JP2007517767A - Methods for inhibiting viral pathogenicity - Google Patents

Abstract

The present invention relates to a method for inhibiting cell activation by a viral erb-B ligand and to a virus which is infected with, is considered to be infected or contains a nucleic acid sequence encoding the viral erb-B ligand in the genome. Features a method of enhancing the immune response of an animal at risk (eg, a human subject).

Description

TECHNICAL FIELD The present invention relates to the control of viral infections and more particularly to the control of poxvirus (eg, smallpox) infections.

background
Effective therapeutics for such infections in view of the tremendous potential for human economic and social devastation caused by viral diseases such as hepatitis B and the use of bioterrorists for diseases such as smallpox It is essential that a prophylactic drug is developed. Since smallpox was thought to have been eradicated from mankind more than 20 years ago, vaccination with the causative virus (decubitus) was abolished at that time. Thus, a relatively large proportion of the current human population has not been vaccinated against disease so far. Moreover, only a limited supply of smallpox vaccines produced prior to the abolition of vaccination is currently available.

SUMMARY The inventors have discovered that compounds that inhibit erb-B protein tyrosine kinases are effective in inhibiting poxvirus infections, and thus these compounds are effective antiviral therapeutics and It is likely to be a preventive drug.

  Example 2 describes a poxvirus protein with relatively high homology to the mammalian protein epiregulin (Epiregulin: EPI), particularly within these epithelial factor (EGF) -like domains. These poxvirus proteins are referred to herein as epiregulin-like growth factor (ELGF). Smallpox (decubitus) virus ELGF is a designated smallpox growth factor (SPGF), and vaccinia ELGF is a designated vaccinia growth factor (VGF).

  One aspect of the present invention is that binding of a recombinant epidermal growth factor (EGF) -like domain of D4R (SPGF derived from a Bangladesh strain of variola) to a cell surface erb-B1 receptor comprises (a) Activation of tyrosine kinase activity of such receptors in expressing cells; (b) enhanced proliferation and / or survival of such cells; (c) rapid internalization of erb-B1 receptors; and ( d) Based on the observation that viral virulence resulting from replication and concomitant enhancement of inflammation results. These findings indicate that SPGF plays a central role in the entry of, and replication within, and subsequent inflammation of the pressure ulcer virus into the host cell (eg, on the surface of or detachment from the pressure ulcer virion). Show that it fulfills.

  In addition, the inventors have shown that protein tyrosine kinase activity stimulated by SPGF binding to cells via the erb-B1 receptor and erb-B1 receptor internalization after SPGF ligation is It was found to be inhibited by tyrosine kinase inhibitors. This inhibition of erb-B1 internalization was apparently due to the inhibition of inducible binding between c-Cbl and erb-B1. Consistent with these findings, the inventors also observed that the quinazoline protein tyrosine kinase inhibitor CI-1033 inhibited clinical symptoms in animals infected with vaccinia virus and reduced viral load. The treatment effect of CI-1033 was enhanced by co-administration of neutralizing vaccinia-specific monoclonal antibody (mAb) (anti-L1R mAb). In addition, the inventors have found that erb-B tyrosine kinase inhibitors reduce the transmission of variola virus from one cell to another.

  Interestingly, CI-1033 also enhanced T cell immunity against vaccinia virus in infected animals, and this enhancement could be further increased by co-administration of anti-L1R mAb to infected animals. found. Furthermore, the inventors, like CI-1033, have shown that mAbs that bind to VGF (SPGF vaccinia homologous species) enhanced T cell immunity against vaccinia virus in animals infected with vaccinia, and this effect It was found that this was further increased by administration of anti-L1R mAb to the animals.

  These findings indicate that inhibitors of erb-B tyrosine protein kinase activity or activation of protein kinase activity may be effective agents against poxvirus infections in animals (eg, human subjects). Show. These inhibitors appear to act at least by inhibiting viral replication in the cell and by allowing the virus to exit the cell and by enhancing immunity against the virus. Inhibitors may also reduce viral entry into cells.

  More particularly, the present invention includes: (a) identifying an animal that may or may have been exposed to a virus comprising a gene encoding a viral erb-B ligand; And (b) treating the animal with a compound that inhibits the activity of erb-B tyrosine kinase or inhibits the activation of erb-B tyrosine kinase. The animal is identified as having been infected with the virus before, during or after step (b). The viral erb-B ligand can be a poxvirus erb-B ligand, such as an orthopox virus (eg, major acne, smallpox, simian virus, 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 (VGF), and erb-B tyrosine kinases are erb-B1, erb-B2 Or erb-B4 tyrosine kinase.

  The compound can be, for example, a non-agonist antibody that binds to erb-B tyrosine kinase, or a non-agonist erb-B ligand, or a non-agonist fragment of erb-B ligand. Alternatively, the compound may be, for example, a compound based on quinazoline (eg, 4-anilino quinazoline), a compound based on pyridopyrimidine, a compound based on quinoline-3-carbonitrile, or a compound based on pyrrolopyrimidine, etc. It can be a small molecule erb-B tyrosine kinase inhibitor. Small molecule erb-B tyrosine kinase inhibitors can be irreversible or reversible inhibitors of erb-B tyrosine kinase activity. The compound based on quinazoline can be, for example, CI-1033, PD168393, PD160678, PD160879, PD174265, PD153035, ZD1839, GW572016, GW974, OSI-774, or AG1478. The compound based on pyridopyrimidine can be, for example, PD69896, PD153717, or PD158780. A compound based on quinoline-3-carbonitrile can be, for example, EKB-569, and a compound based on pyrrolopyrimidine can be, for example, CGP59326A.

  In this method, the animal can be a human. The method comprises an intracellular mature virus particle of an antibody having the ability to substantially neutralize one or more forms of the virus, such as an orthopox virus (eg, large pox, small pox, vaccinia, or monkey pox virus). It may further comprise administering to the animal an antibody that binds to an (IMV) form, an extracellular enveloped virus (EEV) form, and / or a cell-bound enveloped virus (CEV) form.

  In this method, treatment with the compound can enhance the immune response to the virus in the animal. The immune response can be a T cell response, such as a CD8 + T cell response or a CD4 + T cell response, and the T cell response can be an interferon-γ (IFN-γ) producing T cell response. Alternatively, the immune response can be an antibody producing B cell response.

  In one embodiment of this method, the virus can be a poxvirus expression vector and (a) a heterologous nucleic acid sequence encoding an immunogen; and (b) operably linked to the heterologous nucleic acid sequence. It may further comprise a transcription regulatory element (TRE). The poxvirus expression vector can be a vaccinia virus expression vector, an attenuated vaccinia virus expression vector, a canary variola virus vector, or a fowlpox virus vector.

  The present invention also provides: (a) identifying an animal susceptible to infection with a virus comprising a gene encoding an erb-B ligand; and (b) a virus that stimulates an immune response against the virus (i) Or treatment with an antibody that substantially neutralizes one or more forms of the vaccine, and (ii) a compound that inhibits erb-B tyrosine kinase or inhibits activation of erb-B tyrosine kinase; And a method comprising: The antibodies that substantially neutralize one or more forms of viruses, animals, erb-B ligands, compounds, and viruses can be any of those described above. Moreover, the method can enhance any of the immune responses detailed above, and the virus can be any of the above poxvirus expression vectors.

  Another aspect of the present invention is: (a) providing an isolated compound that binds to erb-B tyrosine kinase and inhibits the activity of erb-B tyrosine kinase or activation of a receptor tyrosine kinase. (B) contacting the compound with a cell expressing erb-B tyrosine kinase; and (c) functionalizing the viral erb-B ligand or ligand before, simultaneously with, or after step (b) An in vitro method comprising the step of contacting a fragment with a cell. The compound can reduce the activation of erb-B tyrosine kinase on the cell by the ligand or fragment. Alternatively, the method can further comprise determining whether the compound reduces activation of erb-B tyrosine kinase on the cell by the ligand or fragment. The viral erb-B ligand, erb-B tyrosine kinase, and compound can be any of those described above.

  The present invention also provides a method for determining whether a compound is an antiviral compound comprising: (a) inhibiting the activity of erb-B tyrosine kinase, or activating erb-B tyrosine kinase. Providing a compound that inhibits; (b) administering the compound to an animal susceptible to infection with a virus comprising a gene encoding an erb-B ligand; (c) prior to step (b) Or after, exposing the animal to the virus; and (d) determining whether the compound reduces the symptoms of viral infection in the animal. The virus can be a pox virus, eg, an orthopox virus such as large ulcer, small ulcer, monkey variola virus, or vaccinia. The animal, erb-B ligand, and compound can be any of those described above.

  Yet another method characterized by the present invention is: (a) identifying an animal susceptible to infection by a virus comprising a gene encoding an erb-B ligand; and (b) an animal, (i) And (ii) treating with an antibody that substantially neutralizes one or more forms of a virus or vaccine that stimulates an immune response against the virus. The animal can be identified as having been infected with the virus before, during, or after step (b). Treatment can enhance the immune response to the virus, and the immune response can be any of those listed above. The virus can be a poxvirus expression vector and (a) a heterologous nucleic acid sequence encoding an immunogen; and (b) a transcriptional regulatory element (TRE) operably linked to the heterologous nucleic acid sequence. Can further be included. The poxvirus expression vector can be a vaccinia virus expression vector, an attenuated vaccinia virus expression vector, a canary variola virus vector, or a fowlpox virus vector. The antibody that binds to the ligand can be a 13E8 monoclonal antibody (ATCC accession number PTA-5040) or an 11D7 monoclonal antibody (ATCC accession number PTA-5039). The antibodies that substantially neutralize one or more forms of viruses, animals, erb-B ligands, compounds, and viruses 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 used in the present invention include those having 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. In general, variant polypeptides having conservative substitutions are less than 40 (eg, 35; 30; 25; 20; 15; 13; 11; 10; 9; 8; 7; 6; 5; 4; 3; 2; or Less than one) conservative substitution. A variant polypeptide has at least 20% of the activity of the wild-type polypeptide (eg, at least: 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; 99.8%; 99.9%; or 100% or more) is all that is needed.

  As used herein, “cell activation” means triggering enhanced cell proliferation or enhanced cell survival. “Enhanced cell proliferation or enhanced cell survival” is increased compared to (a) low levels of cell proliferation or cell survival; or (b) substantially no cell proliferation or cell survival. Means that.

  As used herein, “enhancing an immune response” is to increase an immune response compared to (a) a lower immune response; or (b) substantially no immune response It means to do.

  The term “isolated compound” as used herein is a component that does not have or naturally accompanies its naturally occurring counterpart, such as pancreas, liver, spleen, ovary, Compounds that are not separated or purified from components in tissues such as testis, muscle, joint tissue, nerve tissue, gastrointestinal tissue, or tumor tissue, or body fluids such as blood, serum, or urine (eg, proteins) Say. Typically, a naturally occurring biological compound is “isolated” when it is at least 70% dry weight by weight of naturally associated proteins and other naturally occurring organic molecules. Is considered. Preferably, the preparation of the compound used in the present invention is at least 80%, more preferably at least 90%, and most preferably at least 99% by dry weight of the compound. The degree of isolation or purity can be measured by any suitable method, for example by column chromatography, polyacrylamide gel electrophoresis, or HPLC (high performance liquid chromatography) analysis. Chemically synthesized compounds (eg, proteins) are separated in nature from the components that naturally accompany it, and synthetic compounds are “isolated” by definition.

  Isolated compounds, and additional agents useful for the present invention can be obtained, for example, by: (i) extraction from a natural source (eg, from a tissue or body fluid); (ii) the compound is If it is a protein, by expression of a recombinant nucleic acid encoding the protein; or (iii) by standard chemical synthesis methods known to those skilled in the art. A protein that is produced in a cellular system that is different from the source from which it is naturally derived is "isolated" because it necessarily does not contain components that naturally accompany it.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Preferred methods and materials are described below, but methods and materials identical or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

  Other features and advantages of the invention, including, for example, inhibiting cell activation, will be apparent from the following specification, drawings, and claims.

DETAILED DESCRIPTION The experimental findings outlined in the summary section above show that compounds that inhibit either erb-B1 protein tyrosine kinase activity or stimulation of erb-B1 protein tyrosine kinase activity are orthopoxvirus-mediated diseases It can be effective in the treatment of and / or prevention of the occurrence of (such as smallpox or vaccinia infections). Although the present invention is not limited by any particular mechanism of action, such compounds may inhibit the entry of viral particles into the host cell, viral replication within the cell, and / or virus release from the cell. As well as acting by enhancing adaptive T cell immunity against the virus. The orthopoxvirus genome may be functionally highly related to inhibiting viral replication and enhancing adaptive immunity in that it contains genes encoding immunosuppressive factors. For example, previous findings showing the role of erbB receptors in infection of cells with hepatitis B virus, EB virus, RNA tumor virus (eg chicken erythroblastosis virus), cytomegalovirus (CMV), and reovirus (See reference in Example 7), in view of this observation, inhibitors of erb-B protein tyrosine kinase are effective against multiple poxviruses as well as a wide variety of other viruses This suggests that it is likely to be a therapeutic and / or prophylactic agent.

  Of particular interest are smallpox (major and small ulcers) and cowpox (vaccinia) viruses. These viruses are large double-stranded DNA viruses that replicate in the cytoplasm of infected cells. Decubitus virus enters the subject via the respiratory tract; human-to-human transmission usually occurs as a result of viral coughing in the oral pharyngeal secretions. Following the 7-19 day incubation period is fever, headache, and back pain. After 2-3 days, the fever falls, a rash appears on the face, trunk, and limbs, progressing to vesicles, pustules, and crusts, lasting for several weeks. Acne infections are fatal in about 40% of unvaccinated humans. Death is mainly due to internal bleeding (disseminated intravascular coagulation) and vascular collapse. Infection with smallpox results in a less severe form of smallpox with reduced mortality.

  With the international eradication of smallpox around 1980, vaccination was abolished. Thus, smallpox protection has not been provided in the United States for about 20 years. As a result, a significant population has not been vaccinated against smallpox so far. In addition, the US currently does not have adequate reserves of smallpox vaccines (attenuated vaccinia virus), and available ones have the ability to adversely affect immunocompromised (eg, AIDS and cancer) patients It is produced by a poorly controlled process that produced vaccinia virus. In addition, in the case of 10-20 second generation, it can result from a single human pressure ulcer infection. These factors make variola virus, especially variola virus, an obvious choice as a biological weapon against the US population. Therefore, it is inevitable that an efficient, safe therapeutic and / or preventive methodology for viruses will be developed as quickly as possible.

  Pressure ulcers are one of the largest and most complex double-stranded DNA viruses and can be seen with a light microscope. Sequence data is available for major variola virus and small variola virus. GenBank pressure ulcer current sequence data includes: (a) registration of 5 nucleotide sequences of major pressure ulcers and registration of 626 amino acid sequences; and (b) registration of 3 nucleotide sequences of small pressure ulcers and 619 amino acids. Array registration. There are two complete major pressure ulcer genome nucleotide sequences, corresponding to Indian strain 1967 (GI: 9627521; 185,578 bp) and Bangladesh strain (GI: 623595; 186,103 bp), and one complete Genomic nucleotide sequence (GI: 5830555; 186,986 bp) is available at GenBank. A comparison of the large and small pressure ulcer genomes shows that they are more than 97% identical throughout the entire genome. Similarly, comparison of any pressure ulcer genome to that of vaccinia (GI: 9790357; 191,737 bp) indicates that they are ˜95% identical.

  There are 197 genes (GI: 9627521) in the genome of Indian acne strain 1967. Little is known about the relationship between these functions and the life cycle of the virus. Data from a computer-based analysis of the genome of variola virus (Ovarian decubitus Indian strain) showed that antibodies (such as co-pending US Application Serial No. 10 /) are considered vaccines against smallpox and effective passive immunoprotective agents. 429,685 (the disclosure of which is incorporated herein by reference in its entirety) has identified various polypeptides encoded by genes in the genome that are potentially useful. One of the polypeptides identified by this analysis is D1L, which is an epidermal growth factor (EGF) -like membrane-bound protein and ≧ 97 relative to the corresponding molecule of other pressure ulcer strains. % Homology, 86-89% identity with the corresponding vaccinia protein, and 30% identity to human epiregulin. As noted above, the EGF-like membrane-bound proteins of these poxviruses are collectively referred to herein as epiregulin-like growth factor (ELGF); ELGFs from pressure ulcer strains are referred to herein as smallpox growths. The factor (SPGF) is referred to and the one derived from the vaccinia strain is referred to as vaccinia growth factor (VGF). In FIGS. 3A and B, SPGF from two pressure ulcer strains, D1L from large pressure ulcer India (FIG. 3A; SEQ ID N0: 1) and D4R from large pressure ulcer Bangladesh (FIG. 3B; SEQ ID NO: 2). The amino acid sequence is shown. Note that these two SPGFs differ only in the two amino acid sites (shown in bold in FIGS. 3A and B and underlined).

  Various aspects of the invention are described below.

Methods of Inhibiting Cell Activation The present invention includes methods of inhibiting the activation of cells that express cell surface erb-B protein tyrosine kinase molecules. In these methods, one or more isolated compounds that inhibit erbB protein tyrosine kinase activity or inhibit activation of erb-B protein tyrosine kinase activity are delivered to a cell of interest. Delivery of one or more compounds to the cell involves contacting the cell with a fragment of a viral erb-B ligand (eg, ELGF) or a functional (ie, binding and activating erb-B) erb-B ligand. It can occur before, simultaneously or after. The method can include identifying cells expressing erb-B protein tyrosine kinase prior to the delivery step. The cell is any cell that expresses an erb-B protein tyrosine kinase molecule, such as: It can be skin epithelial cells (eg, keratinocytes), or genitourinary epithelial cells (eg, bladder or uterine epithelial cells); or fibroblasts. The cell can be a normal cell or a malignant cell. The erbB protein tyrosine kinase is an erb-B1, erb-B2, or erb-B4 protein tyrosine kinase, a homodimer of any of these three proteins, or erb-B1, erb-B2, erb-B3, and It can be a heterodimer between any two of the erb-B4 protein tyrosine kinases.

  Viral erb-B ligand (EBL) can be derived from any virus that contains a nucleic acid (DNA or RNA) that encodes a protein whose genome binds to erb-B protein tyrosine kinase. For example, the virus EBL can be any virus listed herein. For example, it can be derived from a poxvirus. The poxviruses of interest (Poxviridae) include orthopoxviruses (eg, pressure ulcers, vaccinia, and monkey pox), avian poxviruses (eg, fowlpox and canary pox), capripoxviruses ( For example, sheep poxvirus), leoporipoxviruses (eg, myxoma and shopevirus), parapoxviruses (eg, orf virus and swinepox), molluscipoxviruses ( Examples include, but are not limited to, infectious molluscum virus, and Yatapoxviruses (eg, Yatasar tumor virus). Pressure ulcers (major pressure ulcers and small pressure ulcers) and vaccinia virus are of particular interest. The poxvirus ELGF can be, for example, SPGF (eg, D1L protein from a large ulcer Indian strain or a D4R protein from a large ulcer Bangladesh strain) or VGF (from a vaccinia virus). Functional fragments of viral EBL are used herein as the fragments are shorter than the full-length wild-type mature protein, but the wild-type mature protein is one or more of the erb-B receptors. At least 20% of the ability to bind (eg, at least: 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 97%; 98%; 99%; 99.5%; 99.7%; 99.8%; 99.9%, or more than 100%). Methods for measuring the ability of a ligand to bind to a receptor are known in the art (see below for examples of such methods). A functional fragment can be or include, for example, amino acids 40-90 of an EGF-like domain, such as SPGF D1L (SEQ ID NO: 1), or D4R (SEQ ID NO: 2). By sequence alignment (see FIG. 2), one of skill in the art will understand that the region of EBL of interest includes or is an EGF-like domain.

  Viral EBL that contacts the cell surface erb-B protein tyrosine kinase can be a component of the viral particle, and in fact, the binding of this form of viral EBL to the cell surface erb-B molecule is responsible for infection of the cell by the viral particle. At least one of the initial steps. Alternatively, viral EBL (or a functional fragment thereof) can be secreted by cells infected with the virus or disrupted from extracellular viral particles. In addition, viral EBL (or functional fragments thereof) can be, for example, partially or fully purified from the virus, or a molecule produced chemically or recombinantly.

  The compound that inhibits erb-B protein tyrosine kinase can be an antibody that binds to erb-B protein. Such an antibody can be a non-agonist antibody, ie, an antibody that does not substantially activate the signal through the receptor to which it binds. Antibodies specific for the ectodomain of erb-B have been described as competing for ligand binding and blocking erb-B1 signaling; in addition, these antibodies are capable of cell cycle arrest and / or cell It has been shown to cause death [Arteaga (2001) J. Clin Oncol. 19 (18) (September 15 supplement): 32s-40s, and references cited therein].

  Another class of related compounds includes antibodies that bind to viral EBL, eg, mAbs that bind SPGF or VGF, such as 3D4R-13E8 and 3D4R-11D7 mAbs (also referred to as 13E8 and 11D7 mAbs, respectively).

  Further, compounds useful for the methods of the invention can be non-agonist erb-B ligands or non-agonist fragments of erb-B ligands that bind to erb-B proteins. 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 for erb-B include, but are not limited to, EGF, epiregulin, transforming growth factor alpha (TGFα), amphiregulin, heparin-binding EGF, betacellulin, and heregulin. [Arteaga et al. (2001) J. Clin. Oncol. 19: 32s-40s]. Other suitable compounds include non-agonist fragments of viral proteins such as ELGF (eg SPGF or VGF).

Small molecule erb-B tyrosine kinase inhibitors Compounds of particular interest are small molecule inhibitors of erb-B protein tyrosine kinase activity. Many such compounds have been described. There are essentially three categories of such compounds. Category A includes compounds that appear to act substantially by competing with the Mg-ATP binding site of the catalytic domain of erb-B protein tyrosine kinase [Al-Abeidi (2000) Oncogene 18: 5690-5701] . Category B includes compounds that apparently act substantially by competing for tyrosine sites on the erb-B protein tyrosine kinase [Posner et al. (1994) Mol. Pharmacol. 45: 673-683, in its entirety. Is hereby incorporated by reference]. Category C includes compounds that appear to act by both mechanisms [Posner et al. (1994) Mol. Pharmacol. 45: 673-683].

  Category A includes compounds based on bicyclic quinazolines (eg 4-anilinoquinazoline), compounds based on pyridopyramidine, compounds based on quinoline-3-carbonitrile, compounds based on pyrrolopyrimidine, pyrazolopyrimidines As well as tricyclic derivatives of quinazoline (imidazoquinazoline, pyrroloquinazoline, and pyrazoloquinazoline), and other bicyclic compounds listed above, but are not limited to [Fry et al. (2003) Exp. Cell Res. 284: 131-139, which is incorporated herein by reference in its entirety].

  Examples of compounds based on quinazoline include: ZD1839 / Iressa [Fry et al. (2003)]; GW572016 (N- {3-chloro-4- [3-fluorobenzyl) 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. 11: 1401-1405, both of which are incorporated herein by reference in their entirety]; GW974 [Cockerill et al. (2001); and Rusnak et al. (2001) Cancer Res. 2001) 61: 7196-7203, which is incorporated herein by reference in its entirety]; PD168393 [Fry et al. (1998) Proc. Natl. Acad. Sci. USA 95: 12022-12027, which is incorporated by reference in its entirety. FIG. 12], PD153035 / ZM252868 / AG1517 [Fry et al. (1994) Science 265: 1093-1095, which is hereby incorporated by reference in its entirety; FIG. 12], and CI- 1033 [Pfizer; Fig. 12]; OSI-774 / CP -358, 774 / Tarceva [Fry et al. (2003); and Stamos et al. (2002) J. Biol. Chem. 277: 46265-46272, incorporated herein by reference in its entirety]; AG1478 [ Levitzki et al. (1995) Science 267: 1782-1788, both of which are incorporated herein by reference in their entirety]; and PD160678, PD160879, PD168393, and PD174265 [Fry et al. (1998) Proc. Natl. Acad. Sci. USA 95: 12022-12027].

  Examples of useful pyridopyrimidine-based compounds are PD69896, PD153717, and PD158780 [Fry et al. (1997) Biochem. Pharmacol. 54 (8): 877-887, which is incorporated herein by reference in its entirety. ]including. A useful quinoline-3-carbonitrile is EKB-569 [Fry et al. (2003)], and a useful pyrrolopyrimidine is CGP59326A / PKI-166 [Lydon et al. (1998) Int. J. Cancer 76 (1): 154-163, which is incorporated herein by reference in its entirety], and useful pyrazolopyrimidines are described, for example, in Traxler et al. [(1997) J. Med. Chem. 40: 3601. -3616, which is incorporated herein by reference in its entirety].

を参照されたい、これらの全てが、参照としてこれらの全体が本明細書に組み入れられる]。 It is clear that all derivatives of all of these classes of compounds with a wide range of substituents show erb-B tyrosine kinase inhibitory activity and are therefore likely candidates for the method of the present invention [e.g. ,

All of which are incorporated herein by reference in their entirety].

Some of the above compounds (eg CI-1033, PD168393, EKB-569, PD1606678, PD 160879, and PD 174265) can be obtained by alkylating the Cys ⁷⁷³ residue of erb-B1, for example, with an acrylamide group Acts as an irreversible inhibitor. A comparison of the relative effectiveness of a series of such acrylamide derivatives can be found in Smaill et al [(1999) J. Med. Chem. 42 (10): 803-1815, incorporated herein by reference in its entirety. ]It is described in. In addition, substitution of these compounds with alkylamine and alkoxyamine side chains (eg, at the 7-position of quinazoline and pyridopyrimidine compounds) has been shown to help solve aqueous solvent solubility problems. [Smaill et al. (2000) J. Med. Chem. 43 (7) 1380-1397, which is incorporated herein by reference in its entirety].

  Category A includes the compound DAPH 1 (4,5-dianilinophthalimido) and its derivatives [Buchdunger et al. (1994) Proc. Natl. Acad. Sci. USA 91: 2334-238, Incorporated herein by reference], and fungal products, quercetin, genistein, and lavendustin A [Levitzki et al. (1995) Science 267: 1782-1788, incorporated herein by reference in their entirety. There is.

  Category B includes compounds AG537 and AG538 [Posner et al. (1994) Mol. Pharmacol. 45: 673-683], and Cushman et al. [(1994) J. Med. Chem. 37: 3353-3362, There are flavone derivatives such as compounds 10a, 10c, and 10m), which are incorporated herein by reference in their entirety.

  Category C includes compounds 1, 2, and 3 of: Geissler et al. [(1990) J. Biol. Chem. 265 (36): 22255-22261, incorporated herein by reference in its entirety]. Thiazolidinediones; 3-substituted 2,2 such as compounds 33 and 34 of Showalter et al. [(1997) J. Med. Chem. 40: 413-426, incorporated herein by reference in its entirety]. Compounds 9, 32, and 33 of '-diselenobis (1H-indole); Thompson et al. [(1993) J. Med. Chem. 36: 2459-2469, which is incorporated herein by reference in its entirety] 2,3-dihydro-2-thioxo-1H-indole-3-alkanonic acid and 2,2'-dihydrobis (1H-indole-3-alkanonic acid); and Traxler et al. [(1995) J. Med. Chem. 38: 2441-2448, which is incorporated herein by reference in its entirety] [(alkylamino) methyl] acrylophenone such as compounds 5, 17, 18, and 19 And there is (alkylamino) propiophenone.

  The term “small molecule erb-B tyrosine kinase inhibitor” as used herein refers to all compounds described above under the heading “small molecule erb-B tyrosine kinase inhibitor”.

  Both large molecule (eg, antibody) and small molecule compounds can be delivered to cells alone or in combination of two or more, eg 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 can do. Such combinations can include only one of the above classes of inhibitors, or combinations including one or more (eg, 2, 3, or 4). Furthermore, in order to enhance the effects of the compounds described above, supplements can be delivered to a cell or cell environment, such as a culture medium or animal containing cells. Such replacement agents that can be used are antibodies that bind to one or more forms of the virus containing the gene encoding the EBL of interest and substantially neutralize the virus. As used herein, “substantially neutralize” means at least 5 times, eg, at least 10 times; 20 times; 50 times; 100 times; 1,000 times; 10,000 times; 100,000 times; Means having the ability to reduce the titer of the virus. Examples of such antibodies include poxvirus intracellular mature virions (IMV) (eg, antibodies specific for the L1R vaccinia protein or its pressure ulcer homologous species), extracellular enveloped virus (EEV), Or a neutralizing antibody that binds to cell-bound outer membrane virus (CEV).

  In certain in vivo embodiments, instead of administering a neutralizing antibody to the subject, the subject can be vaccinated with an appropriate antigen source to produce the neutralizing antibody in situ. . Suitable antigens and methods of immunization are described in the section on enhancing immune responses below.

  All of the antibodies described above can be polyclonal antibodies or mAbs, and can be any wide range of tumors such as humans, non-human primates (eg monkeys or chimpanzees), cows, horses, goats, sheep, pigs, cats, It can be derived from dogs, rabbits, guinea pigs, hamsters, gerbils, rats, mice, or chickens.

As used herein, the term “antibody” refers not only to a complete antibody (eg, IgM, IgG, IgA, IgD, or IgE) molecule, but also to an antigen-binding fragment, such as Fab, F (ab ') ₂ Also refers to 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. Also included are chimeric antibodies, such as humanized antibodies.

Antibody fragments that contain the binding domain of the molecule can be generated by known techniques. For example: F (ab ′) ₂ fragments can be generated by pepsin digestion of antibody molecules; and Fab fragments can be produced by reducing disulfide bridges of F (ab ′) ₂ fragments or with papain and reducing agents. It can be made by treating an antibody molecule. See, for example, National Institutes of Health, 1 Current Protocols In Immunology, Coligan et al., Ed. 2.8, 2.10 (Wiley Interscience, 1991). scFv fragments can be generated, for example, as described in US Patent No. 4,642,334, which is hereby incorporated by reference in its entirety.

  For example, chimeric humanized monoclonal antibodies are described in Robinson et al., International Patent Application No. PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al. European Patent Application No. 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al., US Pat. 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. Immunol. 139, 3521-26; Sun et al. (1987) PNAS 84, 214-18; Nishimura et al. (1987) 47,999-1005; Wood et al. (1985) Nature 314, 446-49; Shaw et al. (1988) J. Natl. Cancer Inst. 80, 1553-59; Morrison, (1985) Science 229, 1202-07; Oi et al. (1986) BioTechniques 4, 214; Winter, US Pat. No. 5,225,539; Jones et al. (1986) Nature 321, 552-25; Veroeyan et al. (1988) Science 239, 1534; And using the method described in Beidler et al. (1988) J. Immunol. 141, 4053-60. It can be produced by known recombinant DNA techniques in the art.

  A fully human antibody (polyclonal or monoclonal) refers to a transgenic animal (eg, a mouse) containing a gene segment that encodes a gene segment that encodes all human immunoglobulin (ie, variable, conjugation, diversity, and constant) regions. It can be produced by immunization (see, eg, US Patent Nos. 5,545,806 and 5,569,825).

  Applicants deposited 3D4R-11D7 and 3D4R-13E8 hybridomas under the Budapest Treaty at the American Type Culture Collection (ATCC), Rockville, MD 20852, U.S.A. The 3D4R-11D7 hybridoma was assigned ATCC accession number PTA-5039, and the 3D4R-13E8 hybridoma was assigned ATCC accession number PTA-5040. Hybridomas deposited at the ATCC are obtained from deposits maintained by Dana Farber Cancer Institute, Inc. prior to the priority date of this application. No matter how long the hybridoma deposits, for a period of 30 years, or for a period of 5 years after the most recent request, or for the duration of a valid patent, will be maintained at the ATCC depository without any restrictions during that period. If the deposit becomes unable to grow, it will be replaced.

  The method of inhibiting cell activation can be in vitro or in vivo.

  In vitro application of the method of the invention will be useful for basic scientific studies on the mechanism of viral infection and cellular resistance to viral infection. In the in vitro methods of the invention, the compound can be cultured with the cell of interest (see above) and any form of viral EBL detailed above. For example, measurement of virus titer, level of cell proliferation / survival, and / or protein phosphorylation is performed after various incubation times using methods described herein and known in the art. Can do.

  In addition, such methods would be useful for screening test compounds for their ability to inhibit viral infection of cells. In such assays, a test compound that binds to an erb-B receptor object of interest can be contacted with a cell that expresses the erb-B receptor by culturing the test compound with the cells. Cells can be contacted with viral EBL or a functional fragment of EBL before, simultaneously with, or after contacting. A determination is then made as to whether the test compound reduces activation of the erb-B receptor by the ligand or functional fragment. Activation of the erb-B receptor can be accomplished by methods known in the art, for example (a) directly by an activated erb-B receptor or in a cascaded manner following activation of the erb-B receptor. Detect or measure tyrosine phosphorylation of erb-B receptor or downstream intracellular protein indirectly phosphorylated by activated protein; (b) detect internalization of erb-B receptor Or (c) or (c) can be measured by detecting or measuring cell proliferation and / or survival.

  The in vitro method of the present invention when the compound to be used is known to activate the erb-B receptor includes the above-described screening assay method for a compound having the ability to inhibit cell activation by viral EBL. Can be a “positive control”.

  The method of the present invention is preferably performed in vivo. These applications will be useful for the treatment and prevention of related viral diseases. They may also be useful to attenuate the side effects of live virus (eg, cowpox) vaccination. Infected animals (eg, human smallpox patients), animals that will be infected with an EBL-expressing virus (eg, human subjects vaccinated with vaccinia virus), or animals at risk of infection (eg, expected bioterrorists) Treatment or prevention from the clinical symptoms caused by the virus can be achieved by administering the compound of interest to a human subject at risk of being infected with the variola virus during the attack.

  As used herein, “prevention” means complete prevention of disease symptoms, delay of onset of disease symptoms, or less serious disease symptoms that occur thereafter. Can do. As used herein, “treatment” can mean the complete disappearance of a symptom of a disease or a decrease in the severity of a symptom of a disease.

  Modifications of the above in vivo methods of the invention can be used as screening assays for compounds that are effective prophylactic and / or therapeutic agents against the virus of interest. In such methods, a test compound that inhibits the activity of erb-B tyrosine kinase or that inhibits activation of erb-B tyrosine kinase is administered to an animal susceptible to infection with a virus containing a gene encoding EBL. Is done. The animals are exposed to the virus before, during or after administration. A determination of the effectiveness of the compound can then be made, for example, by body fluid (eg, blood, urine, or mucosa), organ or tissue (eg, lung, spleen, or liver tissue), or lavage fluid (eg, lung, intestine, bladder, or This is done by detecting or measuring changes in the virus titer in the vaginal lavage fluid. Alternatively, a reduction in one or more symptoms of viral infection (eg, fever, rash, limb paralysis) can be detected or measured.

, Horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, mice, and birds such as chickens, turkeys, and canaries.

In Vivo Approach In a preferred in vivo approach, the isolated compound itself is administered to the subject. Usually, the compound is suspended in a pharmaceutically acceptable carrier (eg, saline) and administered orally or by intravenous (iv) infusion, or subcutaneously, intramuscularly, intrathecally. It is injected intracavity, intraperitoneally, into the rectum, into the vagina, into the nasal cavity, into the stomach, into the trachea, or into the lungs. They can be delivered directly into the lung, for example, if the infection is in the lung, for example if the infection is in the lung. The required dose will depend on the choice of route of administration; the nature of the formulation; the nature of the patient's illness; the size, weight, surface area, age, and sex of the subject; other agents being administered; Dependent. A suitable dose is in the range of 0.0001 to 100.0 mg / kg. The wide variation in dosage required is anticipated in light of the variety of compounds available and the different efficiencies for various routes of administration. For example, oral administration may require a higher dose than administration by iv injection. These dose level variations can be adjusted using standard empirical routines for optimization, as is well known in the art. Administration can be one or more times (eg, 2, 3, 4, 6, 8, 10, 20, 50, 100, 150, or more times). Encapsulating the compound in a suitable delivery vehicle (eg, polymeric microparticles or implantable devices) will increase the efficiency of delivery, particularly with respect to oral delivery.

  Where the useful compound is a protein, these may be expressed in an expression vector containing a nucleic acid encoding such a protein, or a cell transformed with such an expression vector (eg, a cell derived from an animal) It will be appreciated that can be delivered to cells by administration in vivo to an animal in which is present. Such methods are well known in the art.

The present invention features a method for enhancing an immune response. The enhanced immune response can be a CD8 ⁺ or CD4 ⁺ T cell response. The enhancement of the T cell response can be a cytokine / lymphokine producing or cytotoxic T lymphocyte (CTL) response. The cytokine / lymphokine-producing reaction is preferably a Th1-type cytokine / lymphokine reaction, such as a reaction that produces interleukin (IL) -12 and IFN-γ. However, under certain circumstances (eg, antibody-to-cell immune responses are preferred), interleukin (IL) -2, IL-3, IL-4, IL-10, IL-13, IL-15, granules It is also desirable to activate reactions involving the production of cytokines or lymphokines such as sphere colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), or granulocyte macrophage-colony stimulating factor (GM-CSF) Will. (1) The method of the present invention has been shown to enhance CD4 ⁺ T cell responses and CD8 ⁺ T cell responses (Examples 6 and 7), and (2) B cell (antibody productivity) responses Is generally required for the activity of CD4 ⁺ helper T cells: Thus, immune responses that can be enhanced by the methods of the present invention include B cell responses.

  These methods of the invention can be in vivo and are essentially similar to the in vivo methods of inhibiting cell activation. In addition, they can be applied to the same subject as the latter method is detailed above. The method can include the following additional steps: performing the reaction by, for example, performing one or more treatments, testing for an immune response in an animal, and / or methods familiar to the art, such as Characterizing as a T cell response, a CD4 + T cell response, a CD8 + T cell response, or an antibody producing B cell response.

  Where required to enhance immune responsiveness, supplements can be used in addition to antibodies (see above) that substantially neutralize one or more forms of the virus of interest. In particular, a method of enhancing an immune response of interest substantially comprises one or more of the above-described antibodies that bind to viral EBL (eg, mAbs 13E8 and 11D7), and one or more forms of the virus of interest. Administering one or more antibodies that neutralize, eg, anti-L1R antibodies.

  Additional supplements useful for enhancing an immune response include, for example, appropriate viral antigens. An antigen can be a component of a virus that is at risk of infecting, subject to, or at risk to the subject and for which the administered compound is a source of viral EBL that inhibits the viral replication enhancing effect. For example, methods of enhancing immunity are when used to enhance the efficiency of vaccination with live virus (eg, vaccination against vaccination against vaccinia virus). However, additional antigens can be administered. Such an antigen can be, for example, a dead virus, a viral protein, or a peptide fragment of a viral protein. The antigen can be used alone or as an adjuvant, such as cholera toxin (CT), E. coli heat-labile toxin (LT), mutant CT (MCT) [Yamamoto et al. (1997) J. Exp. Med. 185: 1203- 1210], and mutant E. coli heat labile toxin (MLT) [Di Tommaso et al. (1996) Infect. Immunity 64: 974-979]. MCT and MLT contain point mutations that substantially reduce toxicity without substantially compromising adjuvant activity compared to that of the parent molecule. Other useful adjuvants include alum, Freund's complete and incomplete adjuvants, and RIBI.

  Other supplements that can be used to further enhance immunity include MIP-1α, MIP-3β, RANTES, 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) -α, β, or γ, granulocyte colony stimulating factor (G-CSF), macrophage colony Includes chemokines, lymphokines, and cytokines such as stimulatory factors (M-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF). In addition, supplements that inhibit the immune response of interest, chemokines, lymphokines, or antibodies specific for cytokines can be used. Thus, for example, if it is required to enhance the immune response of a cell, antibodies specific for IL-4 or IL-10 may be used as a supplement. On the other hand, if it is required to enhance the antibody-producing B cell response, antibodies specific for IL-12 or IFN-γ may be used as a supplement.

  All methodologies (or obvious variations thereof) for the delivery and administration of the compounds described above in the section on methods of inhibiting cellular activation are for the delivery and administration of all supplements described herein. It is understood that can be used for. These supplements can be administered before, after, or simultaneously with the compound. They can be administered by the same or different route as the compound and at the same or different frequency.

  These methods of the invention can be applied to gene immunization. In such methods, a poxvirus expression vector comprising a heterologous nucleic acid sequence encoding an immunogen of interest is transformed into a suitable animal (eg, human, non-human primate (eg, monkey or chimpanzee), cow, horse, goat. , Sheep, pigs, cats, dogs, rabbits, guinea pigs, hamsters, gerbils, rats, mice, chickens, or any other species detailed herein) by methods known in the art Is administered at a frequency determined to be Within the vector, the heterologous nucleic acid sequence is operably linked to a transcriptional regulatory element (TRE). TREs include promoters and enhancers and are known in the art. As used herein, an expression control sequence (eg, TRE) “operably linked” to a coding sequence is incorporated into the genetic construct so that it efficiently controls the expression of the coding sequence. It is done. Poxvirus vectors of interest include vaccinia vectors, attenuated vaccinia vectors, canary acne vectors, and infectious epithelioma vectors.

  The route of administration will preferably be skin (eg, by random cuts), intramuscular, subcutaneous, intravenous, or intraperitoneal, or mucosa (eg, intranasal or rectal). Prior to, simultaneously with, or after administration of the vector, one or more of the erb-B inhibitor compounds described above are administered to the animal. Any of the above supplements can also be administered to the animal. Also, the frequency of administration of the compound and supplement can be readily determined by one of techniques in the art.

  Administration of an erb-B tyrosine kinase inhibitor enhanced the CD4 + and CD8 + immune responses of animals infected with poxvirus (see Examples 6 and 7), so the above gene immunization using poxvirus vectors The stratification strategy will be effective in enhancing both cellular responses (CD8 + and CD4 + T cells) or B cell mediated responses to the immunogen encoded by the heterologous nucleic acid sequence in the poxvirus vector used for immunization. They can be used to generate a therapeutic, prophylactic, or non-therapeutic / non-prophylactic immune response against a wide variety of immunogens. These methodologies allow the use of replication-competent poxvirus vectors (eg, vaccinia vectors in humans) and provide a stronger response than non-replicating poxvirus vectors (eg, canary acne vectors in humans). Inducing, however, raises concerns about possible out of control infection of host animals (eg, immunocompromised patients) for genetic immunization. Accordingly, a replicating poxvirus vector containing a sequence encoding the immunogen of interest is administered to the subject. The vector can be sufficiently replicated for sufficient time to generate sufficient immunogen to initiate a strong immune response in the subject. To establish an effective time, methods well known in the art can be used. One or more erb-B inhibitor compounds are then administered to the subject (with or without one or more supplements). This administration serves both to reduce the titer of the viral vector in the subject and to enhance the immune response to the immunogen.

  The immunogen of interest can be, for example, a microbial (eg, viral, bacterial, fungal, yeast, or protozoan) antigen, a parasite (eg, nematode) derived antigen, a tumor antigen, or a research, therapeutic, prophylactic, Alternatively, it can be a protein against what is required to produce antibodies (polyclonal or monoclonal) for diagnostic purposes.

  Examples of related microorganisms derived from nucleic acid sequences encoding immunogens that can be obtained include Mycobacteria tuberculosis, Salmonella enteriditis, Listeria monocytogenes, M. leprae, Staphylococcus aureus ( Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Borrelia burgdorferi, Actinobacillus pleuropneumoniae, Helicobacter pylori, Meningococcus, Enterocoli Yersinia enterocolitica, Bordetella pertussis, Porphyromonas gingivalis, Mycoplasma, Histoplasma capsulatum, Cryptococdia neoformans Candida alb icans), Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Entamoeba histolytica, Toxoplasma brucei, Toxoplasma brucei, Toxoplasma brucei Toxoplasma gondii), Leishmania major, human immunodeficiency virus 1 and 2, influenza virus, measles virus, rabies virus, hepatitis virus A, B, and C, rota Including but not limited to viruses, papillomaviruses, respiratory syncytial viruses, feline immunodeficiency viruses, feline leukemia viruses, simian immunodeficiency viruses, and all poxviruses detailed herein. Examples of related microbial immunogens include the B subunit of E. coli heat-labile enterotoxin [Konieczny et al. (2000) FEMS Immunol. Med. Microbiol. 27 (4): 321-332], heat shock proteins such as Y enterocolitica heat shock protein 60 [Konieczny et al. (2000) supra; Mertz et al. (2000) J. Immunol. 164 (3): 1529-1537], and Mycobacterium tuberculosis heat shock protein hsp60 and hsp70; trachoma pathogen outer membrane protein [Ortiz et al. (2000) Infect. Immun. 68 (3): 1719-1723]; B. burgdorferi outer surface protein [Chen et al. (1999 Arthritis Rheum. 42 (9): 1813-1823]; L. major GP63 [White et al. (1999) Vaccine 17 (17): 2150-2161 (and Vaccine 17 (20-21): 2755 published errata)]; N. meningitidis meningococcal serotype 15 PorB protein [Delvig et al. (1997) Clin. Immunol. Immunopathol. 85 (2): 134 -14 2]; P. gingivalis 381 Hippocampal protein [Ogawa, (1994) J. Med. Microbiol. 41 (5): 349-358]; E. coli outer membrane protein F [Williams et al. (2000) Infect. Immun. 68 (5): 2535-2545]; influenza virus hemagglutinin and neuraminidase; retrovirus (eg, HIV) surface glycoprotein (eg, HIV gp160 / 120), or retrovirus tat or gag protein Including, but not limited to.

  Examples of tumors from which a nucleic acid encoding an immunogen inserted into a poxvirus vector can be obtained include neural tissue cancer, melanoma, breast cancer, lung cancer, gastrointestinal cancer, egg sac cancer, testicular cancer, lung cancer, prostate cancer, Includes cervical cancer, bladder cancer, vaginal cancer, liver cancer, kidney cancer, bone cancer, blood cell cancer, and vascular tissue cancer. Examples of related tumor antigens are prostate specific membrane antigen (PSMA) [Israeli et al. (1993) Cancer Res. 53 (2): 227-230], mucins such as MUC-1, or on the surface of tumor cells Any other antigen expressed in

  The heterologous nucleic acid sequence of the poxvirus vector can encode a full-length immature protein, a full-length mature protein, a segment of a protein that includes an immunogenic epitope, or a peptide fragment that includes a T cell epitope. These can be a single polypeptide sequence or multiple (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, 25, 30 or more) polypeptides Can be coded. Where the heterologous nucleic acid sequence encodes a plurality of polypeptides, the polypeptides may include one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10) microbial organisms and / or Or it can be derived from a tumor.

  The poxvirus vector / erb-B inhibitor strategy described above is understood not to be limited to genetic immunization, but instead of immunogens, for example, cytokines or growth factors (any of those listed herein) Etc.), heterologous nucleic acid sequences encoding drugs or prodrugs or therapeutic molecules such as adenosine deaminase that are deficient in the subject of interest can generally be applied to poxvirus vector gene therapy. .

  Pox viruses and their uses are described in further detail in Paoletti [(1996) Proc. Natl. Acad. Sci. USA 93: 11349-11353], which is hereby incorporated by reference in its entirety.

  Methods for testing whether a particular regimen is therapeutic or prophylactic for a particular disease are known in the art. Where a therapeutic effect is being tested, a test population that exhibits symptoms of the disease (eg, a human or laboratory animal with smallpox) is treated with a test regimen that includes any of the strategies described above. Control groups showing disease symptoms are also treated with placebo or a different regimen. The disappearance or reduction of disease symptoms in the test subject will indicate that the study regimen is an effective treatment.

  By applying the same strategy to subjects prior to the onset of disease symptoms (eg, experimental animals prior to random infection with vaccinia virus), the test regimen can be tested for effectiveness as a prophylaxis . In this situation, prevention or delay of the onset of disease symptoms is tested.

  The following examples are meant to illustrate the invention and are not limiting.

Examples Example 1. Materials and Methods
Production of recombinant EGF-like domain of SPGF D4R
A polypeptide containing the EGF-like domain of SPGF D4R (amino acid residues 40-90 of SEQ ID NO: 2) is expressed as inclusion bodies in E. coli DL21 cells and the resulting protein is human EGF and mouse epiregulin (EPI ) Compared to similar EGF-like domains made from For convenience, these EGF-like domains are referred to as recSPGF, recEGF, and recEPI, respectively. Inclusion bodies were dissolved in 6M guanidine-HCl solution and the polypeptide was purified by Ni ²⁺ -NTA column chromatography utilizing the N-terminal tag sequence of the polypeptide containing 6 histidine residues. The partially purified EGF-like domain was refolded in redox buffer (2 mM reduced and 1 mM oxidized glutathione) and the polypeptide was further purified using RP-HPLC (reverse phase high performance liquid chromatography). RecSPGF (8 kDa) was compared to recEGF (6 kDA) and recEPI (5 kDa) by 15% sodium SDS-PAGE; the protein was stained with Coomassie blue (FIG. 1).

Human keratinocytes and fibroblast mitogen assay Normal primary human dermal fibroblast cell line R2F [Tubo et al. (1987) Oncogene Re.1: 407-421] is a 15% calf serum (HyClone). , Inc., Logan, UT) and “complete fibroblast medium” consisting of DMEM / F12 medium (Life Technologies, Inc., Carlsbad, Calif.) Supplemented with 10 ng / ml EGF. The normal primary human epidermal keratinocyte strain N [Schon et al. J. Invest. Dermatol. 107: 428-438] was previously described [Rheinwald et al. (2002) Mol. Cell Biol. 22: 5157-5172]. In “complete keratinocyte medium” consisting of GIBCO keratinocyte serum-free medium (Life Technologies, Inc.) supplemented with 30 μg / ml bovine pituitary extract (BPE), 0.2 ng / ml EGF, and 0.3 mM Cacl ₂ Cultured.

  The mitogenic assay did not contain EGF and reduced the amount of serum (and NPE for BPE so that their proliferation in response to a concentration range of added erb-B ligand could be assessed. Was performed by seeding the cells at low density in the relevant medium.

3000 R2F cells were seeded in 9 cm ² culture wells in DMEM / F12 medium containing 1% calf serum. The next day, and 4 days after plating, the wells received either medium supplemented with recEGF or recSPGF (at various concentrations) or complete fibroblast medium.

1000 N cells were seeded in 9 cm ² culture wells in GIBCO keratinocyte serum-free medium containing BPE (15 μg / ml) and 0.3 mM Cacl ₂ . The next day, and 4 days after plating, the wells received either medium supplemented with recEGF or recSPGF (at various concentrations) or complete keratinocyte medium.

Six days after seeding, the cells were detached from the bottom of the culture well by trypsinization and counted. The growth rate was measured as population doubling per day: [log ₂ (number of cells after 6 days / number of cells scattered)] / 6.

  For tyrosine kinase inhibition experiments, human foreskin fibroblasts (SC-J) were plated in 6-well tissue culture plates and “starved” overnight in 2% FBS (fetal bovine serum) DMEM medium. " Cells were pretreated with tyrosine kinase inhibitors for 1 hour at 37 ° C. and then stimulated with different amounts of recSPGF. After 18 hours, the cells were collected and fixed in 80% ice-cold ethanol for 1 hour at 4 ° C. After washing once with phosphate buffered saline (PBS), the cells were stained with a solution of 2.5 μg / ml propidium iodide containing 50 μg / ml RNase A for 30 minutes at 37 ° C. Cell cycle data was obtained by FFC analysis and analyzed using Cell Quest® (Becton Dickinson, San Jose, Calif.) And Modfit® (Becton Dickinson) software.

Biosynthesis of RecSPGF and binding to epithelial cells was analyzed by FFC
RecSPGF was biotinylated using the ECL protein biotinylation module (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's protocol. 1 × 10 ⁵ MB468 (or MB453) epithelial cells were used for each test sample. For direct binding assays, an aliquot of one cell was incubated with 1 μg anti-erb-B1 blocking antibody (mAb 528; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) For 1 hour at 4 ° C. Different concentrations of biotinylated recSPGF in 50 μl FFC buffer (1 × PBS / 2.5% FCS / 0.02% NaN ₃ ) solution were added to the samples and then further incubated at 4 ° C. for 45 minutes. For competition assays, cells were incubated for 20 minutes at 4 ° C. with 50 μl FFC buffer containing different amounts of unlabeled recSPGF, recEPI, or recEGF. Biotinylated recSPGF (25 ng) was added to all samples, which were then incubated at 4 ° C. for 30 minutes. Cells were washed in FFC buffer and bound biotinylated recSPGF was detected by FFC with phycoerythrin (PE) -conjugated streptavidin (Molecular Probes, Eugene, OR). The data collected was mean fluorescence intensity (MFI). Inhibition of ligand binding is expressed as a ratio of (MFI _exp -MFI _min / MFI _max -MFI _min ) where MFI _exp is the MFI detected in the experimental sample, where MFI _min is the biotinylated recSPGF MFI detected in the negative control sample not added, and MFI _max is the MFI detected in the positive control sample to which biotinylated recSPGF was added but no inhibitor was added.

Cell lysate preparation, immunoprecipitation, and Western blotting
HeLa cells (80% -90% confluent) in 10 cm culture dishes were stimulated with various erb-B1 ligands at a concentration of 50 ng / ml for 10 minutes at 37 ° C. The dish was washed once with ice-cold PBS. Cells were then lysed with 1 ml lysis buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.35 trypsin inhibitor unit / ml aprotinin, 5 μg / ml leupeptin , 10 mM NaF, 10 mM β-glycerophosphate, and 1 mM Na ₃ VO ₄ ) in a dish directly at 4 ° C. for 30 minutes. The lysate was transferred to a microtube and after centrifugation, 0.5 ml lysate supernatant was added to 1 μg goat anti-EGFR polyclonal antibody (Santa Cruz Biotechnology, Inc.) and 10 μl gamma-binding plus ™ beads (Amersham Biosciences). ) At 4 ° C. overnight. The beads were washed 3 times and the protein was eluted from the beads directly in 2 × SDS-PAGE loading buffer. An aliquot of whole cell lysate or immunoprecipitated sample was resolved on 7.5% SDS-PAGE and the resulting gel was blotted onto a PVDF (polyvinylidene fluoride) membrane. Membranes in Tris-buffered saline / Tween-20 (TBST) with 2.5% bovine serum albumin (for erb-B receptor) or TBST with 2% gelatin (for 4G10) at 37 ° C. for at least 30 minutes Blocked. Membranes are incubated overnight at 4 ° C. with primary antibody according to manufacturer's instructions, washed, and 1: 10,000 anti-goat IgG (for erb-B receptor) or anti-mouse IgG2b (for 4G10) HRPO (Horseradish peroxidase) was incubated with the conjugate for 1 hour at room temperature. After extensive washing, the membrane was developed using a chemiluminescent reagent kit (Perkin Elmer Biosystems, Wellesley, Mass.) And MR film (Eastman Kodak Co., Rochester, NY).

  For tyrosine kinase inhibition experiments, HeLa cells were treated with inhibitors at 50 nM for 30 minutes at 37 ° C., followed by stimulation with recSPGF (50 ng / ml). Cell lysates are prepared and goat anti-erb-B1 polyclonal antibody (Santa Cruz Biotechnology, Inc.) and gamma-conjugated plus beads or anti-c-Cbl antibody-coated beads (Santa Cruz Biotechnology, Inc.) at 4 ° C. overnight for immunoprecipitation (as above). The beads were washed and eluted in 2 × SDS-PAGE loading buffer. Whole cell lysates or immunoprecipitates were analyzed by Western blotting with anti-EGFR or anti-c-Cbl polyclonal antibodies.

Chemical crosslinking of recSPGF and erb-B receptors
The 2.25 × 10 ⁶ of MB468 or MB453 cells were pretreated with 30min between 0.04% NaN ₃ at 4 ° C. in order to inhibit receptor internalization. Cells were then incubated with biotinylated recSPGF (1 μg / ml) for 30 minutes at 4 ° C. The culture dish was washed with ice-cold DMEM, and bis (sulfosuccinimidyl) -suberate (BS ³ ) crosslinker was added at a concentration of 1 mM. The reaction mixture was incubated for an additional hour at 4 ° C. with occasional shaking. After washing twice with ice-cold PBS, 1 ml lysis buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1% TritonX-100, 1 mM phenylmethylsulfonyl fluoride, 0.35 trypsin inhibitor unit / ml aprotinin , And 5 μg / ml leupeptin) were added directly to the dish followed by shaking at 4 ° C. for 30 minutes. The lysate is transferred to a microtube and centrifuged, and then the lysate supernatant is treated with 1 μg anti-erb-B1, anti-erb-B3, or anti-erb-B4 goat polyclonal antibody (Santa Cruz Biotechnology), or 1:40. It was subjected to immunoprecipitation (IP) with diluted anti-erb-B2 (Cell Signaling Technology, Beverly, Mass.) Rabbit polyclonal antibody and 10 μl of gamma-conjugated plus beads. After spinning the sample at 4 ° C. for 5 hours, the beads were washed 4 times with 1 × TBS / 1% Triton X-100 and then boiled in 2 × SDS-PAGE loading buffer. Western blotting with SDS-PAGE and streptavidin-HRPO was used to detect biotinylated recSPGF.

Affinity of recSPGF binding to EGFR
using lactoperoxidase catalyzed method for ¹²⁵ I- labeling RecSPGF, for ¹²⁵ I- labeling RecEGF, using chloramine T catalyzed process. The specific activity of labeled recSPGF was 0.54pm / cpm, and that of recEGF was 0.34pm / cpm. MB468 cells were seeded at a concentration of 3 × 10 ⁴ /0.1 ml / 96 well. ¹²⁵ I-labeled recSPGF or recEGF was added at various concentrations to 50 μl of binding medium solution of cells (L15 medium containing 0.1% NaN ₃ ) at 4 ° C. and incubated for 5 hours. The supernatant was collected and the cells were quickly washed twice with 60 μl ice-cold binding medium. The supernatant and washings were then combined. 50 μl of 0.5N NaOH was added to each well and the plates were incubated for 1 h at room temperature to lyse adherent cells and washed once more with 60 μl 0.5N NaOH. The solubilized solution and the washing solution were combined. Gamma count was used to determine the amount of free ligand in the culture supernatant and the amount of bound ligand in the cell lysate. Receptor number and affinity were determined by Scatchard analysis (eg, Current Protocols in Immunology. Eds. John E. Coligan et al., John Wiley & Sons. (2001)).

Erb-B1 internalization
HeLa cells were cultured on chamber slides and stimulated with Erb-B1 ligand (50 ng / ml) at 37 ° C. for 10 minutes. 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. Cells were incubated for 30 minutes at room temperature with 1% BSA / PBS mAb (Santa Cruz Biotechnology, Inc.), 1 μg anti-erb-B1. Bound anti-erb-B1 mAb was detected by staining with anti-mouse Ig-FITC (fluorescein isothiocyanate) conjugate.

  A431 epidermoid carcinoma cells were pretreated with 50 nM inhibitor for 30 minutes at 4 ° C. 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. After streptavidin-PE staining, MFIs of various samples were obtained by FFC.

Cells and viruses Vaccinia virus strain WR was grown in L929 cells and virus titer assays were performed in Vero cells as previously described [Selin et al. (1994) J. Exp. Med. 179: 1933- 1943; Selin et al. (1998) J. Exp. Med. 188: 1705-1715]. For all animal infections, crude vaccinia virus from tissue culture supernatant was used.

Vaccinia virus infection
6-8 week old male C57BL / 6 (B6) mice were injected intraperitoneally (ip) with 200 μg of test antibody and / or 1 mg of CI-1033 in PBS. The antibody was administered only once, but CI-1033 was given daily for a period of 6-10 days of the experiment. Six hours after antibody and initial CI-1033 administration, mice were infected intranasally with 4 × 10 ⁴ PFU (plaque forming units) of vaccinia virus. Mice were weighed daily and observed for symptoms of infection (1 = normal, 2 = disturbed fur; 3 = 2 plus curled posture; 4 = 3 plus little movement; 5 = 4 plus lethargy Minimal response). Virus titers were determined at the indicated times.

RNase Protected Cytokine Assay One lobe of the lung from each mouse sacrificed at various days after infection with vaccinia virus was disrupted in RLT buffer (Qiagen, Valencia, CA) through an 18 gauge needle. The lysate was further homogenized using the QIAshredder ™ module (Qiagen) and purified using the RNeasy mini ™ kit (Qiagen) according to the manufacturer's instructions. The same amount of total RNA (2 μg / mouse) was pooled from 3 mice at each time point (4, 6, and 8 days). 6 μg of RNA was hybridized with probes made from mCK-2b ™ template set (BD Biosciences-Pharmingen) according to the manufacturer's instructions and the protected fragments were resolved on a DNA sequencing gel. Autoradiograms were made by exposing the gel to X-ray film.

Intracellular cytokine staining Single cell suspensions were prepared from the spleens of infected mice treated in various ways and erythrocytes were removed by lysis using 0.84% NH ₄ Cl solution. Vaccinia virus-specific, IFN-γ-producing, CD8 ⁺ T cells, vaccinia virus-infected fibroblasts (MC57G), uninfected control MC57G fibroblasts, or anti-CD3 (145-2C11) mAb bound to a plate And then detected by FFC using Cytofix / Cytoperm Kit Plus ™ (GolgiPlug ™, BD Biosciences-Pharmingen) at 5 μg / ml.

Briefly, MC57G cells were infected with 5 × 10 ⁶ PFU virus for 2 h to infect vaccinia virus strain WR (MOI (infection efficiency) = 1.2). Thereafter, 1-2 × 10 ⁶ spleen cells from experimental mice were combined with 2.5 × 10 ⁵ vaccinia-infected MC57G (VV-MC57G) cells or uninfected MC57G cells in 96 well plates with 10 U / ml human recombinant IL- Incubated for 5 hours at 37 ° C. in the presence of 2 (BD Biosciences), and 0.2 μl GolgiPlug ™ reagent. Control cultures to detect all recently activated CD8 ⁺ T cells included anti-CD3 mAb. In these cultures, splenocytes were incubated with purified anti-mouse CD3 _ε mAb (145-2C11) coated at the bottom of the culture well (at a concentration of 5 μg / ml) (under the same conditions described above). .

After preincubation with 1 μL Fc Block ™ reagent (2.4G2 mAb) in a 96-well plate in 100 μl FFC buffer (HBBS (Hank parallel salt solution), 2% FCS, 0.1% NaN ₃ ) Cells were stained with a combination of fluorescent dye-labeled mAbs specific for CD8α (mAb 53-6.7) and CD44 (mAb IM7) (20 min, 4 ° C.). Thereafter, the cells were fixed and permeabilized to allow the anti-IFN _γ mAb to reach inside the cells (XMG1.2; BD Biosciences). Freshly stained samples were analyzed using Becton Dickinson FACS Calibur and CellQuest software (San Jose, CA).

Testing the effect of CI-1033 on pressure ulcer plaques and comet formation Confluent BSC-40 cell monolayers with various concentrations of CI-1033 in RPMI + 2% FBS (RPMI-2%) solution at room temperature 3 times Pretreated for 30 minutes or mock pretreated under identical culture conditions. Seven concentrations of CI-1033 were evaluated, and three mock pretreated controls for each concentration of CI-1033 were performed on seven individual 6-well tissue culture plates. Monolayers were infected with a suspension of pressure ulcer strain Solaimen so that 50 plaques were observed in each well. Plates were incubated for 1 hour at 35 ° C. in a 6% CO ₂ atmosphere and rocked at 15 minute intervals to ensure uniform infection of the monolayer. The inoculum was removed and the monolayer was rinsed with 1 × RPMI-2%. Monolayers were covered with medium without CI-1033 or at the appropriate concentration and incubated for 4 days at 35 ° C. in a 6% CO ₂ atmosphere. Plaques were analyzed by immunohistochemical staining. A comet was defined as larger than two consecutive small plaques combined into a large plaque and a comet type. The numbers of plaques and comets in the presence of different concentrations of CI-1033 were compared to control plates using the Wilcoxon rank sum test. Seven randomly selected mock-treated wells (selected from each of 7 plates) were used for comparison.

Example 2
The EGF-like domain of SPGF stimulates cell proliferation and / or cell survival Figure 2 shows the alignment of human and mouse epiregulin amino acid sequences, and orthopoxvirus homologous species D1L, pressure ulcer, camel camellia, vaccinia, and CMP11R, C11R, and D3R derived from monkeypox virus are shown. Note the high homology (~ 90%) between viral proteins with ~ 30% identity with mammalian proteins. Within the EGF-like domain itself, the virtual identity of the viral protein sequence is evident (Figure 2). In the 51-residue EGF-like domain (residues 40-90 of D1L (SEQ ID NO: 1) and D4R (SEQ ID NO: 2)), D4R differs from vaccinia homologous species by only 3 residues (C11R SEQ ID NO: 4). D1L (Figure 3A; SEQ ID NO: 1) from a large acne Indian strain differs from the corresponding molecule (Figure 3B; SEQ ID NO: 2) D4R from a large acne Bangladesh strain by only two amino acids.

  A series of functional studies were performed with recSPGF, recEPI, and recEGF. As shown by the following experiments, recSPGF stimulates cell proliferation or cell survival. Human primary fibroblasts (SC-J) were starved in medium containing 2% fetal bovine serum (FBS) for 6 hours and then stimulated with various concentrations of recSPGF, recEPI, or recEGF for 18 hours. Cell cycle analysis was performed using propidium iodide stained FFC. From the FFC data, the proportion of cells in the S phase of the cell cycle was calculated for each sample and plotted as a function of ligand concentration (FIG. 4). recSPGF showed lower potency than recEGF or recEPI (assessed by maximum percentage of cells induced in S phase), but recSPGF activity reached a plateau at a lower molar concentration than other growth factors .

  FIG. 5 shows that SPGF is a long-term growth factor for both primary human keratinocytes (N cells) and human fibroblasts (R2F). Cells 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”) The cells were cultured for 7 days, and the total viable cells were counted every day. Data are expressed as average population doublings per day.

Example 3
SPGF binds to the Erb-B1 receptor
There are four subtypes of EGFR: erb-B1, erb-B2, erb-B3, and erb-B4. To which erb-B receptor is determined whether binding to SPGF, two binding ^{experiments, MB453 (erb-B1 -,} -B2 +.-B3 +, -B4 +) and MB468 (erb-B1 ^{+ , -B2 -, -B3 +, -B4} -) was performed by recSPGF which is biotinylated using epithelial cell line. The first experiment was a cross-linking analysis. MB453 or MB468 cells were incubated with biotinylated recSPGF. Crosslinking of recSPGF and erb-B receptor, was carried out with a crosslinking agent BS ^3. A lysate was then prepared and subjected to immunoprecipitation, which was treated with HRPO-conjugated streptavidin (“Sav-HRPO”) (FIG. 6, upper panel) or with each erb-B receptor-specific antibody (FIG. 6). , Middle panel) by Western blotting. The expression of erb-B receptors 1-4 in these two cell lines is shown in the table shown in the lower panel of FIG. The upper panel of FIG. 6 demonstrates that recSPGF binds to a detectable extent only to erb-B1.

  Consistent with the chemical cross-linking results, FIG. 7 shows that anti-erb-B1 mAb blocks recSPGF binding to MB468 epithelial cells. Since MB453 cells do not express erb-B1, binding of recSPGF to them was not detected (FIG. 7).

  Binding inhibition experiments indicate that SPGF binding to erb-B1 is slightly weaker than EGF but stronger than epiregulin (Figure 8).

Measurement of the affinity of recSPGF binding to EGFR1-expressing cells was obtained by Scatchard analysis (FIG. 9). MB468 cells were seeded in the wells of a 96 well microtiter tissue culture plate. ¹²⁵ I-labeled recSPGF was added at various concentrations and the plates were incubated at 4 ° C. for 5 hours. Affinity (Kd) and number of receptors were calculated as described in Example 1. In this analysis, there is about 5.5 x 10 ⁴ high affinity (Kd = 0.14 nM) erb-B1 receptor and 8.8 x 10 ⁶ low affinity (Kd = 258 nM) erb-B1 receptor per MB468 cell Showed that.

  The erb-B1 receptor is usually found in epithelial cells as evidenced by the almost uniform pattern of linear fluorescence obtained when staining unstimulated cells with an antibody specific for erb-B1 (Figure 10A). It was distributed fairly uniformly on the surface. However, 10 minutes after the addition of recSPGF, recEPI, or recEGF, the receptor was rapidly internalized (FIGS. 1OB, 1OC, and FIG. 10D, respectively).

  The ability to activate IMV invasion via rapid internalization of erb-B1 activated by recSPGF and the formation of cell protrusions containing actin and ezrin by tyrosine protein kinase phosphorylation [Locker et al. (2000 Mol. Biol. Cell 11: 2497-2511] both contribute to facilitate the entry of pressure ulcer virions into cells. Naturally, the initial binding of a decubitus virion to the cell surface prior to invasion can undergo at least in part the SPGF-erb-B1 interaction. In addition to allowing virion entry into the cell, the above activation event makes the host cell a more efficient viral replication “factory”.

Example 4
The EGF-like domain of SPGF stimulates protein tyrosine phosphorylation in cells
RecSPGF stimulated phosphorylation of EGF receptors and their cellular substrates. Western total of 4G10 anti-phosphotyrosine monoclonal antibody from whole cell lysates from HeLa cells treated with no treatment (-) or recSPGF ("SPGF"), recEPI ("EPI"), or recEGF ("EGF") Blot method (WB) was used (FIG. 11A). In addition, HeLa cell lysate was immunoprecipitated (IP) with anti-EGFR polyclonal antibody and subjected to WB with anti-phosphotyrosine antibody (FIG. 11B). The tyrosine residues of both EGFR itself and its various cell substrates were phosphorylated in a similar manner, even if not identical, by the three growth factors tested.

Example 5
Stimulation of protein tyrosine phosphorylation by SPGF's EGF-like domain is inhibited by quinazoline-based compounds
To evaluate the effect of several quinazoline-based tyrosine kinase inhibitors on SPGF-stimulated activity, a series of experiments were performed. FIG. 12 shows the chemical structure of three 4-anilinoquinazoline tyrosine kinase inhibitors (PD 153035, PD 168393, and CI-1033) as well as the general 4-anilinoquinazoline structure. The structure of the erb-B1 receptor kinase domain alone and in complex with one of such inhibitors shows how these 4-anilinoquinazolines bind to the ATP binding pocket of the kinase domain [Stamos et al. (2002) J. Biol. Chem. 277: 46265-46272]. As shown in FIG. 12, PD153035 and PD168393 have the same 4- (3′-bromo-aniline) ring, but their R3 and R4 groups attached to the quinazoline ring are different. In particular, PD168393 has an acrylamide in position 6 that can alkylate erb-B1 Cys ⁷⁷³ , so that the inhibitor binds erb-B1 irreversibly in a 1: 1 molar ratio. The tyrosine kinase active erb-B2 and erb-B4 molecules have cysteine residues corresponding to Cys ⁷⁸⁴ and Cys ⁷⁷⁸ , respectively, and can be targeted for modification. In contrast, PD153035 binds in a reversible manner primarily via hydrophobic forces. CI-1033, like PD168393, has an acrylamide adduct at position 6 (R3), and therefore binds erb-B1 irreversibly via Cys ⁷⁷³ , like PD168393. CI-1033 additionally has a soluble morpholine side chain at position 7 (R4). CI-1033 exhibits IC ₅₀ values of 0.8, 19, and 7 nM for erb-B1, erb-B2, and erb-B4, respectively [Allen et al. (2002) Sem. Oncol. 29: 11- twenty one].

  In the first experiment, two quinazoline based compounds (PD168393 and PD153035) and pyridopyrimidine based compound (PD158780) were tested for their ability to inhibit tyrosine phosphorylation. All three compounds were obtained from Calbiochem, San Diego, CA, and their chemical structure is shown in the Calbiochem catalog. HeLa cells were seeded into wells of 24-well tissue culture plates and inhibitor compounds were added to a final concentration of 50 nM. Cells were incubated for 30 minutes and recSPGF (50 ng / ml) was added to all wells except the negative control sample (shown as (−) in FIG. 13); the sample shown as (+) in FIG. Received but not received the inhibitor. Cells were then incubated for an additional 10 minutes at 37 ° C. and then washed and lysed. Cell lysates were separated by 10% SDS-PAGE and SDS-PAGE gels were blotted onto PVDF membranes. The membrane was exposed to 4G10 anti-phosphotyrosine mAb and its binding was detected by HRPO-conjugated anti-mouse IgG2b polyclonal antibody. The membrane was developed by ECL (enhanced chemiluminescence). The data in FIG. 13 shows that tyrosine phosphorylation activated by recSPGF is inhibited by all three compounds (PD168393, PD153035, and PD158780).

  The effect of tyrosine kinase inhibitors on recSPGF-mediated cell proliferation and / or increased cell survival was tested. As shown in FIG. 14A, after overnight stimulation of human fibroblasts with various concentrations of recSPGF, the number of cells entering S phase approached 8%. Pretreatment of cells with the indicated inhibitors at 50 nM for 1 hour at 37 ° C. blocks this increase in DNA synthesis. FIG. 14B shows that the same compounds that inhibited tyrosine phosphorylation of erb-B1 (150 kD band) and additional substrates of that phosphorylation (120 kD, 80 kD, 60 kD and 55 kD) were activated by recSPGF in HeLa cells It shows that. The Src family PTK specific inhibitor PP2 had little ability to block phosphorylation of these erb-B1 substrates, even when used at 10 μM. In contrast, addition of PD168393 or CI-1033 at a concentration of 50 nM prevented phosphorylation triggered primarily by recSPGF, but only a partial effect was observed with PD153035. These results suggest that reversible inhibitors of kinase activity are less efficient at blocking protein tyrosine phosphorylation compared to irreversible inhibitors at the 50 nM concentration tested. Similar inhibition of tyrosine kinase phosphorylation was also observed with phosphorylation activated by vaccinia virus growth factor (VGF) (data not shown).

  FIG. 10B shows the distribution of erb-B1 receptors on human HeLa epithelial cells detected by immunofluorescence microscopy before and after incubation with recSPGF at 37 ° C. for 15 minutes. In the absence of growth factors, the distribution of erb-B1 is mainly restricted to the membrane. In contrast, after SPGF exposure, erb-B1 molecules are rapidly internalized and appear as punctate intracellular fluorescent aggregates. To determine whether tyrosine kinase inhibitors affect SPGF capacity and downmodulate erb-B1, A431 cells were pretreated with inhibitors, incubated with biotinylated recSPGF, and then fixed . The erb-B1 ligand bound to the cell surface was then visualized with streptavidin-PE and fluorescence was quantified by FFC analysis. FIG. 14C shows that in the absence of inhibitor, the MFI was ˜110; in contrast, the irreversible erb-B inhibitor increased the MFI ˜3-fold (300-350) and was reversible The target erb-B1 inhibitor PD153035 increased the MFI to 240. The src family kinase inhibitor PP2 only slightly affected the copy number of the erb-B1 surface.

  The erb-B receptor is removed by two pathways: 1) Ligand-dependent endocytosis and degradation involving the c-Cbl ubiquitin ligase mechanism [Levkowitz et al. (1999) Mol. Cell 4: 1029- 1040], and 2) shuffling of stress-induced erb-B receptor-bound chaperones and the involved proteasomal proteinases [Xu et al. (2001) J. Bio. Chem. 276: 3702-3708]. Recent studies show that inhibition of erb-B2 kinase by CI-1033 promotes erb-B2 down-regulation via a second process [Citri et al. (2002) EMBO J. 21: 2407- 2417]. However, given that CI-1033 enhances erb-B1 surface expression, this finding suggests that the effect of CI-1033 on ligand-specific SPGF erb-B1 expression is on constitutive erb-B2 expression. Suggests dissimilarity.

  To test whether 4-anilinoquinazoline affects the interaction of c-Cbl with the erb-B receptor, lysed HeLa cells pretreated with inhibitors or untreated recSPGF-stimulated HeLa cells. The lysate was immunoprecipitated with anti-erb-B1 antibody. Western blotting is then performed either with anti-erb-B antiserum to quantify the expression of erb-B1 molecules or with anti-c-Cbl antibodies to assess the effect of compounds on erb-B1 binding. went. As shown in FIG. 14D, compared to unstimulated cells (−), RecSPGF-triggered HeLa cells (+) have less total erb-B1 due to rapid intracellular degradation. Consistent with the FFC analysis (Figure 14C), PP2 had little effect on this process. On the other hand, pretreatment with PD153035, PD168393, and CI-1033 increased the total erb-B1 protein that was immunoprecipitated despite the addition of recSPGF; over that obtained from control cells that were not recSPGF stimulated It showed a several-fold increase over erb-B1. The latter result implies constitutive internalization or degradation blockage of erb-B1 by inhibitors. More importantly, recSPGF-induced c-Cbl binding to erb-B was blocked by 4-anilinoquinazoline but not by PP2. The decrease in c-Cbl / erb-B1 complex formation was secondary to the decrease in total cellular c-Cbl levels as shown by parallel c-Cbl immunoprecipitation and Western blotting. Instead, these erb-B kinase inhibitors prevented inducible c-Cbl binding to erb-B1 after SPGF binding.

Example 6
In Vivo Antiviral Effects of Tyrosine Kinase Inhibitors Disruption of Vaccinia WR VGF gene has been shown to reduce vaccinia virus virulence in vivo and slow viral growth up to 10,000-fold [Buller et al. (1988) J. Virol. 62: 866-874]. As shown above, CI-1033 blocks SPGF-stimulated erb-B1-driven cell proliferation or survival, receptor-mediated tyrosine phosphorylation, internalization, and degradation, so erb -B1 kinase inhibitors appeared to be able to reduce orthopox growth factor activity in vivo. To test this possibility, we investigated the effect of CI-1033 on the clinical course of B6 mice lethally given vaccinia WR intranasally [Chen et al. (2001) Nat. Immunol. 2: 1067- 1076]. As shown in FIG. 15A, all untreated animals died by 7 days after infection with fulminant acute pneumonia. In contrast, ip started with CI-1033 at 50 mg / kg daily starting 6 h before infection prevented death. The therapy was as effective as a single 200 μg ip dose of anti-L1R vaccinia virus mAb (7D11), known to neutralize vaccinia intracellular mature virus particles (IMV) [Hooper et al. al. (2000) Virol. 266: 329-339]. The combination of CI-1033 and anti-L1R was also protected. Clinical monitoring of animals after infection showed that anti-L1R mAb or CI-1033 treatment significantly reduced overall symptoms but was not as efficient as combination therapy (FIG. 15B).

  The animal cohort was sacrificed 6 days after infection and these lungs were examined. The difference in total lung weight between treatment groups is shown in FIG. 15C. Untreated animals had multiple hemorrhages and edematous lungs with approximately twice the weight of normal, non-infectious B6 mice. This condition was somewhat attenuated by treatment with anti-L1R or CI-1033 alone, but was dramatically improved by the combination of CI-1033 plus anti-L1R. Consistent with pathological findings, vaccinia virus titers in the lung were slightly reduced by anti-L1R or CI-1033 alone when assayed on day 8, but essentially eliminated by combination therapy It was done. Since all untreated animals died by day 8, the 7 day viral lung titer was used for comparison. FIG. 15D also shows: (i) immunotherapy with anti-SPGF mAb (13E8), which cross-reacts with VGF (see pending US Application Serial No. 10 / 429,685); and (ii ) Illustrates the results of double immunotherapy with anti-L1R plus anti-SPGF mAbs. Two additional course record reductions in viral titer observed with anti-L1R plus CI-1033 treatment (compared to anti-L1R mAb plus anti-SPGF mAb treatment) compared to the reversible binding of mAb: This may be the result of irreversible binding of CI-1033 [Fry et al. (1998) Proc. Natl. Acad. Sci. USA 95: 12022-12027]. Irreversible binding of the inhibitor will result in a more complete blockage of the erb-B1 pathway than reversible binding of the inhibitor.

  In a combined anti-L1R mAb plus anti-SPGF mAb immunotherapy experiment (see pending U.S. Application Serial No. 10 / 429,685), virus titer did not decline until 6 days after infection. This slow time course suggested that an adaptive T cell response to the virus was generated rather than the virus neutralizing effect of the antibody. Although not shown, similar kinetics were observed with anti-L1R mAb plus CI-1033 combination therapy. These findings suggest that CI-1033, either alone or in combination with anti-L1R mAbs, will increase T cell responses. To test this concept, T cell-regulated cytokine production in the lung was tested by RNase protection analysis at 4, 6, and 8 days after infection. As shown in FIG. 16A, in mice given CI-1033 alone, levels of interleukin-1β (IL-1β), interleukin-1 receptor antagonist (IL-1Ra), and IFN-γ mRNA Increased compared to untreated mice and peaked at 6 days. For example, the level of IFN-γ RNA was 4.1 times greater at day 6 than in the lungs of CI-1033 treated mice compared to that of control infected animals. However, effects on the levels of interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 12 (IL-12), or macrophage migration inhibitory factor (MIF) mRNA given CI-1033 Neither was seen in the lungs of other mice.

Consistent with the levels of lung cytokines, systemic antigen-specific T cell responses to VV-MC57G cells were evaluated in control untreated mice as assessed by intracellular IFN-γ production of splenic CD8 ⁺ CD44 ⁺ T cells As compared to CI-1033 treated mice (FIG. 16B). The proportion of IFN-γ producing CD8 ⁺ CD44 ⁺ T cells activated by VV-MC57G cells increased 10-fold 8 days after infection. This increase corresponded to a 21-fold increase in the absolute number of IFN-γ producing CD8 ⁺ CD44 ⁺ T cells activated by VV-MC57G cells (1.85 × 10 ⁵ vs 8.5 × 10 ³ cells); , Due to lymphopenia in untreated vaccinia virus-infected mice. In the same experiment, a 22.7 and 48-fold increase was observed for anti-L1R and anti-L1R + CI-1033 treated mice, respectively. Anti-L1R treatment produced more IFN-γ cytokine RNA in the lung than anti-L1R + CI-1033 dual therapy, indicating a sustained viral load with the former treatment (FIG. 15D). ). There was a slight but significant increase in the ratio of IFN-γ producing CD4 ⁺ CD44 ⁺ T cells after anti-L1R and anti-L1R + CI-1033 treatment of mice (data not shown).

Example 7
Enhancement of in vivo immunity against vaccinia virus by antibodies specific for VGF In the ongoing US Application No. 10 / 429,685, as with CI-1033, mAbs that are specific for pressure ulcer SPGF D4R and cross-react with VGF (13E8) was shown to enhance the protective effect of anti-L1R mAb. As used herein, with respect to CI-1033 (see Example 6), mAb 13E8 enhances T cell immunity against vaccinia virus in vivo and increases the enhancement of T cell immunity obtained by anti-L1R mAbs in vivo Is shown.

Since vaccinia virus is regulated in vivo by IFN-γ producing T cells, its levels peak 6-8 days after infection [Ruby et al. (1991) Lymphokine Cytokine Res. 10: 353-358]. , 13E8 and anti-L1R mAb found kinetics of virus reduction (co-current US Application No. 10 / 429,685) suggested that adaptive immunity is involved. Thus, the CD8 ⁺ T cell responses were examined on day 7 treated with below mice infected with vaccinia virus: (a) control 1A3 mAb, (b) 13E8 mAb , (c) anti L1R mAb; or (d ) Combination of 13E8 and anti-L1R mAbs. Intracellular in freshly isolated splenic T cells stimulated in vitro for 5 h with VV-MC57G stimulator cells or with anti-CD3 mAbs (which stimulate all recently activated T cells) IFN-γ levels were analyzed. The experiment showed increased IFN-γ responses of CD44 ⁺ CD8 ⁺ T cells in the treatment group infected with vaccinia in the following order compared to naive uninfected mice: 1A3 mAb (control), 13E8 mAb, anti-L1R mAb, and anti-L1R mAb + 13E8 mAb (Figure 17C). In this and other experiments, the spleen expanded in the anti-L1R mAb and anti-L1R mAb + 13E8 mAb groups, resulting in, for example, vaccinia virus specific per spleen of mice treated with a combination of anti-L1R mAb + 13E8 mAb The total number of IFN-γ producing cells was 11 times higher than 1A3-treated mice (0.1 marked 3.3 × 1.4 × 10 ⁵ vs. 0.3 ± 0.1 × 10 ⁵ , n = 3 per group).

There was a small but significant increase in the ratio of IFN-γ producing CD4 ⁺ CD44 ⁺ T cells after anti-L1R and anti-L1R + 13E8 mAb treatment in mice (data not shown).

Enhanced levels of activated IFN-γ producing spleen T cells in anti-L1R + 13E8 mAb-treated mice suggests a reservoir of cells capable of migrating to the lung to combat lung infection; CD44 ( Note that activation markers (up-regulated by T cell stimulation) are recyclable hyaluronan receptors involved in leukocytes homing to the site of inflammation [DeGrendele et al. (1997) Science 278: 672 -675]. To test this possibility, lungs were collected from uninfected or 7 days infected, and mAb-treated mice and examined by hematoxylin-eosin staining of tissue sections. Lungs from infected control mAb-treated mice were necrotic bronchiole epithelium compared to lungs from uninfected mice where the alveolar space was easily distinguishable and bronchiole epithelium was untreated And characterized by severe alveolar edema. This reflected that the weight of the lung infected with vaccinia virus was approximately twice that of the uninfected lung (.35 ± .04 g vs. .21 ± .01 g, n = 3). Alveolar cavities were removed in that they were filled with acidophilic substances; there was slight, if any, parenchymal cell infiltration. The degree of cell invasion was somewhat increased in 13E8 mAb treated mice, more increased in anti-L1R mAb treated mice, and most dramatically increased in mice treated with combined mAbs. In the double mAb treatment group, the alveolar space was open and the bronchiole epithelium was normal. Most of the cells that infiltrated were CD8 ⁺ T cells. Leukocytes isolated from pooled lungs of 3 anti-L1R mAb treated mice are 25% CD8 ⁺ and 5% CD4 ⁺ T cells, isolated from anti-L1R mAb plus 13E8 mAb treated mice There were 34% CD8 ⁺ and 8% CD4 ⁺ T cells. Thus, the non-neutralizing 13E8 mAb enhances vaccinia virus clearance associated with increased spleen and lung T cell responses.

  Innate immunity, the systemic inflammatory component of the so-called acute phase response, is rapidly induced when the integrity of the organism is disrupted [Yoo et al. (2003) Proc. Natl. Acad. Sci USA 100: 1157-1162]. Sentinel cells, including epithelial cells, produce IL-1β and / or IL-6 during this process [Schluns et al. (1997) J. Immunol. 1158: 2704-2712]. Blocking erb-B1 stimulation by 13E8 mAb will help avoid subsequent cytokine dysregulation and associated immune suppression by virally encoded molecules [Diehl et al. (2002) Mol. Immunol. 39: 531-536; Tanaka et al. (2001) J. Mol. Cell Cardiol. 33: 1627-1635]. Furthermore, EGF and TGFα (both erb-B ligands) can induce exudates in vivo [Ohmura et al. (1990) Cancer Res. 50: 4915-4917]. ELGF, such as SPGF and VGF, may contribute to similar pathologies, and its inhibition by anti-ELGF antibodies (eg, l3E8 mAb) constitutes further therapeutic activity of antibodies against such orthopoxvirus infections I think that.

Example 8
Protein tyrosine kinase inhibitors inhibit intracellular formation and cell-to-cell propagation of variola virus To examine the effect of CI-1033 on variola virus proliferation, confluent monolayers of BSC-40 cells can be Approximately 50 plaque forming units of pressure ulcer strain Solaimen were infected in the presence or absence of an erb-B inhibitor and cultured in vitro for 4 days. As shown in FIG. 18A, increasing the concentration of CI-1033 drastically reduced individual plaque size and comet formation, but had a minimal effect on plaque number. The effects on plaque size and comet formation were titratable (FIGS. 18A, 18B, and 18C); at the highest concentration of CI-1033 (10 μM), only very small plaques appeared faint. The total number of plaques at each concentration was not significantly different when compared to 7 randomly selected control wells (FIGS. 18A and 18B).

  Comets show extracellular outer membrane virus (EEV) formation and dissemination [Payne (1980) J. Gen. Virol. 50: 89-100]. FIG. 18C shows a statistically significant decrease in the number of comets (p <0.05) at CI-1033 concentrations of 500 nM and above. These aggregated results suggest that CI-1033 is not a primary infection but rather blocks EEV viral morphogenesis. In particular, the release of EEV is sensitive to inhibition.

  A less significant effect on plaque size is seen and appears qualitatively at higher concentrations of CI-1033, indicating an effect on cell-bound inner and outer membrane virus (CEV) formation. Orthopoxviruses use both microtubules and actin filaments to emerge [Hollinshead et al. (2001) J. Cell Biol. 154: 389-402; Smith et al. (2002) J. Gen. Virol .83: 2915-2931], CEV induces the actin tail to drive itself out of the cell. Since erb-B1 activation reorganizes the actin microfilament system [Lynch et al. (2003) J. Biol. Chem. 278: 21805-21813], it is a cell-associated virus and an outer membrane virus. It is likely that both releases are blocked, delaying the propagation of secondary virus between cells, and thus limiting the size of an individual's plaque.

  The more important related effects on the widespread transmission of viruses via EEV (as shown by inhibition of comet formation) suggest a possible additional interference mechanism. Since erb-B endocytosis is also linked to the actin cytoskeleton [Lynch et al.], erb-B1 kinase inhibition may also help maintain surface EGFR expression via this mechanism. unknown. CI-1033 blocks erb-B activation by trans-stimulation of erb-B1 via cellular erb-B ligands or other receptors, as well as effects on cytoskeletal elements used for downstream poxvirus emergence Is likely to underlie a significant effect on plaque morphogenesis not observed with anti-SPGF mAbs (data not shown).

  In short, combined immunotherapy / chemotherapy is very effective at reducing viral titer and stimulating T cell immunity. The present invention is not limited by any particular mechanism of action, but the latter has prevented the generation of anti-inflammatory viral products, as well as the round after viral DNA replication in epithelial cells dependent on viral growth factors. [Alcami (2003) Nat. Rev. Immunol. 3: 36-50; Seet et al. (1998) Ann. Rev. Immunol. 21: 377-423]. The use of blocking host cell signaling pathways as a target for antiviral chemotherapy is typically distinct from other approaches directed against the pathogen itself. One advantage of a strategy targeted to the host is that it cannot develop drug resistance. In addition, unlike anti-SPGF mAbs, erb-B tyrosine kinase inhibitors may be used in various erb-Bs where cross-reactivity with other closely related virulence factors (ie, VGF versus SPGF) is not guaranteed. It can potentially block immunologically different ligands for protein tyrosine family numbers. For example, VGF and myxoma virus growth factor (MGF) bind to different erb-B receptors [Tzahar et al. (1998) EMBO J. 17: 5948-5963], and this broader target activity Related.

  It appears likely that CI-1033 alone would be sufficient to delay “actual” infection by orthopox (eg, natural or bioterrorist epidemic). The dose of inhalation of pox particles is likely to be significantly less than that used in this study. Its CI-1033 increases T cell response and also reduces the overall symptoms of infection: (a) as an immunostimulant in combination with vaccinia vaccination; or (b) treatment of vaccination complications Use in is also suggested.

  In addition to poxviruses and these growth factors for erb-B, hepatitis B virus and EB virus also have genes encoding products that dysregulate erb-B1 transcription [Menzo et al. (1993) Virol 196 878-882; Miller et al. (1995) J. Virol. 69: 4390-4398]. In addition, RNA-type tumor viruses such as chicken erythroblastosis virus also utilize erb-B1 signaling [Adelsman et al. (1996) J. Virol. 70: 2533-2544]. Furthermore, functional EGF receptors have been reported to be required for effective reovirus and human cytomegalovirus (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 associated with erb-B mediated signaling networks. Epithelial cells that constitutively or inducibly express erb-B receptors are components of the innate sentinel cell line in the body [Yoo et al. (2003) Proc. Natl. Acad. Sci USA 100: 1157 -1162]. erb-B inhibitors can be applied more broadly in infections than orthopoxvirus infections. The development of chemical inhibitors of cellular signaling pathways utilized by viral virulence factors will generally lead to new approaches to the control of infectious diseases.

  A number of aspects of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other aspects are within the scope of the claims.

Coomassie blue-stained SDS-PAGE of a recombinant form of the epidermal growth factor (EGF) -like domain of mouse epiregulin (“Epi”), human EGF, and variola strain of smallpox Bangladesh (“SPGF”) It is a photograph of a sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel. These recombinant proteins are referred to herein as recEGF, recEPI, and recSPGF, respectively. The gel position of the molecular weight (MW) marker is shown in the upper left of the photo and the value is shown as MW × 10 ⁻³ . Human epiregulin (SEQ ID NO: 6) and mouse epiregulin (SEQ ID NO: 7), and orthopoxvirus homologous species D1L (SEQ ID NO: 7) derived from Indian variola strain, camel camellia, vaccinia, and simian virus In depiction of amino acid sequence alignments (for optimal homology) of ID NO: 1), CMP11R (SEQ ID NO: 3), C11R (SEQ ID NO: 4), and D3R (SEQ ID NO: 5) is there. The region corresponding to the leader sequence and transmembrane region is shown, and the most conserved segment of the EGF-like domain is framed and labeled “EGF”. FIGS. 3A and 3B are depictions of the amino acid sequences of SPGFs of the Indian variola strain (D1L) (SEQ ID NO: 1) and the variola strain Bangladesh (D4R) (SEQ ID NO: 2), respectively. The different positions of the two sequences are shown in bold and underlined. Of S-phase human primary fibroblasts (SC-J) in the cell cycle after 18 hours of culture with the indicated concentrations of recEGF (“EGF”), recEPI (“EPI”), and recSPGF (“SPGF”) It is a line graph which shows a ratio. In standard tissue culture medium with 10% fetal bovine serum (FBS) (“complete”) or with 2% FBS and the indicated concentrations of recSPGF (“SPGF”) or recEGF (“EGF”) FIG. 5 is a bar graph showing proliferation of primary human keratinocytes (“N keratinocytes”) and primary human fibroblasts (“R2F fibroblasts”) during culture in any of the tissue culture media. 2 is a pair of photographs of a Western blot of immunoprecipitates from MB453 cells (“453”) or MB468 cells (“468”). Immunoprecipitates, were prepared by incubating cells with recSPGF which is biotinylated, and exposure of the cells to the cross-linking agent BS ³ (a recSPGF molecules biotinylated bound to erb-B receptors on the cell surface Lysing cells and erb-B receptors, ie erb-B1 (“B1”), erb-B2 (“B2”), erb-B3 (“B3”), or erb -Prepared by immunoprecipitation ("IP") of an aliquot of lysate with an antibody specific for B4 ("B4"). Two duplicate Western blots were made from the immunoprecipitates; one stained with horseradish peroxidase conjugated streptavidin (“Sav-HRPO”) (FIG. 6, upper panel), the other with each erb-B Staining was performed with a receptor-specific antibody (FIG. 6, middle panel). The lower panel of FIG. 6 shows 4erb-B receptor expression by MB453 (“453”) and MB468 (“468”) cells. Biotinylated recSPGF (detected by phycoerythrin (PE) -conjugated streptavidin) in the absence (white circles) and presence (“+ mAb”; black circles) of blocking antibodies specific for erb-B1 (“SPGF” ) Is a line graph showing the binding of the indicated amount (“ng”) to MB468 cells (“versus 468”). Data is shown as mean fluorescence intensity (“MFI”), which was determined by fluorescence flow cytometry (FFC). In addition, the binding of recSPGF to MB453 cells that do not express erb-B1 (“pair 453”) was tested (“pair 453”; white triangle). Relative ability of the indicated concentrations of recSPGF (“SPGF”), recEPI (“EPI”), and recEGF (“EGF”) to inhibit the binding of biotinylated recSPGF to MB468 cells (expressed as% inhibition) It is a line graph which shows. Scatchard plot of recSPGF binding to MB468 cells. Analysis of SPGF on cells is about 5.5 × 10 ⁴ high-affinity receptor (“K _D = 0.14 nM”) and about 8.8 × 10 ⁶ low-affinity receptor (“K _D = 258 nM”) cells Existence above. 10A-D are unstimulated (“(−)”; FIG. 1A), or recSPGF (“SPGF”; FIG. 10B), recEPI (“EPI”: FIG. 10C), or recEGF (“EGF”; Figure 10D) Erb-B1 receptor (mouse antibody specific for erb-B1 receptor and fluorescein isothiocyanate (FITC) -conjugated anti-mouse Ig antibody on either HeLa cells stimulated for 10 minutes with either Is a series of fluorescence micrographs showing the cell distribution of those detected by. FIG. 11A shows cells of HeLa cells that are either untreated (“(-)”) or stimulated with recSPGF (“SPGF”), recEPI (“EPI”), or recEGF (“EGF”). It is the photograph of the western blot of the whole lysate. Western blots were stained with mAbs specific for phosphotyrosine residues ("WB: 4G10"). The position of the molecular weight (MW) marker on the gel is shown in the upper left of the photograph and the value is shown to be MW × 10 ⁻³ . FIG. 11B shows cells of HeLa cells that are either untreated (“(-)”) or stimulated with recSPGF (“SPGF”), recEPI (“EPI”), or recEGF (“EGF”). It is a photograph of the western blot of the lysate immunoprecipitate. Immunoprecipitates were prepared with antibodies specific for erb-B1 (“IP: EGFR”). Western blots were stained with mAbs specific for phosphotyrosine residues ("WB: 4G10"). 12A-12D shows the general chemical structure of a 4-anilinoquinazoline compound showing the related atom numbering (“generic 4-anilinoquinazoline”; FIG. 1D); as well as three specific 4- 2 is a depiction of an anilinoquinazoline compound, PD153035 (FIG. 12A), PD168393 (FIG. 12B), and CI-1033 (FIG. 12C). Untreated ("(-)") or pre-exposed to an erb-B protein tyrosine kinase inhibitor, or three erb-B protein tyrosine kinase inhibitors, PD153035, PD158780, and FIG. 6 is a photograph of a Western blot of whole cell lysates of HeLa cells stimulated with either recSPGF (“SPGF”) pre-exposed to one of PD168393. Western blots were stained with mAbs specific for phosphotyrosine residues. The position of the molecular weight (MW) marker on the gel is shown in the upper left of the photograph and the value is shown to be MW × 10 ⁻³ . FIG. 14A shows cells after pretreatment without pretreatment (“SPGF alone”) or stimulated overnight with PD153035, CI-1033, or PS168393; and the indicated concentrations of recSPGF (“ng / ml”); It is a line graph which shows the ratio of the human primary fibroblast (SC-J) of the S phase of a cycle. FIG. 14B shows untreated (“(−)”) or pre-exposed to erb-B protein tyrosine kinase inhibitor (“(+)”) or Src family protein tyrosine Whole cell potential of HeLa cells stimulated with recSPGF (“SPGF”), pre-exposed to kinase inhibitor PP2 or erb-B protein tyrosine kinase inhibitor, PD153035, PD16839, or CI-1033 It is the photograph of the western blot of a lysate. Western blots were stained with mAbs specific for phosphotyrosine residues ("WB: PY (4G10)"). The position of the molecular weight (MW) marker on the gel is shown in the upper left of the photograph and the value is shown to be MW × 10 ⁻³ . The arrow in the upper right of the photo indicates the position of protein phosphorylation affected by erb-B1 protein tyrosine kinase inhibition. FIG. 14C is a bar graph showing erb-B1 receptor expression levels (of MFI as determined by FFC) on A431 cells after: pretreatment without inhibitors, or PP2, PD153035, PD168393 Or pretreatment with CI-1033; and 5 min incubation with biotinylated recSPGF. FIG. 14D shows cell lysis of HeLa cells that were not pretreated (“(+)”) or pretreated with either PP2, PD153035, PD168393, or CI-1033, and then stimulated with recSPGF for 5 minutes. FIG. 3 is a series of photographs of Western blots of product immunoprecipitates (“IP”). Cells were also run with recSPGF ("(-)") controls that were not pretreated or stimulated. Immunoprecipitates (“IP”) were prepared with either an antibody specific for erb-B1 (“EGFR”) or an antibody specific for c-Cbl (“c-Cbl”). Western blots were stained with mAbs specific for erb-B1 (“EGFW”; upper panel) or c-Cbl (middle and lower panels) (“WB”). FIG. 15A is challenged intranasally with vaccinia virus and untreated (“control”; open circle), pre-treated with anti-L1R mAb (“anti-L1R”; filled circle), or treated daily with CI-1033 Mice ("CI-1033"; open triangles) or mice pretreated with anti-L1R mAb once and treated daily with CI-1033 ("CI-1033 + anti-L1R"; black inverted triangles) Is a line graph showing survival over time (in percentage of mice in various treatment groups). FIG. 15B is a line graph showing the clinical scores (derived from those described in Example 1) of the mouse experimental group described in FIG. 15A. The same symbols used in FIG. 15A are used to indicate various experimental groups. FIG. 15C shows no treatment (“control”), pretreatment with anti-L1R mAb (“anti-L1R”), daily treatment with CI-1033 (“CI-1033”), 6 days after lethal challenge with vaccinia virus, or 1 is a bar graph showing the average weight of all lungs from any mouse pretreated with anti-L1R mAb and treated daily with CI-1033 (“CI-1033 + anti-L1R”). FIG. 15D shows pretreatment with anti-L1R mAb (“anti-L1R”), daily treatment with CI-1033 (“CI-1033”), pretreatment with 13E8 mAb, anti-L1R, 8 days after infection against vaccinia virus. Mean viral titer in the lungs of either pre-treated with mAb and 13E8 mAb, or pretreated with anti-L1R mAb and treated daily with CI-1033 (“CI-1033 + anti-L1R”) (“Log ₁₀ PFU”; PFU (plaque forming unit)). Since all mice in the control group of this experiment (“none”; hatched bars) (mice infected with virus but not pre-treated or treated) died on day 8 of the experiment, the control group was , Derived from a separate experiment of mice sacrificed on day 7. Figure 16A shows no infection ("0"); after indicated days of infection against vaccinia virus ("4", "6" or "8"): Rnase protection using RNA prepared from mouse lungs 2 is a photograph of an autoradiogram from an assay. Infected animals are untreated (“control”), pretreated with anti-L1R mAb (“anti-L1R”), treated daily with CI-1033 (“CI-1033”), or anti-L1R mAb Once treated with CI-1033 ("CI-1033 + anti-L1R") daily. As shown in the upper left of the photograph, lung RNA was hybridized with a cytokine-specific probe of the mCK-2b template set (BD Biosciences-Pharmingen, San Diego, Calif.). The protected fragment was lysed on a DNA sequencing gel, which was then exposed to a radiograph. Cytokine mRNA whose levels were increased by CI-1033 (alone or with anti-L1R mAb) is indicated by the arrow in the upper right of the photo. FIG. 16B shows in vitro: levels of intracellular interferon-γ in mouse spleen CD8 ⁺ cells stimulated with normal MC57G cells (“MC57G”); or MC57G cells infected with vaccinia virus (“VV infected MC57G”) ( A series of two-dimensional FFC profiles showing levels of “IFN-γ (log)”), or cell surface CD44 (“CD44 (log)”). Experimental groups were infected with vaccinia virus (5 days before sacrifice and assay), untreated (“control”), pretreated once with anti-L1R mAb (“anti-L1R”), or daily with CI-1033 Treated ("CI-1033") or pretreated once with anti-L1R mAb and treated daily with CI-1033 ("CI-1033" + "anti-L1R"). Data shown are from representative mice from each experimental group of mice. Intracellular interferon-γ of mouse splenic CD8 ⁺ cells stimulated in vitro with normal MC57G cells (“MC57G”); vaccinia virus-infected MC57G cells (“VV-infected MC57G”); or anti-CD 3 mAb (“anti-CD3”) A series of two-dimensional FFC profiles showing the level of “IFN-γ (log)”) and the level of cell surface CD44 (“CD44 (log)”). Experimental groups were either untreated mice (“naive”) or infected with vaccinia virus (5 days before sacrifice and assay), untreated (“control”), or pre-treated with anti-L1R mAb (“anti-antibody”). L1R "), pre-treated with 13E8 mAb (" 13E8 ") or pre-treated with anti-L1R mAb and 13E8 mAb (" anti-L1R + 13E8 "). Data shown are from representative mice from each experimental group of mice. FIG. 18A shows the pressure ulcer strain solymen obtained by culturing BSC-40 cell monolayers either in the absence (“−”) or presence (“+”) of CI-1033 at the indicated concentrations. It is a series of photographs showing immunostaining. FIG. 18B shows plaques obtained by culturing BSC-40 cell monolayers either in the absence (“0”) or presence (“CI-1033”) of the indicated concentrations of CI-1033. It is a bar graph which shows the effect of CI-1033 with respect to a number. FIG. 18C shows the number of comets obtained by culturing BSC-40 cell monolayers either in the absence (“0”) or presence (“CI-1033”) of CI-1033 at the indicated concentrations. It is a bar graph which shows.

Claims (61)

(A) identifying an animal that may or may have been exposed to a virus comprising a gene encoding a viral erb-B ligand; and
(B) treating the animal with a compound that inhibits the activity of erb-B tyrosine kinase or inhibits the activation of erb-B tyrosine kinase;
Including methods.   2. The method of claim 1, wherein the animal is identified as having been infected with the virus before, during, or after step (b).   2. The method of claim 1, wherein the viral erb-B ligand is a poxvirus erb-B ligand.   4. The method according to claim 3, wherein the poxvirus is an orthopoxvirus.   5. The method of claim 4, wherein the orthopoxvirus is variola major.   5. The method of claim 4, wherein the orthopoxvirus is variola minor.   The method according to claim 4, wherein the orthopoxvirus is a simian virus.   5. The method of claim 4, wherein the orthopoxvirus is vaccinia.   2. The method of claim 1, wherein the erb-B ligand is an epiregulin-like growth factor (ELGF).   10. The method of claim 9, wherein ELGF is smallpox growth factor (SPGF).   10. The method of claim 9, wherein ELGF is vaccinia growth factor (VGF).   2. The method of claim 1, wherein the erb-B tyrosine kinase is erb-B1 tyrosine kinase.   2. The method of claim 1, wherein the compound is a non-agonist antibody that binds to erb-B tyrosine kinase.   2. The method of claim 1, wherein the compound is a non-agonist erb-B ligand or a non-agonist fragment of erb-B ligand.   2. The method of claim 1, wherein the compound is a small molecule erb-B tyrosine kinase inhibitor.   16. The method of claim 15, wherein the small molecule erb-B tyrosine kinase inhibitor is a quinazoline-based compound.   16. The method of claim 15, wherein the small molecule erb-B tyrosine kinase inhibitor is a pyridopyrimidine based compound.   16. The method of claim 15, wherein the small molecule erb-B tyrosine kinase inhibitor is a quinoline-3-carbonitrile-based compound.   16. The method of claim 15, wherein the small molecule erb-B tyrosine kinase inhibitor is a pyrrolopyrimidine based compound.   17. A method according to claim 16, wherein the quinazoline based compound is 4-anilinoquinazoline.   16. The method of claim 15, wherein the small molecule erb-B tyrosine kinase inhibitor is an irreversible inhibitor of erb-B tyrosine kinase activity.   24. The method of claim 21, wherein the irreversible inhibitor is CI-1033.   17. The method of claim 16, wherein the quinazoline based compound is PD168393, PD160678, PD160879, or PD174265.   17. The method of claim 16, wherein the quinazoline based compound is PD153035, ZD1839, GW572016, GW974, OSI-774, or AG1478.   18. The method of claim 17, wherein the pyridopyrimidine based compound is PD69896, PD153717, or PD158780.   19. The method of claim 18, wherein the compound based on quinoline-3-carbonitrile is EKB-569.   20. The method of claim 19, wherein the pyrrolopyrimidine based compound is CGP59326A.   2. The method of claim 1, wherein the animal is a human.   2. The method of claim 1, further comprising administering to the animal an antibody having the ability to substantially neutralize one or more forms of the virus.   30. The method of claim 29, wherein the antibody binds to an orthopoxvirus, an intracellular mature virus particle (IMV) form, an extracellular membrane virus (EEV) form, or a cell-associated enveloped virus (CEV) form.   32. The method of claim 30, wherein the orthopoxvirus is variola.   32. The method of claim 30, wherein the orthopoxvirus is smallpox.   32. The method of claim 30, wherein the orthopoxvirus is vaccinia.   32. The method of claim 30, wherein the orthopoxvirus is a simian virus.   2. The method of claim 1, wherein the virus is a poxvirus expression vector and further comprises: (a) a heterologous nucleic acid sequence encoding an immunogen; and (b) operably linked to the heterologous nucleic acid sequence. , A transcriptional regulatory element (TRE).   36. The method of claim 35, wherein the poxvirus expression vector is a vaccinia virus expression vector or an attenuated vaccinia virus expression vector.   36. The method of claim 35, wherein the poxvirus expression vector is a canary variola virus or fowlpox virus vector.   3. The method of claim 2, wherein the treatment enhances an immune response to the virus in the animal.   40. The method of claim 38, wherein the immune response is a T cell response.   40. The method of claim 39, wherein the T cell response is a CD8 + T cell response.   40. The method of claim 39, wherein the T cell response is a CD4 + T cell response.   40. The method of claim 39, wherein the T cell response is an interferon-γ producing T cell response.   40. The method of claim 38, wherein the immune response is an antibody producing B cell response. (A) identifying an animal susceptible to infection with a virus comprising a gene encoding an erb-B ligand; and
(B) the animal is (i) an antibody that substantially neutralizes one or more forms of a virus or vaccine that stimulates an immune response to the virus, and (ii) an erb-B tyrosine kinase, or treating with a compound that inhibits activation of erb-B tyrosine kinase;
Including methods. (A) providing an isolated compound that binds to erb-B tyrosine kinase and inhibits activity of tyrosine kinase activity or activation of receptor tyrosine kinase;
(B) contacting the compound with a cell expressing erb-B tyrosine kinase; and
(C) contacting the cell with the viral erb-B ligand or a functional fragment of the ligand before, simultaneously with, or after step (b);
An in vitro method comprising:   46. The method of claim 45, wherein the compound decreases activation of erb-B tyrosine kinase on the cell by the ligand or fragment.   45. The method of claim 44, further comprising determining whether the compound reduces activation of erb-B tyrosine kinase on the cell by the ligand or fragment. A method for determining whether a compound is an antiviral compound comprising the following steps:
(A) providing a compound that inhibits erb-B tyrosine kinase activity;
(B) administering the compound to an animal susceptible to infection with a virus comprising a gene encoding erb-B ligand;
(C) exposing the animal to the virus before, during or after step (b); and
(D) determining whether the compound reduces the symptoms of viral infection in the animal. (A) identifying an animal susceptible to infection with a virus comprising a gene encoding erb-B ligand; and
(B) treating the animal with (i) an antibody that binds to a ligand, and (ii) an antibody that substantially neutralizes one or more forms of a virus or vaccine that stimulates an immune response against the virus;
Including methods.   50. The method of claim 49, wherein the animal is identified as having been infected with the virus before, during, or after step (b).   51. The method of claim 50, wherein the treatment enhances an immune response against the virus.   52. The method of claim 51, wherein the immune response is a T cell response.   53. The method of claim 52, wherein the T cell response is a CD8 + T cell response.   53. The method of claim 52, wherein the T cell response is a CD4 + T cell response.   53. The method of claim 52, wherein the T cell response is an immune response of interferon-γ producing T cells.   52. The method of claim 51, wherein the immune response is an antibody producing B cell response.   51. The method of claim 50, wherein the virus is a poxvirus expression vector and further comprises: (a) a heterologous nucleic acid sequence encoding an immunogen; and (b) operably linked to the heterologous nucleic acid sequence. , A transcriptional regulatory element (TRE).   58. The method of claim 57, wherein the poxvirus expression vector is a vaccinia virus expression vector or an attenuated vaccinia virus expression vector.   57. The method of claim 56, wherein the poxvirus expression vector is a canary variola virus or fowlpox virus vector.   50. The method of claim 49, wherein the antibody that binds to the ligand is a 13E8 monoclonal antibody (ATCC Accession No. PTA-5040).   50. The method of claim 49, wherein the antibody that binds to the ligand is an 11D7 monoclonal antibody (ATCC Accession No. PTA-5039).

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