImM phenylmethylsulfonyl fluoride, 0.35 trypsin inhibitor units/ml aprotinin, 5 ⁇ g/ml leupeptin, 10 mM NaF, 10 mM ⁇ -glycerophosphate and 1 mM Na 3 VO- ⁇ ) at 4°C for 30 minutes.
- the lysates ' were transferred to microfuge tubes and, after centrifugation, 0.5 ml lysate supernatant was immunoprecipitated with 1 ⁇ g of goat anti-
- EGFR polyclonal antibody (Santa Craz Biotechnology, Inc.) and 10 ⁇ l gamma-bind plusTM beads (Amersham Biosciences) at 4°C overnight.
- the beads were washed three times and the proteins were eluted from the beads directly in 2 x SDS-PAGE loading buffer. Aliquots of whole cell lysates or immunoprecipitated samples were resolved by 7.5% SDS-PAGE and the resulting gel was blotted onto a PVDF (polyvinylidene fluoride) membrane.
- PVDF polyvinylidene fluoride
- the membrane was blocked in Tris-buffered saline/Tween-20 (TBST) containing 2.5% bovine serum albumin (for erb-B receptors) or TBST containing 2% gelatin (for 4G10) at 37°C for at least 30 minutes.
- TBST Tris-buffered saline/Tween-20
- the membranes were incubated with primary antibody overnight at 4°C according to the manufacturer's instructions, washed, and incubated with 1 : 10,000 anti-goat IgG (for erb-B receptors) or anti-mouse IgG2b (for 4G10) HRPO (horseradish peroxidase) conjugates at room temp for 1 hour.
- Cell lysates were prepared and subjected to immunoprecipitation (as described above) with goat anti-erb-Bl polyclonal antibody (Santa Craz Biotechnology, Inc.) and gamma-bind plus beads or anti-c-Cbl antibody-coated beads (Santa Craz Biotechnology, Inc.) at 4°C overnight using standard methods. Beads were washed and eluted in 2 X SDS-PAGE loading buffer. Whole cell lysates or immunoprecipitates were analyzed by western blotting with either anti-EGFR or anti-c-Cbl polyclonal antibodies.
- 1 ml lysis buffer 25 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100, ImM phenylmethylsulfonyl fluoride, 0.35 trypsin inhibitor units/ml aprotinin and 5 ⁇ g/ml leupeptin was added directly to the dishes followed by shaking at 4°C for 30 minutes.
- the lysates were transfe ⁇ ed to microfuge tubes and, after centrifugation, the lysate supernantants were subjected to immunoprecipitation (IP) with 1 ⁇ g anti-erb-Bl, anti-erb-B3, or anti-erb-B4 goat polyclonal antibodies (Santa Craz Biotechnologies) or a 1:40 dilution of anti-erb-B2 (Cell Signaling Technology, Beverly, MA) rabbit polyclonal antibody, and 10 ⁇ l of gamma-bind plus beads.
- IP immunoprecipitation
- the beads were washed 4 times with 1 x TBS/ 1% Triton X-100 and then boiled in 2 x SDS-PAGE loading buffer. SDS-PAGE and western blotting with streptavidin-HRPO were used to detect the biotinylated recSPGF.
- Chloramine T-catalyzed method was used for 125 I-labeling of recEGF.
- the specific activity of labeled recSPGF was 0.54 pm/cpm and that of recEGF was 0.34 pm/cpm.
- MB468 cells were plated at a concentration of 3 x 10 /0.1ml/96 well.
- 125 I- labeled recSPGF or recEGF was added at various concentrations to the cells in 50 ⁇ l of binding medium (L15 medium with 0.1%KaN 3 ) at 4°C and incubated for 5 hours. Supernatants were harvested and the cells were quickly washed twice with 60 ⁇ l ice-cold binding medium. The supernatants and washes were then combined.
- Erb-Bl intemalization HeLa cells were cultured on chamber slides and stimulated with Erb-Bl ligands (50 ng/ml) at 37°C for 10 minutes. The cells were then fixed with 3.7% formaldehyde for 10 minutes and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. The cells were incubated with l ⁇ g of anti-erb-Bl mAb (Santa Craz Biotechnology, Inc.) in 1% BSA/PBS at room temperature for 30 minutes. Bound anti-erb-Bl mAb was detected by staining with an anti-mouse Ig-FITC (fluorescein isothibcyanate) conjugate. A431 epidermal carcinoma cells were pretreated with 50 nM inhibitors at 4°C for
- the cells were incubated with biotinylated recSPGF (100 ng) for 30 minutes at 4°C. After washing, the cells were incubated at 37°C for 5 minutes and fixed in formaldehyde for 5 minutes at room temperature. Subsequent to streptavidi ⁇ -PE staining, the MFIs of the various sample were obtained by FFC.
- Vaccinia virus infection 6-8 week-old male C57BL/6 (B6) mice were injected intraperitoneally (i.p.) with 200 ⁇ g test antibody and/or 1 mg CI-1033 in PBS. Antibody was administered only once but the CI- 1033 was given each day for the 6-10 day duration of the experiment.
- RNase protection cytokine assay One lobe of a lung from each mouse sacrificed at various days post-infection with vaccinia virus was dismpted in RLT buffer (Qiagen, Valencia, CA) through an 18 gauge needle. Lysates were further homogenized using QIAshredderTM modules (Qiagen) and purified using an RNeasy MiniTM kit (Qiagen) according to the manufacturer's instructions. Equal amounts of total RNA (2 ⁇ g/mouse) were pooled from three mice at each time point (days 4, 6 and 8).
- RNA sequencing gel Six ⁇ g of RNA were hybridized with probes made from the mCK-2bTM template set (BD Biosciences-Pharmingen) according to the manufacturer's instructions and protected fragments were resolved on a DNA sequencing gel. An autoradiogram was generated by exposing the gel to x-ray film.
- Intracellular cytokine staining Single cell suspensions were prepared from spleens of infected mice treated in various ways and erythrocytes removed by lysis using a 0.84% NH 4 CI solution.
- Vaccinia virus-specific, IFN- ⁇ producing, CD 8+ T cells were detected by FFC after stimulation in vitro with vaccinia virus-infected fibroblasts (MC57G), uninfected control MC57G fibroblasts, or plate bound anti- CD3 (145-2C11) mAb at 5 ⁇ g/ml using the Cytofix/Cytoperm Kit PlusTM (with GolgiPlugTM, BD Biosciences-Pharmingen).
- Control cultures to detect all recently activated CD8 + T cells contained anti-CD3 mAb.
- splenocytes were incubated (under the same conditions described above) with purified anti-mouse CD3 ⁇ mAb (145-2C11) coated onto the bottoms of culture wells (at a concentration of 5 ⁇ g/ml).
- Fc BlockTM reagent (2.4G2 mAb) in 96- well plates containing 100 ⁇ l of FFC buffer (HBBS (Hank's Balanced Salt Solution), 2% FCS, 0.1% NaN 3 ), the cells were stained (20 minutes, 4°C) with combinations of fluorochrome-labeled mAbs specific for CD8 ⁇ (mAb 53-6.7) and CD44 (mAb IM7). Subsequent fixation and permeabilization of the cells were performed to allow intracellular access to the anti-IFN ⁇ mAb (XMG1.2; BD Biosciences). Freshly stained samples were analyzed using Becton Dickinson FACSCalibur and CellQuest software (San Jose, CA).
- Confluent BSC-40 cell monolayers were either pretreated with various concentrations of CI-1033 in RPMI + 2% FBS (RPMI-2%) in triplicate for 30 minutes at room temperature or mock pretreated under identical culture conditions. Seven concentrations of CI-1033 were evaluated and three mock pretreated controls per concentration of CI-1033 were performed in 7 individual 6-well tissue culture plates. The monolayers were infected with a suspension of variola strain Solaimen such that ⁇ 50 plaques were observed in each well. Plates were incubated at 35°C in an atmosphere of 6% CO2 for 1 hour and rocked at 15 minute intervals to insure an even infection of the monolayer.
- the inoculum was removed and the monolayer was rinsed lx with RPMI-2%.
- the monolayers were over-laid with media without or with CI-1033 at the appropriate concentrations and incubated at 35 °C in an atmosphere of 6% CO2 for 4 days.
- Plaques were analyzed by immunohistochemical staimng. Comets were defined as greater than two successively smaller plaques in comet-shaped association with a large plaque.
- the number of plaques and comets in the presence of different concentrations of CI-1033 were compared to control plates by using a Wilcoxon rank-sum test. Seven randomly selected mock-treated wells, one selected from each of seven plates, were used for comparison.
- Example 2 The EGF-like Domain of an SPGF Stimulates Cell Growth and/or Cell Survival Fig. 2 shows the amino acid sequence alignments of human and murine epiregulin and the orthopox viral orthologs D1L, CMPl 1R, CI 1R, and D3R from variola, camelpox, vaccinia, and monkeypox viruses, respectively. Note the high (-90% or greater) homology among viral proteins with -30% identity with the mammalian proteins. Within the EGF-like domain itself, the virtual identity of the viral protein sequences is evident (Fig. 2).
- D4R differs from the vaccinia ortholog (CI 1R; SEQ ID NO:4) at only 3 residues.
- DIL from the variola major India strain (Fig. 3 A; SEQ ID NO:l) differs by only two amino acids from D4R, the corresponding molecule from the variola major Bangladesh strain (Fig. 3B; SEQ ID NO:2).
- recSPGF stimulates cell growth or cell survival.
- SPGF is a long-term growth factor for both primary human keratinocytes (N cells) and human fibroblasts (R2F).
- N cells primary human keratinocytes
- R2F human fibroblasts
- the cells were cultured in standard tissue culture medium containing 10% FBS ("Complete”) or in tissue culture medium containing 2% FBS and the indicated concentrations of recSPGF (“SPGF”) or recEGF (“EGF”) for 7 days, and total viable cell counts were performed daily. Data are expressed as the mean population doublings per day.
- Example 3 SPGF Binds to the Erb-Bl Receptor
- erb-Bl There are four subtypes of EGFR: erb-Bl, erb-B2, erb-B3, and erb-B4.
- erb-B receptor binds SPGF
- two binding experiments were performed with biotinylated recSPGF using the MB453 (erb-Bl " , -B2 + , -B3 + , -B4 + ) and MB468 (erb-Bl + , -B2-, -B3 + , -B4 _ ) epithelial cell lines.
- the first experiment was a cross-linking analysis.
- MB453 or MB468 cells were incubated with biotinylated recSPGF.
- Crosslinking of erb-B receptors with recSPGF was performed with the crosslinking agent BS 3 . Lysates were then prepared and subjected to immunoprecipitation, which was followed by western blotting with HRPO-conjugated streptavidin ("Sav-HRPO") (Fig. 6, upper panel) or with each of the erb-B receptor-specific antibodies (Fig. 6, middle panel).
- Sav-HRPO HRPO-conjugated streptavidin
- Fig. 6, middle panel HRPO-conjugated streptavidin
- the expression of erb-B receptors 1-4 in these two cell lines is shown in the table presented in the bottom panel of Fig. 6.
- the upper panel of Fig. 6 demonstrates that recSPGF binds detectably to erb-Bl only.
- Fig. 7 shows that an anti-erb-Bl mAb blocks recSPGF binding to MB468 epithelial cells. Since MB453 cells do not express erb-Bl, no binding of recSPGF to them was detected (Fig. 7). Binding inhibition experiments indicate that binding of SPGF to erb-Bl is slightly weaker than EGF but stronger than epiregulin (Fig. 8). Affinity measurements of recSPGF binding to EGFR1 -expressing cells were obtained by
- MB468 cells were plated into the wells of 96-well microtiter tissue culture plates. I-labeled recSPGF was added at various concentrations, and the plates were incubated at 4°C for 5 hours.
- the erb-Bl receptor is normally distributed fairly uniformly over the surface of epithelial cells as evidenced by a largely homogeneous pattern of linear fluorescence obtained when unstimulated cells are stained with antibody specific for erb-Bl (Fig. 10A). However, 10 minutes after addition of recSPGF, recEPI, or recEGF, the receptor was rapidly internalized (Figs. 10B, 10C, and Fig. 10D, respectively). Both the rapid intemalization of erb-Bl activated by recSPGF and the ability of tyrosine protein kinase phosphorylation to activate IMV entry via formation of actin- and ezrin- containing cellular protmsions [Locker et al. (2000) Mol. Biol.
- Cell 11:2497-2511 serve to facilitate entry of a variola virion into a cell.
- the initial binding of the variola virion to the cell surface prior to entry can be via, at least in part, a SPGF-erb-Bl interaction.
- the above-described activation events also render host cells more efficient viral replication "factories.”
- Example 4 The EGF-like Domain of an SPGF Stimulates Protein Tyrosine Phosphorylation in Cells RecSPGF stimulated phosphorylation of EGF receptors and their cellular substrates.
- Example 5 Stimulation of Protein Tyrosine Phosphorylation by the EGF-like Domain of an SPGF is Inhibited by Quinazoline-based Compounds
- Fig. 12 shows the chemical stractures of three 4-anilinoquinazoline tyrosine kinase inhibitors (PD 153035, PD 168393, and CI-1033) as well the generic 4-anilinoquinazoline structure.
- PD153035 and PD168393 have an identical 4-(3'- bromo-aniline) ring but differ in their R3 and R4 groups attached to the quinazoline ring.
- PD 168393 has an acrylamide at position 6 which can alkylate erb-Bl Cys 7 73 so that the inhibitor i ⁇ eversibly binds to erb-Bl at a 1 :1 molar ratio.
- the tyrosine kinase active erb-B2 and erb-B4 molecules have a comparable cysteine residues at Cys78 and Cys778 5 respectively, which can be targeted for modification.
- PD153035 binds in a reversible manner, primarily via hydrophobic forces.
- CI-1033 like PD168393, has the acrylamide adduct at the 6 position (R3) and thus, like PD168393, binds i ⁇ eversibly to erb-Bl via Cys 773 .
- CI-1033 has, in addition, the solubilizing morpholine side chain at the 7 position (R4).
- CI-1033 demonstrates IC 5 n values of 0.8, 19 and 7 nM for erb-Bl, erb-B2 and erb-B4, respectively [Allen et al. (2002) Sem. Oncol.29:11-21].
- the cells were then cultured at 37°C for a further 10 minutes and then, after washing, lysed.
- the cell lysates were resolved by 10% SDS-PAGE and the SDS-PAGE gel was blotted onto a PVDF membrane. The membrane was exposed to the
- Fig. 14B shows that these same compounds inhibited tyrosine phosphorylation of erb-B 1 (150kD band) as well as additional substrates (120kD, 80kD, 60kD and 55kD) phosphorylation of which was activated by recSPGF in HeLa cells.
- the Src family PTK-specific inhibitor PP2 had little ability to block the phosphorylation of these erb-Bl substrates, even when used at 10 ⁇ M.
- 10B shows the distribution of erb-Bl receptors in human HeLa epithelial cells prior to and after incubation with recSPGF for 15 minutes at 37°C, as detected by immunofluorescence microscopy.
- the distribution of erb-Bl is primarily confined to the membrane.
- erb-Bl molecules are rapidly internalized, appearing as punctate intracellular fluorescent aggregates.
- erb-Bl ligand was then visualized with streptavidin-PE and fluorescence was quantitated by FFC analysis.
- Fig. 14C shows that, in the absence of inhibitors, the MFI was -110; in contrast, the hreversible erb-B inhibitors augmented the MFI -3 fold (300-350) and the reversible erb-Bl inhibitor PD 153035 increased the MFI to 240.
- the src family kinase inhibitor PP2 only modestly affects erb-Bl surface copy number.
- the erb-B receptors are eliminated by two pathways: 1) ligand-dependent endocytosis and degradation involving a c-Cbl ubiquitin ligase mechanism [Levkowitz et al. (1999) Mol. Cell 4:1029-1040] and 2) stress-induced shuffling of chaperones associated with the erb-B receptors and involving proteasomal proteinases [Xu et al. (2001) J. Bio. Chem. 276:3702-3708]. Recent studies show that erb-B2 kinase inhibition by CI- 1033 promotes down-regulation of erb-B2 via the second process [Citri et al. (2002) EMBO J. 21.2407-2417].
- PD153035, PD168393 and CI-1033 pretreatment augmented the total erb-Bl protein immunoprecipitated despite recSPGF addition; note the several-fold increase in erb-Bl over that obtained from the recSPGF-unstimulated control cells. The latter result implies blockade of constitutive intemalization or degradation of erb-Bl by the inhibitors. More importantly, recSPGF-induced c-Cbl association with erb-B was blocked by the 4-anilinoquinazolines but not by PP2.
- CI-1033 blocks SPGF-stimulated erb-Bl driven cell growth or survival, receptor-mediated tyrosine phosphorylation, intemalization and degradation, it seemed possible that the erb-Bl kinase inhibitor might attenuate orthopox growth factor activity in vivo.
- the effect of CI-1033 on the clinical course of B6 mice given a lethal intranasal vaccinia WR challenge [Chen et al. (2001) Nat. Immunol. 2:1067-1076] was examined. As shown in Fig. 15 A, all untreated animals died by day 7 after infection from a fulminant acute pneumonia. In contrast, daily i.p.
- CI-1033 administration of CI-1033 at 50 mg/kg beginning 6 h prior to infection prevented death.
- This treatment was as effective as a single 200 ⁇ g i.p. dose administration of anti-LlR vaccinia viras mAb (7D11) known to neutralize vaccinia intracellular mature virions (IMV) [Hooper et al. (2000) Virol. 266:329-339].
- Combined administration of CI-1033 and anti-LlR was also protective.
- Clinical monitoring of animals post-infection showed that anti-LlR mAb or CI-1033 treatment reduced symptomology significantly but not as efficiently as the combination therapy (Fig. 15B).
- a cohort of animals was sacrificed at 6 days post-infection and their lungs were examined.
- Fig. 15C Differences in gross lung weight between the treatment groups are shown in Fig. 15C.
- Untreated animals had edematous lungs with multiple hemo ⁇ hages and weighing approximately two times those of normal, uninfected B6 mice. This pathology was somewhat attenuated by anti-LlR or CI-1033 treatment alone but dramatically ameliorated by the combination of CI-1033 plus anti-LlR. Consistent with the pathological findings, vaccinia viras titers in lungs were modestly reduced by anti-LlR or CI-1033 alone but essentially eliminated by the combination treatment when assayed on day 8. As all untreated animals died by day 8, day 7 viral lung titers were used for comparison. Fig.
- 15D also illustrates the results of: (i) immunotherapy with the anti-SPGF mAb (13E8), which crossreacts with VGF (see co- pending U.S. Application Serial No. 10/429,685); and (ii) dual immunotherapy with anti-LlR plus anti-SPGF mAbs.
- the additional two log reduction in viral titer observed with anti-LlR plus CI-1033 treatment may be a consequence of the i ⁇ eversible binding of CI-1033 [Fry et al. (1998) Proc. Natl. Acad. Sci. USA 95: 12022-12027] compared with the reversible binding of the mAb.
- Irreversible binding of an inhibitor may result in a more complete blockade of the erb-Bl pathway than reversible binding of an inhibitor.
- viral titers did not decline until six days after infection. This slow time course was suggestive of the development of an adaptive T cell response to the viras rather than a virus-neutralizing effect of the antibodies.
- similar kinetics were observed with the anti-LlR mAb plus CI-1033 combination therapy.
- T cell-regulated cytokine production in lungs was tested by RNase protection analysis at days 4, 6 and 8 post-infection.
- IL-1 ⁇ interleukin- 1 ⁇
- IL-lRa interleukin-1 receptor antagonist
- IFN- ⁇ mRNA IFN- ⁇ mRNA
- Example 7 Enhancement of In Vivo Immunity to Vaccinia Virus by Antibody Specific ' for VGF
- a mAb (13E8) specific for the variola SPGF D4R and cross-reactive with VGF enhanced the protective effect of the anti-LlR mAb.
- mAb 13E8 enhances T cell immunity to vaccinia viras in vivo and increases the enhancement of T cell immunity obtained with the anti-LlR mAb in vivo.
- vaccinia virus is controlled in vivo by IFN- ⁇ -producing T cells, the level of which peaks between days 6 and 8 post-infection [Ruby et al. (1991) Lymphokine Cytokine Res. 10:353-358], the kinetics of viras reduction seen with the 13E8 and anti-LlR mAbs (copending U.S. Application No. 10/429,685) suggested that adaptive immunity was involved.
- CD8 + T cell responses were examined in day 7 vaccinia virus-infected mice treated with: (a) the control 1A3 mAb, (b) the 13E8 mAb, (c) the anti-LlR mAb; or (d) a combination of the 13E8 and anti-LlR mAbs.
- Intracellular IFN- ⁇ levels were analyzed in freshly isolated spleen T cells stimulated in vitro for 5 h with W-MC57G stimulator cells or with anti-CD3 mAb, which stimulates all recently activated T cells.
- the experiments showed increasing IFN- ⁇ responses in the CD44+CD8+ T cells in the vaccinia-infected treatment groups compared to naive uninfected mice, in the following order: 1A3 mAb (control), 13E8 mAb, anti-LlR mAb, and anti-LlR mAb + 13E8 mAb (Fig. 17C).
- mice The enhanced level of activated IFN- ⁇ -producing splenic T cells in anti-LlR + 13E8 mAb-treated mice suggested a reservoir of cells with potential to migrate to the lung to combat pulmonary infection; note that CD44, an activation marker up-regulated upon T cell stimulation, is a recyclable receptor for hyaluronan that is involved in leukocyte homing to sites of inflammation [DeGrendele et al. (1997) Science 278:672-675]. To test this possibility, lungs were collected from uninfected or from day 7-infected and mAb-treated mice and examined by hematoxylin and eosin staining in tissue sections.
- the alveolar spaces were obliterated in that they were filled with eosinophilic material; few, if any, infiltrating parenchymal cells were present.
- the non-neutralizing 13E8 mAb enhances clearance of vaccinia viras in association with augmented T cell responses in the spleen and lung.
- the systemic inflammatory component of innate immunity is rapidly induced when an organism's integrity is breached [Yoo et al. (2003) Proc. Natl. Acad. Sci USA 100:1157-1162].
- Sentinel cells including epithelial cells, produce IL-l ⁇ and/or IL-6 during this process [Schluns et al. (1997) J. Immunol. 1158:2704-2712].
- Blockade of erb-Bl stimulation by the 13E8 mAb may help avoid subsequent cytokine dysregulation, and consequent immunosuppression, by virally encoded molecules [Diehl et al. (2002) Mol.
- EGF and TGF ⁇ both erb-B ligands, can induce effusions in vivo [Ohmura et al. (1990) Cancer Res. 50:4915-4917].
- ELGF such as SPGF and VGF, may contribute analogous pathology, the inhibition of which by anti-ELGF antibodies (e.g., thel3E8 mAb) would constitute an additional therapeutic activity of such antibodies against orthopox viras infection.
- a Protein Tyrosine Kinase Inhibitor Inhibits Intracellular Formation and Intercellular Spread of Variola Viras To examine the effect of CI-1033 on variola viras growth, a confluent monolayer of BSC-40 cells was infected with approximately 50 plaque forming units of variola strain Solaimen in the presence or absence of various concentrations of the erb-B inhibitor and cultured in vitro for 4 days. As shown in Fig. 18 A, increasing concentrations of CI-1033 dramatically reduce the size of the individual plaques and comet formation but had a minimal effect on plaque number. The effect on plaque size and comet formation was titrable (Figs.
- Fig. 18A, 18B and 18C show a statistically significant decrease in the number of comets (p ⁇ 0.05) at CI-1033 concentrations of 500 nM or greater.
- combination immunotherapy/chemotherapy is highly efficient at reducing viral titer and stimulating T cell immunity. While the invention is not limited by any particular mechanism of action, it is possible to speculate that the latter may be a consequence of preventing the elaboration of anti-inflammatory viral products as well as later rounds of viral DNA replication in epithelial cells dependent on the viral growth factor [Alcami (2003) Nat. Rev. Immunol. 3:36-50; Seet et al. (1998) Ann. Rev. Immunol. 21:377-423].
- the use of host cellular signaling pathway blockade as a target for anti-viral chemotherapy is distinct from other approaches typically directed against pathogens themselves.
- One advantage of the host-targeted strategy is that drag resistance cannot develop.
- VGF vs. SPGF closely-related pathogenic factors
- erb-B tyrosine kinase inhibitors are potentially able to block immunologically distinct ligands to various erb-B protein tyrosine family numbers.
- This broader target activity is relevant since, for example, VGF and myxoma vims growth factor (MGF) bind distinct erb-B receptors [Tzahar et al. (1998) EMBO J. 17:5948-5963].
- CI-1033 augments T cell responses while reducing infectious symptomotology also suggests its use: (a) as an immune stimulant in combination with vaccinia vaccination; and/or (b) in treatment of complications of vaccination.
- orthopox e.g., natural or biote ⁇ orist spread of smallpox
- hepatitis B vims and Epstein-Barr viras have genes that encode products that dysregulate erb-Bl transcription [Menzo et al. (1993) Virol 196:878-882; Miller et al. (1995) J. Virol. 69:4390-4398].
- RNA tumor viruses such as avian erythroblastosis viras exploit erb-Bl signaling [Adelsman et al. (1996) J. Virol. 70:2533-2544].
- EGF receptors have been reported to be necessary for efficient reovirus and human cytomegaloviras (CMV) infection of host cells [Strong et al. (1993) Virol. 197:405-411; Wang et al. (2003) Nature 424:456-461]. These findings collectively suggest that multiple viral infections may be coupled to the erb-B-mediated signaling network.
- Epithelial cells which constitutively orinducibly express erb-B receptors, are components of the innate sentinel cell system in the body [Yoo et al. (2003) Proc. Natl. Acad. Sci USA 100:1157-1162].
- erb-B inhibitors will have a wider application in infectious diseases than in orthopox viras infection.
- Development of chemical inhibitors of cellular signaling pathways exploited by viral pathogenic factors may offer a new approach toward infectious disease control in general.
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US45365103P | 2003-03-11 | 2003-03-11 | |
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PCT/US2004/007537 WO2005003325A2 (en) | 2003-03-11 | 2004-03-11 | Inhibition of viral pathogenesis |
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CN103058938A (en) * | 2011-10-18 | 2013-04-24 | 南京大学 | 4-aniline quinazoline compound substituting cinnamic acid, and preparation method and application thereof |
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US20030022226A1 (en) * | 2000-02-11 | 2003-01-30 | Hooper Jay W. | Prophylactic and therapeutic monoclonal antibodies |
Non-Patent Citations (7)
Title |
---|
BULLER R M ET AL: "Deletion of the vaccinia virus growth factor gene reduces virus virulence." JOURNAL OF VIROLOGY MAR 1988, vol. 62, no. 3, March 1988 (1988-03), pages 866-874, XP002419531 ISSN: 0022-538X * |
EPPSTEIN D A ET AL: "Epidermal growth factor receptor occupancy inhibits vaccinia virus infection." NATURE 1985 DEC 19-1986 JAN, vol. 318, no. 6047, 19 December 1985 (1985-12-19), pages 663-665, XP001248472 ISSN: 0028-0836 * |
FAUCI ANTHONY S ET AL: "Host-based antipoxvirus therapeutic strategies: turning the tables." THE JOURNAL OF CLINICAL INVESTIGATION FEB 2005, vol. 115, no. 2, February 2005 (2005-02), pages 231-233, XP002419534 ISSN: 0021-9738 * |
KIM MIKYUNG ET AL: "Biochemical and functional analysis of smallpox growth factor (SPGF) and anti-SPGF monoclonal antibodies." THE JOURNAL OF BIOLOGICAL CHEMISTRY 11 JUN 2004, vol. 279, no. 24, 11 June 2004 (2004-06-11), pages 25838-25848, XP002419533 ISSN: 0021-9258 * |
MARSH Y V ET AL: "Vaccinia virus and the EGF receptor: a portal for infectivity?" JOURNAL OF CELLULAR BIOCHEMISTRY AUG 1987, vol. 34, no. 4, August 1987 (1987-08), pages 239-245, XP002419517 ISSN: 0730-2312 * |
See also references of WO2005003325A2 * |
YANG HAILIN ET AL: "Antiviral chemotherapy facilitates control of poxvirus infections through inhibition of cellular signal transduction." THE JOURNAL OF CLINICAL INVESTIGATION FEB 2005, vol. 115, no. 2, February 2005 (2005-02), pages 379-387, XP002419532 ISSN: 0021-9738 * |
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WO2005003325A3 (en) | 2006-03-23 |
WO2005003325A2 (en) | 2005-01-13 |
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