WO2008140577A2 - Cd44 pathway antagonists for treatment of adenoviral infection - Google Patents

Abstract

New methods and compositions for the treatment and prevention of adenoviral disease are described. In some aspects, adenoviral infections may be treated with CD44 pathway antagonists such as a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor or an antibody that binds to human CD44. Treatment methods of the invention may be used for a variety of adenoviral disease such as systemic infections or local infections of the eye.

Description

DESCRIPTION

CD44 PATHWAY ANTAGONISTS FOR TREATMENT OF ADENOVIRAL INFECTION

BACKGROUND OF THE INVENTION

This application claims the benefit of the filing date of U.S. Provisional Patent

Application Serial No. 60/866,157, filed November 16, 2006, the contents of which is hereby specifically incorporated by reference in its entirety.

A. Field of the Invention

The invention generally concerns the fields of virology and molecular biology. Specifically, the invention primarily concerns methods and compositions for the treatment and prevention of adenoviral disease.

B. Description of Related Art

Adenovirus infection poses a significant cause of morbidity and mortality in both developing countries and the Western world. Adenovirus infection was considered such a problem that the U.S. military developed a vaccine to prevent infections that would often spread rapidly through military personnel (McNeill et al, 2000). Since these vaccinations were discontinued in 1996, adenoviral respiratory infections have remerged as a major health issue among military trainees. Adenoviral infections of the eye are also highly prevalent and contagious. In some cases, these eye infections can become serious and lead to blindness resulting in the patient requiring a corneal transplant. Furthermore, adenoviral infections can cause very serious systemic disease in immune compromised individuals. Pediatric bone marrow transplant patients in particular are very susceptible to disease and, since there are few effective therapies, these diseases are often fatal. Despite the significant health complications associated with adenoviral infection, there are no effective treatment therapies for infected subjects. Thus, methods for treating adenoviral infection are currently in great need.

Human adenoviruses are associated with a variety of human diseases including respiratory, ocular, and gastrointestinal infections. Pathogenic adenovirus infections

..I.. can cause a variety of symptoms in humans from flu-like illness to gastroenteritis or in some cases even hepatitis and encephalitis.

Furthermore, ocular adenoviral infections resulting in keratoconjunctivitis and conjunctivitis are very common and highly infectious. These infections occasionally lead to permanent vision loss. Treatment options are exceedingly limited, and are generally directed at relieving symptoms with measures such as artificial tears and anti-inflammatory agents.

In immune compromised individuals, systemic infection with adenovirus currently has no effective antiviral treatment and is frequently fatal. Adenovirus infections may thus be lethal to immune compromised patients who have received chemotherapy, bone marrow transplants, solid organ transplants, or who suffer from advanced HIV infection. Pediatric bone marrow transplant patients are particularly susceptible to adenovirus, with as many as 10-30% developing adenovirus infections, nearly a quarter of which are fatal (Flomenber et ah, 1994; La Rosa et ah, 2001). Despite this, there are no anti-viral compounds that are effective against adenovirus infections.

In addition to the cellular adenoviral receptor CAR another membrane glycoprotein CD44 seems to play a role in adenoviral infection. In particular, binding of hyaluronan to CAR significantly enhances adenoviral infection. CD44 is involved in a number of signal transduction cascades and certain proteolytic factors may modulate CD44 signaling. For example, gamma-secretase, a complex composed of presenilin 1 (PSl), presenilin 2 (PS2), nicastrin, PEN-2 and APH-I, mediates the cleavage of the inner membrane portion of CD44 (Murakami et ah, 2003; De Stooper, 2003). Additionally, matrix metalloproteinases (MMPs), a large family of zinc dependent proteinases, has been shown to cleave CD44 and modulate its signaling (Nakamura et ah, 2004). Thus far, however, there have been no available methods for modulating CD44 signaling to inhibit adenoviral infection. SUMMARY OF THE INVENTION

The inventors have identified methods of treating or preventing pathogenic adenoviral infection in a subject that involve modulation of CD44 signaling.

In a first embodiment, there is provided a method for treating a subject with an adenoviral infection comprising of administration of an effective amount of a CD44 pathway antagonist. For example, the CD44 pathway antagonist maybe selected from the group consisting of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor and an antibody that binds to human CD44. In some aspects, a subject may be administered two or more of these CD44 pathway antagonists. In certain particular aspects, a subject having an adenoviral infection has been diagnosed with an adenoviral infection. Methods for diagnosing such infections are well known in the art and may involve antigen detection (e.g., by ELISA), virus isolation, detection of viral nucleic acid (e.g., by PCR) or detection of infection by detection of seroconversion in a subject. In some aspects, the invention involves a method for inhibiting adenoviral infection. As used herein inhibition of adenoviral infection may be defined as inhibiting adenoviral entry into cells, inhibiting adenoviral gene expression, inhibiting adenoviral replication or inhibiting adenovirus induced cell death.

In another aspect of the invention there is provided a method for preventing adenovirus infection in a subject comprising administering to the subject an effective amount of a CD44 pathway antagonist selected from the group consisting of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor and an antibody that binds to human CD44. In some cases, the subject may be at risk for developing an adenoviral infection. For instance, an "at risk" subject may be in close proximity to a person with an adenoviral infection or may be highly susceptible to such an infection

(e.g., the subject may be immune compromised). In some aspects a CD44 pathway antagonist of the invention may be administered to prevent an adenoviral infection or adenoviral disease in an uninfected subject. Thus, methods of the invention may, in some cases, be defined as a method for reducing the incidence of adenoviral disease in a susceptible population of subjects by administering a CD44 pathway antagonist of the invention. In some cases, the invention concerns CD44 antagonists such as MMP inhibitors. The MMP inhibitor may be a small molecule, a peptide, a polypeptide, a protein, an antibody, an antibody fragment, a DNA, or an RNA. For example, in some aspects, an MMP inhibitor may be an inhibitor of MMP-I (interstitial collagenase), MMP-2 (Gelatinase A), MMP-3 (Stomelysin-1), MMP-7 (Matrilysin), MMP-8 (Neutraphil collagenase), MMP-9 (Gelatinase B), MMP-IO (Stromelysin-2), MMP-I l (Stromelysin-3), MMP-12 (Metalloelastase), MMP-13 (Collagenase-3), MMP-14 (MTl-MMP), MMP-15 (MT2-MMP), MMP-16 (MT3-MMP), MMP-17 (MT4-MMP), MMP- 18 (Collagenase-4), MMP- 19, MMP-20 (Enamelysin), MMP-21 (MT5-MMP), MMP-23 or MMP-24. A MMP inhibitor may be a general or broad spectrum MMP inhibitor; however an MMP inhibitor may also be a selective MMP inhibitor. As used herein the term "selective inhibitor" refers to an inhibitor that has significant inhibitory activity on a sub group of MMP enzymes. For example, AG3340 is a selective MMP inhibitor with activity against MMP-I, MMP-3, MMP-9, MMP-13 and MMP-14 (Hoekstra et ah, 2001). In some cases an MMP inhibitor may also be defined as a tetracycline compound, such as doxycycline, CMT-I or CMT-8. However, in certain aspects, a non-tetracycline MMP inhibitor may be employed. In still further aspects a MMP inhibitor may be defined as a specific class of inhibitor. For example, in some cases an MMP inhibitor may be a zinc chelator, such as the inhibitors that comprise a hydroxamic acid group. A number of MMP inhibitors are known in the art and any may be used according to the instant invention. For example, in some cases the MMP inhibitor is Marmastat, Batimastat (BB-94), AG3340 (prinomastat), BAY 12-9566, MMI270, COL-3 (metastat), BMS-275291, CP-471,358, AE-94 (neovastat), TAPI-O or TAPI-I. In some cases, a MMP inhibitor of the invention is a MMP-14 inhibitor.

In further embodiments the invention concerns CD44 antagonists such as a gamma-secretase inhibitor. A variety of gamma secretase inhibitors are known in the art. For example, the gamma-secretase inhibitor may be a small molecule, a peptide, a polypeptide, a protein, an antibody, an antibody fragment, a DNA, or an RNA. It has recently been shown that by attaching the piperidine core of some gamma- secretase inhibitors to a cyclopropylcarbamate group their inhibitory activity can be greatly enhanced (Asberom et ah, 2006). Thus, in some aspects, a gamma-secretase inhibitor may comprise a cyclopropylcarbamate group. In still further aspects the gamma secretase inhibitor may be a memapsin 2 inhibitor (Cheng et al, 2004). Furthermore, in some cases, a gamma-secretase inhibitor may be a modified aldehyde dipeptide or tripeptide. In certain very specific aspects a gamma-secretase inhibitor for use in the invention may be DAPT, III-31-C, Compound E, Isocoumarins, D- Helical peptide 294, Epoxide, (Z-LL)₂-ketone (Kornilova et al 2003), LY450139 dihydrate, LY-411,575 (Wong et al, 2004) MRK-560 (Best et al, 2006), or L- 685,458. Thus, in a very specific embodiment the gamma-secretase inhibitor is DAPT.

In still further embodiments the invention concerns CD44 antagonists such as antibodies that bind to human CD44. As used herein the term antibody can mean a polyclonal antibody, a monoclonal antibody, a single chain antibody, a humanized antibody, a Fab fragment, F(ab')2 fragment, single domain antibodies or an antibody paratope peptide. The antibody may be an antibody conjugate, such as an antibody conjugated to a toxin. Thus, in some particular aspects, a CD44 antibody of the invention is a monoclonal or humanized antibody. Methods for making antibodies or modifying known antibodies {e.g., humanizing antibodies) are well know in the art and described further herein. In still further particular aspects, an antibody of the invention binds to human CD44 at the same region as hyaluronan, thus in some cases such an antibody may be defined as competing with hyaluronan for CD44 binding. In yet further cases, an antibody may bind to the same epitope on human CD44 as the BRIC235 antibody. Thus, in certain cases, an antibody for use in the invention may compete with BRIC235 for CD44 binding. In some very specific aspects an antibody of the invention may comprise the variable regions (the heavy and light chain variable regions (VH and VL)) of the BRIC235 antibody. Thus, methods of the invention may employ a BRIC235 antibody (available from INTERNATIONAL BLOOD GROUP REFERENCE LABORATORY) or a fragment thereof.

In some aspects the invention involves method for treating a subject, such as a human subject, with an adenoviral infection. In some cases a subject may present with an adenoviral disease such as acute febrile pharyngitis, pharyngoconjuctival fever, acute respiratory disease, pneumonia, epidemic keratoconjunctivitis, conjunctivitis (e.g., pink eye), pertussis-like syndrome, acute hemorrahagic cystitis, gastroenteritis, meningoencephalitis, hepatitis or myocarditis. However, in some aspects a subject may be an immunocompromised subject, for instance with a persistent subclinical adenoviral infection. Thus, in some aspects, a subject for treatment by the methods of the invention may be an immune compromised subject such as a cancer patient, an organ transplant recipient, a bone marrow transplant patient or a subject with HIV infection.

Methods of the invention may involve administering a CD44 pathway antagonist locally or systemically. For instance, in some cases methods of the invention involve administration topically, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage. However, in certain aspects, methods of the invention may involve administration of CD44 pathway antagonists directly to the sites of adenoviral transmission such as the eye, mouth, or upper respiratory track. For example, administration may be via an eye drop, a mouthwash or a nasal spray. Thus, in methods wherein CD44 pathway antagonists are delivered to the eye, administration may be via topical, subconjunctival, periocular, retrobulbar, subtenon, intracameral, intravitreal, intraocular, subretinal, posterior juxtascleral or suprachoroidal administration.

In still further embodiments, methods of the invention may comprise administering a second therapy for an adenoviral disease prior to, after or concomitantly with a CD44 pathway antagonist of the invention. For example, low molecular weight hyaluronan {e.g., hyaluronic acid, U.S. Patent Publ. 20030186936 incorporated herein by reference), HPMPA ((S)-9-(3-hydroxy-2- phosphonylmethoxypropyl) adenine), cidofovir (2'-nor-cyclic guanosine monophosphate) or ribavirin may be administered in combination or in conjunction with a CD44 pathway antagonist of the invention.

In still another embodiment of the invention there is provided a pharmaceutical composition comprising (a) CD44 pathway antagonist selected from the group consisting of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor and an antibody that binds to human CD44 and (b) a pharmaceutically acceptable carrier. The skilled artisan will understand that the pharmaceutically acceptable carrier will be modified depending upon the selected route of administration but will typically comprise a sterile aqueous solution comprising salts, a pH buffering agent and a preservative, wherein said carrier is a sterile aqueous solution formulated for ocular administration. As used herein, the term aqueous solution refers to a solution predominantly comprised of water. Such a solution may also comprise lipid molecules, for example in the case of hydrophobic CD44 pathway antagonist liposomes or micelles may be used to solublize the antagonist. Additionally, it is contemplated that thickening agents may be added to an aqueous solution to increase the viscosity of the solution. This aspect may be particularly preferred in the case wherein the pharmaceutical compositions are formulated as eye drops. In still a further aspect of the invention there is provided a pharmaceutical composition comprising (a) a combination of at least two CD44 pathway antagonists selected from the group consisting of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor and an antibody that binds to human CD44 and (b) a pharmaceutically acceptable carrier.

In some further embodiments, there is provided a pharmaceutical composition of the invention comprised in a bottle said bottle comprising an exit portal that enables drop-wise administration of the composition. In some cases, a pharmaceutical composition comprised in a bottle comprises multiple doses however in certain aspects a bottle comprises a single dose unit for administration to one or two eyes, preferable a single dose unit is comprised in 1-2 drops of the formulation. As used herein the term "bottle" refers to any fluid container such as an ampoule, dropper or syringe.

In still other aspects of the invention, there is provided a pharmaceutical composition of the invention comprised in a bottle said bottle comprising an exit portal that disperses the composition into a mist or aerosol. Furthermore, the exit portal of the bottle may be designed to fit with the nostril of the human subject to enable intranasal administration of the composition.

In yet another aspect of the invention, there is provided a method of increasing the expression of a transgene at a site in a subject comprising administering to the subject an adenoviral vector comprising a trans gene encoding a therapeutic polypeptide and a pharmaceutically effective amount of an agent that increases the expression and/or activity of a MMP or an agent that increases the expression and/or activity of gamma secretase. The subject may be any subject, such as a human subject. The site may be a site of disease in a subject, and the transgene to be expressed may be a therapeutic gene. In a particular aspect, the subject may be a patient with cancer, and the site may be a cancer in breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colorectal cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, leukemia, or cancer of the eye. In a particular aspect, the cancer is retinoblastoma.

The transgene may be a therapeutic gene, such as a tumor suppressor gene. Tumor suppressor genes are genes that normally restrain cell growth but, when missing or inactivated by mutation, allow cells to grow uncontrolled. One of the best known tumor suppressor genes is p53, which plays a central role in cell cycle progression, arresting growth so that repair or apoptosis can occur in response to DNA damage. Other examples of therapeutic genes include MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-I, TRP-2, MAGE-I, MAGE-3, BAGE, GAGE-I, GAGE-2, pl5(58), CEA, RAGE, NY-ESO (LAGE), SCP-I, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-3, MAGE-4, MAGE-5, MAGE-6, pl85erbB2, pl80erbB-3, c- met, mn-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β- Catenin, CDK4, Mum-1, pi 6, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein , β-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-I, RCASl, SDCCAG 16, TA-90 (Mac-2 binding protein\cyclophilin C- associated protein), TAAL6, TAG72, TLP, TPS, INGl, mamaglobin, cyclin Bl, SlOO, BRCAl, BRCA2, a tumor immunoglobulin idiotype, a tumor T-cell receptor clonotype, MUC-I, epidermal growth factor receptor, and mda-7.

In some embodiments, the subject may be administered an agent that increases the expression of an MMP or gamma secretase. The agent that increases the expression of an MMP or gamma secretase may be a small molecule, a peptide, a polypeptide, a protein, an antibody, an antibody fragment, a DNA, a RNA, a siRNA or shRNA. In a particular aspect, the subject is administered an agent that increases the activity of a MMP or a gamma secretase. The agent that increases the expression of an MMP or gamma secretase may be a small molecule, a peptide, a polypeptide, a protein, an antibody, an antibody fragment, a DNA, an RNA, a siRNA or shRNA. Examples of such agents include but are not limited to fϊbronectin, ursolic acid, interferon beta, sex-steroids, secreted protein acidic and rich in cystein (SPARC), platelet-aggregating factor (PAF), thrombin, nickel, hydrocortisone, and forskolin among others.

Embodiments discussed in the context of a method and/or composition of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.

As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.

FIG. 1 : PMA and vitreous enhance Ad5-Luc transduction of HeLa cells.

HeLa cells were either preincubated with PMA or DMSO (at the concentration used to dissolve the PMA) before being exposed to 5% vitreous and Ad5-Luc. Following transduction cell lysates were assessed for luciferase activity and the results were graphed.

FIG. 2: Vitreous enhancement of Ad5-Luc in Y79 Cells is inhibited by the metalloproteinase inhibitor TAPI-O. Y79 cells were exposed to different concentrations of vitreous (as indicated) in either the presence or absence of TAPI-O

(1 μM) and transduced with Ad5-Luc. Following transduction the luciferase activity in the cell lysates was determined and graphed.

FIG. 3: Vitreous enhancement of Ad5-Luc in Y79 cells is inhibited by the γ- secretase/PSl inhibitor DAPT. Y79 cells were transduced with Ad5-Luc either in the presence or absence of DAPT and/or 10% vitreous. Following transduction cell lysates were assessed for luciferase activity and the results were graphed.

FIG. 4: Vitreous Enhancement of HD-CMV/β-Gal in HEK293 cells requires

CD44. HEK293 cells were transduced with a β-galactosidase expressing adenoviral vector (HD-CMV/β-Gal) either with or without 5% vitreous. Transduction was studied in the presence of different antibodies (ROSl : control IgG; BRIC222: anti CD44; BRIC235: anti-CD44 at the HA binding epitope). Following transduction cells were harvested and β-Gal activity was assessed and plotted.

FIG. 5: Knock-down of MMP 15 in Y79 Cells Decreases Vitreous Enhancement of Ad-Mediated Transgene Expression. To determine if MMP 15 is associated with the TAPI-I /TAPI-O sensitive vitreous enhancement of Ad-mediated transgene expression observed in the retinoblastoma cell line Y79, its expression was knock-down by using a MMP 15 shRNAmir. As a control, Y79 cells were also engineered with a non- silencing shRNAmir. Cells were transduced with an Ad5-Luc vector in the presence or absence of 5% vitreous. Following transduction the luciferase activity in the cell lysates was determined and graphed.

FIG. 6: Effect ofCD44 Antagonists on Ad-Mediated Transgene Expression on Human Conjunctiva Explants. In these experiment human conjunctiva explants were plated in a 96-well plate with DMEM culture media supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin antibiotics and transduced with an Ad5- Luc vector in the presence or absence of BRIC235 (a human CD44 blocking antibody), TAPI-O or TAPI-I (both are non-specific MMP inhibitors), and DAPT (a potent γ-Secretase inhibitor). Tissue cells were lysed and luciferase activity determined. The inhibition of luciferase activity was determined by using the following mathematical formula: %Inhibition = [(Activity in Media - Activity in Antagonist)/ Activity in Media] x 100. Graphed results represent average values from two independent experiments.

FIG. 7: Vitreous enhancement of adenovector-mediated transgene expression can be inhibited by the metalloproteinase inhibitor TAPI-O. Y79 cells were exposed to different concentrations of vitreous (as indicated) in either the presence or absence of TAP-I (1 μM) and transduced with Ad5-Luc. Following transduction the luciferase activity in the cell lysates was determined and graphed.

FIG. 8: Vitreous enhancement of adenovector-mediated transgene expression can be inhibited by the γ-secretase/PSl inhibitor DAPT. Y79 cells were transduced with Ad5-Luc either in the presence or absence of DAPT and/or 10% vitreous.

Following transduction cell lysates were assessed for luciferase activity and the results were graphed.

FIG. 9: The inhibitors TAPI-I and DAPT can inhibit adenovector-mediated transgene expression in human conjunctiva explants. Conjunctiva was harvested from two human eyes using sterile techniques and kept in DMEM culture media supplemented with 1% Penicillin/Streptomycin and 5% fetal bovine serum. Samples were divided into small pieces and placed in wells in a 96-well plate with fresh culture media (10OuL media per well) and transduced with Ad5-luc in the presence or absence of TAPI-I (lOuM), DAPT (50OnM), or a 1 :1000 dilution of DMSO.

Following transduction the luciferase activity in the cell lysates was determined and graphed. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The instant invention provides new methods for treating adenoviral disease with CD44 pathway antagonists. Since few antiviral drugs have demonstrated efficacy against adenoviruses, these new methods provide powerful new treatments for adenoviral disease. For instance, serious adenoviral eye infections may be treated using one or more CD44 pathway antagonists. Such treatments may reduce the spread of highly contagious eye disease such as pink eye and may reduce the incidence of vision loss associated with keratoconjunctivitis. Furthermore, CD44 pathway antagonism may be used to treat immune deficient individuals who are particularly susceptible to adenoviral disease. Furthermore, the methods of the invention may be used to curtail the spread of adenoviral epidemics that often occur in situations of high population density, such as in military training camps. Thus, the instant invention represents a major advance in the treatment and prevention of human adenoviral disease.

A. CD44 Pathway Antagonists

CD44 protein is a cell-surface glycoprotein that is involved in a variety of cell- related processes, including cell-cell interactions, cell migration, and cell adhesion. It participates in a wide variety of cellular functions including lymphocyte activation, tumor metastasis, and hematopoiesis. A "CD44 pathway antagonist" is any molecule that can reduce the expression or activity of CD44.

CD44 is a single chain molecule comprising a conserved amino terminal extracellular domain, a nonconserved membrane proximal region, a variable region expressing various combinations of variant exons, a conserved transmembrane spanning domain and a conserved cytoplasmic tail. The genomic sequence of mouse and human CD44 includes 5 constant exons at the 5' terminus, and 5 constant exons at the 3' end.

The CD44 N-terminus contains the ligand binding site of the molecule.

Hyaluronic acid (HA) is the principal ligand of CD44, but other extracellular matrix

(ECM) components (e.g. laminin, collagen, fibronectin and chondroitin sulfate) as well as non-ECM constituents (mucosal vascular addressin, serglycin, osteopontin and class II invariant chain) can also interact with the CD44 receptor. Marked accumulation of CD44, and sometimes hyaluronic acid, is detected in areas of intensive cell migration and cell proliferation, as in wound healing, tissue remodeling, inflammation (including auto inflammation), morphogenesis and carcinogenesis.

1. MMP Inhibitors

As discussed above, an MMP inhibitor is an example of a CD44 pathway antagonist. An MMP inhibitor is any chemical compound that is effective in inhibiting the biological activity of a matrix metalloproteinase such as collagenase, stromelysin, gelatinase, or elastase. Numerous compounds are known to be matrix metalloproteinase inhibitors, and any of such compounds can be utilized in the method of this invention. An MMP inhibitor may also be an inhibitor of one or more of the various signaling compounds and/or of the transcription factors (e.g., cJUN and cFOS, which together lead to the production of MMPs) by which MMPs are produced naturally. MMPs are zinc-dependent proteases that hydrolyze collagens, proteoglycans, and glycoproteins. The classes include gelatinase A and B, stromelysin- 1 and -2, fibroblast collagenase, neutrophil collagenase, matrilysin, metalloelastase, and interstitial collagenase. These enzymes are implicated with a number of diseases which result from breakdown of connective tissues, such as rheumatoid arthritis, osteoarthritis, osteoporosis, multiple sclerosis, and even tumor metastasis.

Retinoids are one class of MMP inhibitors. The inhibitors of MMPs can act directly on the MMPs and/or on the transcription factors AP-I and NF-kappaB by which MMPs are produced naturally. E5510 has been described as inhibiting NF- kappaB activation. Retinoids are such as those disclosed in U.S. Pat. No. 4,877,805 and the dissociating retinoids that are specific for AP-I antagonism. Other retinoids, besides retinol, include natural and synthetic analogs of vitamin A (retinol), vitamin A aldehyde (retinal), vitamin A acid (retinoic acid (RA)), including all-trans, 9-cis, and 13-cis retinoic acid), etretinate, and others as described in U.S. Pat. No. 4,887,805, and U.S. Pat. No. 4,888,342.

MMPs are also inhibited by BB2284, GI 129471, and TIMPs (tissue inhibitors of metalloproteinases), which inhibit vertebrate collagenases and other metalloproteases, including gelatinase and stromelysin. Still other compounds useful for the present invention include hydroxamate and hydroxy-urea derivatives such as Galardin, Batimastat, and Marimastat.

Other examples of MMP inhibitors include genistein and quercetin (as described in U.S. Pat. No. 5,637,703, U.S. Pat. No. 5,665,367, and FR- A-2,671,724, the disclosures of which are incorporated herein by reference) and related compounds as well as other antioxidants such as NAC (N-acetyl cystein) and others. Other examples of MMP inhibitors are set forth elsewhere in this specification.

2. Gamma Secretase Inhibitors

A gamma secretase inhibitor is another example of a CD44 pathway antagonist. Gamma secretase is a multi-subunit protease complex and integral membrane protein that cleaves transmembrane proteins at residues within the transmembrane domain. Amyloid precursor protein, a large integral membrane protein that produces a short amino acid peptide called amyoid beta, is a substrate of gamma secretase. The abnormally folded fibrillar form of amyoid beta is the primary component of amyoid plaques, which are found in the brains of patients with Alzheimer's disease. Gamma secretase is also critical in the processing of the Notch protein. A gamma secretase inhibitor is any agent that can inhibit the activity of and/or reduce the expression of gamma secretase.

Non-limiting examples of gamma secretase inhibitors include talsaclidine (Hock et al. 2003), Xanomeline, L-689660, L-685458, McN-A-343, CDD-0097, fenchylamine, MG132, WPE-111-31C, MW-11-36C/26A, MW-167, CM-265, lactacystin, DNPSl, DAM, and DAPT (Lanz et al. 2003).

Additional examples of gamma secretase inhibitors can be found in the following U.S. Patent Application Pub. Nos., each of which is hereby specifically incorporated by reference: 20070197581, 20070088063, 20070072227, 20070049612, 20060264474, 20060205666, 20060189666, 20060069147, 20060046984, 20050261276, 20050143369, 20040049038, 20030126380, 20030211559, and 20020013315. 3. AntiCD44 Antibodies

Another example of a CD44 pathway is an antibody that binds to CD44. Any such antibody is contemplated for inclusion in the methods and compositions of the present invention. In particular embodiments, the antibody binds to human CD44. Non-limiting examples of such antibodies are discussed in the following published U.S. Patent Applications, each of which is herein specifically incorporated by reference: 20070230061, 20070189964, 20060019340, 20050271672, 20050271672, 20050244413, 20050215464, 20050147606, and 20040126379. Additional information regarding antibodies is set forth in the specification below.

B. Adenoviruses

Adenoviruses comprise linear double stranded DNA, with a genome ranging from 30 to 35 kb in size (Reddy et al, 1998; Morrison et al, 1997; Chillon et al, 1999). There are over 50 serotypes of human adenovirus, and over 80 related forms which are divided into six families based on immunological, molecular, and functional criteria (Wadell et al, 1980). Physically, adenovirus is a medium-sized icosahedral virus containing a double-stranded, linear DNA genome which, for adenovirus type 5, is 35,935 base pairs (Chroboczek et al, 1992). Adenoviruses require entry into the host cell and transport of the viral genome to the nucleus for infection of the cell and replication of the virus.

Salient features of the adenovirus genome are an early region (El, E2, E3 and

E4 genes), an intermediate region (pIX gene, Iva2 gene), a late region (Ll, L2, L3, L4 and L5 genes), a major late promoter (MLP), inverted-terminal-repeats (ITRs) and a ψ sequence (Zheng, et al, 1999; Robbins et al., 1998; Graham and Prevec, 1995). The early genes El, E2, E3 and E4 are expressed from the virus after infection and encode polypeptides that regulate viral gene expression, cellular gene expression, viral replication, and inhibition of cellular apoptosis. Further on during viral infection, the MLP is activated, resulting in the expression of the late (L) genes, encoding polypeptides required for adenovirus encapsidation. The intermediate region encodes components of the adenoviral capsid. Adenoviral inverted terminal repeats (ITRs; 100 to 200 bp in length) are cis elements, function as origins of replication and are necessary for viral DNA replication. The ψ sequence is required for the packaging of the adenoviral genome.

The mechanism of infection by adenoviruses, particularly adenovirus serotypes 2 and 5, has been extensively studied. A host cell surface protein designated CAR (Coxsackie Adenoviral Receptor) has been identified as the primary binding receptor for these adenoviruses. Interaction between the fiber knob and CAR is sufficient for binding of the adenovirus to the cell surface. However, subsequent interactions between the penton base and additional cell surface proteins, members of the α_v integrin family, are necessary for efficient viral internalization. Disassembly of the adenovirus begins during internalization; the fiber proteins remain on the cell surface bound to CAR. The remainder of the adenovirus is dissembled in a stepwise manner as the viral particle is transported through the cytoplasm to a pore complex at the nuclear membrane. The viral DNA is extruded through the nuclear membrane into the nucleus where viral DNA is replicated, viral proteins are expressed, and new viral particles are assembled. Specific steps in this mechanism of adenoviral infection may be potential targets to modulate viral infection and gene expression.

1. Engineered Adenoviruses and Adenoviral Vectors

In particular embodiments, an adenoviral expression vector is contemplated for the delivery of expression constructs. "Adenovirus expression vector" or "Adenoviral vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein. Thus, an Adenoviral vector may include any of the engineered vectors that comprise Adenoviral sequences.

An adenovirus expression vector according to the present invention comprises a genetically engineered form of the adenovirus. The nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the known serotypes and/or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain one adenovirus vector for use in the present invention. This is because adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

Advantages of adenoviral gene transfer include the ability to infect a wide variety of cell types, including non-dividing cells, a mid-sized genome, ease of manipulation, high infectivity and they can be grown to high titers (Wilson, 1996).

Further, adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner, without potential genotoxicity associated with other viral vectors. Adenoviruses also are structurally stable (Marienfeld et al., 1999) and no genome rearrangement has been detected after extensive amplification (Parks et al. , 1997; Bett et al. , 1993).

Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo (U.S. Pat. Nos. 5,670,488; 5,932,210; 5,824,544). This group of viruses can be obtained in high titers, e.g., 10. sup.9 to 10. sup.11 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells.

Although adenovirus based vectors offer several unique advantages over other vector systems, they often are limited by vector immunogenicity, size constraints for insertion of recombinant genes, low levels of replication, and low levels of transgene expression. A major concern in using adenoviral vectors is the generation of a replication-competent virus during vector production in a packaging cell line or during gene therapy treatment of an individual. The generation of a replication- competent virus could pose serious threat of an unintended viral infection and pathological consequences for the patient. Armentano et al., describe the preparation of a replication-defective adenovirus vector, claimed to eliminate the potential for the inadvertent generation of a replication-competent adenovirus (U.S. Pat. No. 5,824,544). The replication-defective adenovirus method comprises a deleted El region and a relocated protein IX gene, wherein the vector expresses a heterologous, mammalian gene. A common approach for generating adenoviruses for use as a gene transfer vector is the deletion of the El gene (El^"), which is involved in the induction of the E2, E3 and E4 promoters (Graham and Prevec, 1995). Subsequently, a therapeutic gene or genes can be inserted recombinantly in place of the El gene, wherein expression of the therapeutic gene(s) is driven by the El promoter or a heterologous promoter. The El^", replication-deficient virus is then proliferated in a "helper" cell line that provides the El polypeptides in trans {e.g., the human embryonic kidney cell line 293). Alternatively, the E3 region, portions of the E4 region or both may be deleted, wherein a heterologous nucleic acid sequence under the control of a promoter operable in eukaryotic cells is inserted into the adenovirus genome for use in gene transfer (U.S. Pat. Nos. 5,670,488; 5,932,210).

Of course, in particular embodiments of the present invention, it is contemplated that low molecular weight or modified hyaluron or vitreous may be used in treatment of such inadvertently produced replication-competent adenovirus. As stated above, vectors contemplated for use in the present invention are replication defective. However, approaches involving replication competent adenoviral vectors, leading to so-called amplification are also contemplated. Thus, particular embodiments are contemplated in which the extent and rate of amplification of replication competent adenoviral vectors is modulated through the application of low molecular weight or modified hyaluron or vitreous.

C. Pharmaceutical Formulations:

The compositions of the present invention are suitable for administration to a subject by any route or mode of administration. Pharmacological therapeutic agents and methods of administration, dosages, etc. are well known to those of skill in the art (see for example, the "Physicians Desk Reference", Goodman & Gilman's "The Pharmacological Basis of Therapeutics", "Remington's Pharmaceutical Sciences", and "The Merck Index, Eleventh Edition", incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject, and such individual determinations are within the skill of those of ordinary skill in the art. 1. Pharmaceutically Acceptable Carriers

Certain embodiments of the present invention involve introducing a pharmaceutically acceptable dose of a therapeutic agent. Pharmaceutical compositions of the present invention comprise a therapeutically or diagnostically effective amount of a CD44 pathway antagonist of the present invention. The phrases "pharmaceutical or pharmacologically acceptable" or "therapeutically effective" or "diagnostically effective" refer to molecular entities and compositions that do not produce an unacceptable adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of therapeutically effective or diagnostically effective compositions will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, "a composition comprising a therapeutically effective amount" or "a composition comprising a diagnostically effective amount" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the present compositions is contemplated.

The compositions of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection. The therapeutic composition may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, subcutaneously, subconjunctival, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art.

The actual required amount of a composition of the present invention administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of active compound. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non- limiting examples, a dose may also comprise from about 0.1 mg/kg/body weight to about 1000 mg/kg/body weight or any amount within this range, or any amount greater than 1000 mg/kg/body weight per administration.

In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including, but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The therapeutic agent may be formulated into a composition in a free base, free acid, neutral or salt form. Pharmaceutically acceptable salts include the salts formed with the free carboxyl groups derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising, but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

Sterile injectable solutions are prepared by incorporating the diagnostic or therapeutic agent in the required amount of the appropriate solvent with various amounts of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof. In particular embodiments, the compositions of the present invention are suitable for application to mammalian eyes. For example, the formulation may be a solution, a suspension, or a gel. In some embodiments, the composition is administered via a bioerodable implant, such as an intravitreal implant or an ocular insert, such as an ocular insert designed for placement against a conjunctival surface. In some embodiments, the therapeutic agent coats a medical device or implantable device.

In preferred aspects the formulation of the invention will be applied to the eye in aqueous solution in the form of drops. These drops may be delivered from a single dose ampoule which may preferably be sterile and thus rendering bacteriostatic components of the formulation unnecessary. Alternatively, the drops may be delivered from a multi-dose bottle which may preferably comprise a device which extracts preservative from the formulation as it is delivered, such devices being known in the art.

In other aspects, components of the invention may be delivered to the eye as a concentrated gel or similar vehicle which forms dissolvable inserts that are placed beneath the eyelids.

Any amount of therapeutic agent may be present in the compositions of the present invention. For example, the therapeutic agent may be present at a concentration of from 0.01 to 100 mg/ml. For example, the therapeutic agent may be present at a concentration of at least 0.01 mg/ml, at least 0.05 mg/ml, at least 0.10 mg/ml, at least 0.50 mg/ml, at least 1.0 mg/ml, at least 2.0 mg/ml, at least 3.0 mg/ml, at least 4.0 mg/ml, at least 5.0 mg/ml, at least 6.0 mg/ml, at least 7.0 mg/ml, at least 8.0 mg/ml, at least 9.0 mg/ml, at least 10.0 mg/ml, at least 12.0 mg/ml, at least 15 mg/ml, at least 17 mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35 mg/ml, at least 40 mg/ml, at least 45 mg/ml, and least 50 mg/ml, at least 60 mg/ml, at least 70 mg/ml, or at least 80 mg/ml. In particular embodiments, the therapeutic agent is present at a concentration of about 10-30 mg/ml.

The amount of therapeutic agent included in the compositions of the present invention will be whatever amount is therapeutically effective and will depend upon a number of factors known to those of ordinary skill in the art, including the identity and potency of the chosen therapeutic agent. In particular embodiments, the total concentration of therapeutic agent is about 10% or less. In more particular embodiments, the total concentration of the therapeutic agent is about 5% or less.

The compositions of the present invention are preferably not formulated as solutions that undergo a phase transition to a gel upon administration to the eye.

In addition to the therapeutic agent, the aqueous compositions of the present invention may contain other ingredients as excipients. For example, the compositions may include one or more pharmaceutically acceptable buffering agents, preservatives (including preservative adjuncts), non-ionic tonicity-adjusting agents, surfactants, solubilizing agents, stabilizing agents, comfort-enhancing agents, polymers, emollients, pH-adjusting agents and/or lubricants.

The compositions of the invention have a pH in the range of 5-9, preferably 6.5-7.5, and most preferably 6.9-7.4.

The formulation should preferably be isotonic or slightly hypotonic in order to combat any hypertonicity of tears caused by evaporation and/or disease. The compositions of the present invention generally have an osmolality in the range of

220-320 mOsm/kg, and preferably have an osmolality in the range of 235-260 mOsm/kg.

2. Ophthalmically Acceptable Carriers Eye drop formulations: For example, specialized devices such as those described in U.S. Patent 7,094,226 may be used for deliver of therapeutic composition to the eye.

The present invention, particularly in some cases, concerns methods and compositions for the treatment of adenoviral eye infections. Preferably, such compositions will be formulated as solutions, suspensions and other dosage forms for topical administration. Aqueous solutions are generally preferred, based on ease of formulation, as well as a patient's ability to easily administer such compositions by means of instilling one to two drops of the solutions in the affected eyes. However, the compositions may also be suspensions, viscous or semi-viscous gels, or other types of solid or semi-solid compositions. Suspensions may be preferred for some CD44 pathway antagonists that are sparingly soluble in water.

The compositions of the present invention may also contain a surfactant. Examples of surfactants include, but are not limited to: Cremophor.RTM. EL, polyoxyl 20 ceto stearyl ether, polyoxyl 40 hydrogenated castor oil, polyoxyl 23 lauryl ether and poloxamer 407 may be used in the compositions. A preferred surfactant is polyoxyl 40 stearate. In general, the surfactant(s) concentration will be about 0.001 to 2.0% w/v. Preferred compositions of the present invention will contain about 0.1% w/v of polyoxyl 40 stearate.

The compositions of the present invention may also include various other ingredients, such as tonicity agents, buffers, preservatives, co-solvents and viscosity building agents.

Various tonicity agents may be employed to adjust the tonicity of the composition, preferably to that of natural tears for ophthalmic compositions. For example, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, dextrose and/or mannitol may be added to the composition to approximate physiological tonicity. Such an amount of tonicity agent will vary, depending on the particular agent to be added. In general, however, the compositions will have a tonicity agent in an amount sufficient to cause the final composition to have an ophthalmically acceptable osmolality (generally about 150-450 mOsm, preferably 250-350 mOsm).

An appropriate buffer system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate or boric acid) may be added to the compositions to prevent pH drift under storage conditions. The particular concentration will vary, depending on the agent employed. Preferably, however, the buffer will be chosen to maintain a target pH within the range of pH 6-7.5.

Antioxidants may be added to compositions of the present invention to protect the protease inhibitor compounds from oxidation during storage. Examples of such antioxidants include, but are not limited to, vitamin E and analogs thereof, ascorbic acid and derivatives, and butylated hydroxyanisole (BHA). Compositions formulated for the treatment or prevention of adenoviral infection may also comprise aqueous carriers designed to provide immediate, short- term relief of dry eye -type conditions. Such carriers can be formulated as a phospholipid carrier or an artificial tears carrier, or mixtures of both. As used herein, "phospholipid carrier" and "artificial tears carrier" refer to aqueous compositions which: (i) comprise one or more phospholipids (in the case of phospholipid carriers) or other compounds, which lubricate, "wet," approximate the consistency of endogenous tears, aid in natural tear build-up, or otherwise provide temporary relief of dry eye symptoms and conditions upon ocular administration; (ii) are safe; and (iii) provide the appropriate delivery vehicle for the topical administration of an effective amount of one or more protease inhibitors. Examples or artificial tears compositions useful as artificial tears carriers include, but are not limited to, commercial products, such as Tears Naturale™ (Alcon Laboratories, Inc., Fort Worth, Tex.). Examples of phospholipid carrier formulations include those disclosed in U.S. Pat. No. 4,804,539 (Guo et ciL), U.S. Pat. No. 4,883,658 (Holly), U.S. Pat. No. 4,914,088 (Glonek), U.S. Pat. No. 5,075,104 (Gressel et al), U.S. Pat. No. 5,278,151 (Korb et al), U.S. Pat. No. 5,294,607 (Glonek et al), U.S. Pat. No. 5,371,108 (Korb et al), U.S. Pat. No. 5,578,586 (Glonek et al ); the foregoing patents are incorporated herein by reference to the extent they disclose phospholipid compositions useful as phospholipid carriers of the present invention.

Other compounds designed to lubricate, "wet," approximate the consistency of endogenous tears, aid in natural tear build-up, or otherwise provide temporary relief of dry eye symptoms and conditions upon ocular administration the eye are known in the art. Such compounds may enhance the viscosity of the composition, and include, but are not limited to: monomeric polyols, such as, glycerol, propylene glycol, ethylene glycol; polymeric polyols, such as, polyethylene glycol, hydroxypropylmethyl cellulose ("HPMC"), carboxy methylcellulose sodium, hydroxy propylcellulose ("HPC"), dextrans, such as, dextran 70; water soluble proteins, such as gelatin; and vinyl polymers, such as, polyvinyl alcohol, polyvinylpyrrolidone, povidone and carbomers, such as, carbomer 934P, carbomer 941, carbomer 940, carbomer 974P. Other compounds may also be added to the ophthalmic compositions of the present invention to increase the viscosity of the carrier. Examples of viscosity enhancing agents include, but are not limited to: polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, various polymers of the cellulose family; vinyl polymers; and acrylic acid polymers. In general, the phospholipid carrier or artificial tears carrier compositions will exhibit a viscosity of 1 to 400 centipoises ("cps").

Topical ophthalmic products are typically packaged in multidose form. Preservatives are thus required to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, chlorobutanol, benzododecinium bromide, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium-1, or other agents known to those skilled in the art. Such preservatives are typically employed at a level of from 0.001 to 1.0% w/v. Unit dose compositions of the present invention will be sterile, but typically unpreserved. Such compositions, therefore, generally will not contain preservatives.

The preferred compositions of the present invention are intended for administration to a human patient suffering from adenoviral infections of the eye.

Preferably, such compositions will be administered topically. For example, 1-2 drops of such compositions will be administered 1-10 times per day for the treatment of the infection.

D. Methods for Producing Antibodies

As described above certain aspects of the invention involve the use anti- human CD44 antibodies. Antibodies can be made by any of the methods that are well known to those of skill in the art. Antibodies for use in the invention may originate from any animal, including birds (e.g., chicken, turkey) and mammals (e.g., rat, rabbit, goat, horse). Furthermore the antibodies may be any of the various immunoglobulin subtypes such as IgA, IgM, IgE, IgY, IgG (i.e., IgGl, IgG2a, IgG2b, IgG3 or IgG4) or a fragment thereof. The following methods exemplify some of the most common antibody production methods. 1. Polyclonal Antibodies

Polyclonal antibodies generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the antigen (e.g., human CD44 or a fragment thereof). However, it is contemplated that intramuscular, intranasal, intradermal and/or intraocular antigen administration may also be used for these purposes. As used herein the term "antigen" refers to any polypeptide that will be used in the production of antibodies.

In the case where an antibody is to be generated that binds to a particular polypeptide, it may be useful to conjugate the antigen or a fragment containing the target amino acid (e.g., a hyaluronan binding domain) sequence to a protein that is immunogenic in the species to be immunized, e.g. keyhole limpet hemocyanin, serum albumin, human thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glytaraldehyde, succinic anhydride, SOCl₂, or R¹ N=C=NR, where R and R¹ are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivatives by, for example, combining 1 mg or 1 μg of conjugate (for rabbits or mice, respectively) with 3 volumes of Freud's complete adjuvant and injecting the solution intradermally at multiple sites. In certain cases, live (e.g., live attenuated) viral antigens may also be used for immunization, or as primer for subsequent booster immunization with a particular antigen of interest. The skilled artisan will understand that the amount of antigen, antibody-antigen complex, or viral titer used in any particular protocol will depend on the antigen, the route of administration and the size (mass and/or surface area) of the animal. Relative antigen amount for various administration methods are exemplified in Harlow and Lane (1988) incorporated herein by reference. Harlow and Lane provided example antigen doses in the case of rabbits and mice, but may also be used to determine doses for larger animals such conversions may be accomplished for example by the calculations described in Frάrάch et al. (1966).

About one month after initial administration the animals are boosted with about 1/5 to 1/10 the original amount of conjugate in Freund's incomplete or complete adjuvant by subcutaneous injection at multiple sites. 7 to 14 days later the animals are bled and the serum is assayed for specific antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the same antigen conjugate, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents, such as alum, or other adjuvants may be used to enhance the immune response.

2. Monoclonal Antibodies

In further embodiments of the invention, a CD44 binding antibody is a monoclonal antibody. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Hence, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. Monoclonal antibodies offer certain advantages over polyclonal antibodies. For example, hybridomas that produce monoclonal antibodies offer an ongoing source of antibodies that will have consistent characteristics (e.g., each batch having similar virus binding or neutralizing activity).

For example, monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler & Milstein (1975), or may be made by recombinant DNA methods (Cabilly et al; U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal is immunized as described above to elicit lymphocytes (i.e., plasma cells) that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding 1986).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-defϊcient cells.

Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-I l mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego,

Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA.

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the target antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson & Pollard (1980). In certain additional cases, the activity of an antibody may be determined. For instance, the neutralizing activity of an antibody maybe determined. Methods for measuring the neutralizing activity of an antibody are well known to those of skill in the art and may involve, for example tissue culture neutralization assays {e.g., by plaque assay or focus forming assay) or infection neutralization assays in animal models.

After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity {e.g., virus binding and/or neutralization), the clones may be subcloned by limiting dilution procedures and grown by standard methods, Goding (1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI- 1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al. (1984), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity for any particular antigen described herein.

Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for the target antigen and another antigen-combining site having specificity for a different antigen. Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry.

For some applications, the antibodies of the invention will be labeled with a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin; biotin (which enables detection of the antibody with an agent that binds to biotin, such as avadin; or an enzyme (either by chemical coupling or polypeptide fusion), such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al. (1962); David et al. (1974); Pain et al. (1981); and Nygren (1982).

The antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, 1987). Such assays may be used, for example, to further characterize the binding specificity of an antibody, such as to determine the binding affinity or epitope specificity of an antibody. Certain particular assays are described below.

a. Competitive binding assay

Competitive binding assays rely on the ability of a labeled standard (which may be a purified target antigen or an immunologically reactive portion thereof) to compete with the test sample analyte for binding with a limited amount of antibody. The amount of antigen in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex. David & Greene, U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an antiimmunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. 3. Antibodies with Species Specific Sequences

As discussed previously, antibodies for use in the methods of the invention may be polyclonal or monoclonal antibodies or fragments thereof. However, in some aspects it is preferred that the antibodies comprise amino acid substitutions such that they comprise primary sequences that are not immunogenic to a given species. For example, amino acids in a murine antibody may be substituted for sequences at the corresponding position in a human antibody (e.g., to generate a humanized antibody). Such antibodies may be preferable in certain cases since they will not produce an antibody directed immune response in a human subject. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non- human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, 1986); Riechmann et al, 1988; Verhoeyen et al, 1988), by substituting the antibody source animal CDRs or CDR sequences for the corresponding sequences of a human or human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (Cabilly, supra), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

In this aspect of the invention, it is important that antibodies be modified (i.e., humanized) with retention of high affinity for the antigen and other favorable biological properties (e.g., virus binding and/or neutralization). To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. For further details see U.S. Pat. No. 5,821,337.

4. Single Chain Antibodies

Single chain antibodies (SCAs) are genetically engineered proteins designed to expand on the therapeutic and diagnostic applications possible with monoclonal antibodies. SCAs have the binding specificity and affinity of monoclonal antibodies and, in their native form, are about one-fifth to one-sixth of the size of a monoclonal antibody, typically giving them very short half-lives. SCAs also have certain differences as compared to a corresponding monoclonal antibody. For example, SCAs may be metabolized at a different rate as compared to a monoclonal antibody. Thus, vaccine compositions that employ SCAs may require greater or lesser amounts of SCAs (e.g., SCA to virus ratios) for maximal effectiveness and safety. One potential benefit of SCAs is that fully-human SCAs may be isolated by generating a human SCA library forgoing the need for costly and time consuming "humanization" procedures.

Single-chain recombinant antibodies (scFvs) consist of the antibody VL and

VH domains linked by a designed flexible peptide tether (Atwell et al., 1999). Compared to intact IgGs, scFvs have the advantages of smaller size and structural simplicity with comparable antigen-binding affinities, and they can be more stable than the analogous 2-chain Fab fragments (Colcher et al., 1998; Adams and Schier, 1999).

The variable regions from the heavy and light chains (VH and VL) are both approximately 110 amino acids long. They can be linked by a 15 amino acid linker or longer with the sequence, for example, which has sufficient flexibility to allow the two domains to assemble a functional antigen binding pocket. In specific embodiments, addition of various signal sequences allows the scFv to be targeted to different organelles within the cell, or to be secreted. Addition of the light chain constant region (Ck) allows dimerization via disulfide bonds, giving increased stability and avidity. Thus, for a single chain Fv (scFv) SCA, although the two domains of the Fv fragment are coded for by separate genes, it has been proven possible to make a synthetic linker that enables them to be made as a single protein chain scFv (Bird et al., 1988; Huston et al., 1988) by recombinant methods. Furthermore, they are frequently used due to their ease of isolation from phage display libraries and their ability to recognize conserved antigens (for review, see Adams and Schier, 1999). Thus, in some aspects of the invention, an antibody may be an SCA that is isolated from a phage display library rather that generated by the more traditional antibody production techniques described above.

5. Bispecific antibodies

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, 1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in PCT application publication No. WO 93/08829 and in Traunecker et al (1991).

According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CHl) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are cotransfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance. In a preferred embodiment of this approach, the bispecifϊc antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation.

6. Conjugate antibodies

Conjugate antibodies such as heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (PCT application publication Nos. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross- linking techniques.

Antibodies or antibody fragments used in practicing the present invention may have additional elements joined or conjugated thereto. For example, a microsphere or microparticle may be joined to the antibody or antibody fragment, as described in U.S. Pat. No. 4,493,825, the disclosure of which is incorporated herein by reference. Additionally, antibodies may be conjugated to nucleic acid sequences. For example, siRNA sequences may be linked to antibodies for the purpose of enhancing or modulating an immune response. In certain other aspects, a nucleic acid expression vector, such as a vector that expresses a viral antigen may be linked to an antibody thereby enabling methods for using nucleic acid vaccination in conjunction with the methods of the invention. E. Treatment and Prevention of Disease 1. Definitions

"Treatment" and "treating" refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, in the context of the present invention, adenoviral keratoconjunctivitis may be treated by topically applying to the ocular surface a pharmaceutically effective amount of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor or an antibody that binds to human CD44 for the purpose of relieving the redness and irritation of the affected eye.

The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well- being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, regarding the treatment of adenoviral keratoconjunictivis, a therapeutic benefit is obtained when there is decreased associated pain or redness of the affected eye.

A "disease" can be any pathological condition of a body part, an organ, or a system resulting from any cause, such as infection, genetic defect, and/or environmental stress. The cause may or may not be known. Examples of a disease or health related condition include adenoviral pneumonia and adenoviral conjunctivitis.

"Prevention" and "preventing" are used according to their ordinary and plain meaning to mean "acting before" or such an act. In the context of a particular disease, those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of blocking the onset of a disease or health-related condition. For example, a first subject who has been exposed to a second subject with adenoviral infection of the eye may administer to the eye a composition as set forth herein for the purpose of blocking the onset of adenoviral infection of the eye.

The subject can be a subject who is known or suspected of being free of a particular disease or health-related condition at the time the relevant preventive agent is administered. The subject, for example, can be a subject with no known disease or health-related condition (i.e., a healthy subject). In some embodiments, the subject is a subject at risk of developing a particular disease or health-related condition. For example, the subject may be an immune compromised subject.

In additional embodiments of the invention, methods include identifying a patient in need of treatment. A patient may be identified, for example, based on taking a patient history or based on findings on clinical examination.

2. Diseases to be Treated or Prevented

Certain embodiments of the present invention are directed to methods of treating or preventing an adenoviral infection in a subject using a pharmaceutically effective amount of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor or an antibody that binds to human CD44 set forth herein. The adenoviral infection can affect any organ or tissue of a subject. In particular embodiments, the subject is a mammalian subject, such as a human subject. In non- limiting examples, the infection may affect the heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries, capillaries, veins, thymus, lymph nodes, bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, nerves, skeletal muscle, smooth muscle, bone, testes, epididymides, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, conjunctiva, cornea, anterior chamber, vitreous, retina, choroid, eyelids, or optic nerve. For example, the infection may be acute febrile pharyngitis, pharyngoconjunctival fever, acute respiratory disease, pneumonia, epidemic keratoconjunctivitis, pertussis-like syndrome, acute hemorrhagic cystitis, gastroenteritis, meningoencephalitis, hepatitis or myocarditis.

3. Dosage

The amount of therapeutic agent to be included in the compositions or applied in the methods set forth herein will be whatever amount is pharmaceutically effective and will depend upon a number of factors, including the identity and potency of the chosen therapeutic agent. One of ordinary skill in the art would be familiar with factors that are involved in determining a therapeutically effective dose of a particular agent. Thus, in this regards, the concentration of the therapeutic agent in the compositions set forth herein can be any concentration. In some particular embodiments, the total concentration of the drug is less than 10%. In more particular embodiments, the concentration of the drug is less than 5%. The therapeutic agent may be applied once or more than once. In non-limiting examples, the therapeutic agent is applied once a day, twice a day, three times a day, four times a day, six times a day, every two hours when awake, every four hours, every other day, once a week, and so forth. Treatment may be continued for any duration of time as determined by those of ordinary skill in the art.

F. Secondary Forms of Therapy

The therapies of the present invention may be used in combination with secondary therapies directed to the treatment or prevention of an adenoviral infection in a subject. For example, therapies directed to diseases of the eye may include other antiviral agents in the form of ointments, lotions or eye drops placed directly into the eye. One of skill in the art will recognize other secondary therapies that may be used in conjugation with the methods and compositions of the present invention. Non- limiting examples of antiviral agents for use as secondary therapies include cidofovir, amantadine, rimantadine, acyclovir, gancyclovir, pencyclovir, famciclovir, foscarnet, ribavirin, or valcyclovir.

G. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 CD44 Modulates Adenoviral Infection

Adenovirus is a common human pathogen that in some cases may cause severe or even life treating disease. Despite its role in human disease there are no well accepted animal model systems for studying the virus. Given this problem, many virologists have turned to adenoviral transduction vector that comprise reporter genes. Once transduced by the vector, human or animal cells may be assessed for the level of reporter gene expression. Thus, factors involved in adenoviral infection may be identified by using this vector transduction model system. Using this system, it has been demonstrated that a CD44 binding molecule, hyaluronan, can modulate adenoviral transduction. Hyaluronan, a CD44 ligand, enhances viral infection by stimulating a CD44 signaling pathway (U.S. Patent Publ. 20030186936). Specifically, intact hyaluronan from vitreous fluid significantly enhances the efficiency of adenoviral transduction. Conversely, it was found that degraded or low molecular weight forms of hyaluronan inhibited adenoviral transduction. Furthermore, the effects of hyaluronan on adenoviral infection seem to be mediated through the CD44 receptor protein, since certain antibodies binding to murine CD44 could block the effects of hyaluronan.

The studies herein employ an adenoviral vector that expresses a reporter gene to assess the efficiency of viral infection (e.g., cell entry and viral gene expression).

These studies demonstrate that CD44 plays a central role in virus infection. To further assess the role of CD44 in adenoviral infection, transduction studies were performed using CD44 positive HeLa cells. Briefly, cells were transduced with an adenoviral vector (Group C, Serotype 5) that expresses luciferase (AD5-Luc). Following transduction HeLa extracts were assessed for luciferase activity to determine transduction efficiency with various treatment conditions. The studies herein have identified a new method that may be used to antagonize the CD44 signaling pathway and thereby inhibit viral infection. Studies shown in FIG. 2 demonstrate that the broad spectrum MMP inhibitor, TAPI-1-0, effectively reduces adenoviral transduction in the presence of vitreous. Interesting, HeLa cells treated with DMSO alone or DMSO plus 5% vitreous were both poorly transduced indicating that vitreous had little effect on adenoviral infection of HeLa cells (FIG. 1).

However, HeLa cells treated with PMA were transduced more efficiently and the addition of PMA also restored the enhancing effect of vitreous on transduction efficiency. Thus, although PMA by itself enhanced the vector transduction, there was even greater enhancement in the presence of 5% vitreous. These results support a model for vitreous enhancement of Ad5-Luc in which there is a CD44 activation/dimerization step followed by hyaluronan binding. Furthermore, as shown in FIG. 3, inhibitors of gamma-secretase, a complex known to cleave CD44, also inhibit both basal and vitreous enhanced adenoviral transduction. Furthermore, an antibody that binds to human CD44 in the hyaluronan binding region is also able to inhibit both baseline and vitreous enhanced adenoviral transduction. Thus, the studies here demonstrate that CD44 pathway antagonists may be used to inhibit adenoviral infection.

Example 2 MMP Inhibitors Reduce Adenoviral Infection

MMPs are known to agonize the CD44 pathway in expressing cells by cleaving the CD44 extra cellular domain. Thus, the effect of MMP inhibitors on adenoviral transduction was assessed. Y79 cell that are susceptible to adenoviral transduction in a manor that is vitreous enhanced were used to determine the effect of

TAPI-O, a broad range MMP inhibitor, on adenoviral transduction. Cells or human conjunctival explants were transduced with an Ad5-Luc vector either in the absence (media) or presence of 1 μM TAPI-O or 10 μM TAPI-O in various amounts of vitreous. Following transduction cell lysates were assessed for luciferase activity.

The results shown in FIG. 2, show that TAPI-O does inhibits adenoviral transduction.

More specifically, vitreous enhancement of vector transduction was inhibited in the presence of TAPI-O. These results are consistent with the hypothesis that cleavage of the CD44 ectodomain by a metalloproteinase is required for vitreous enhancement of

Ad5-Luc transgene expression and thus adenoviral infection (FIG. 2).

Example 3 γ-Secretase Inhibitors Reduce Adenoviral Infection

Another factor that may agonize the CD44 signaling pathway is the γ- secretase/PSl complex which is known to cleave the CD44 intracellular domain

(Murakami et al, 2003). Given the role of γ-secretase in the CD44 pathway, the effect of γ-secretase inhibitors on adenoviral transduction was assessed. Y79 cells or human conjunctival explants were transduced with Ad5-Luc in the absence (media) or presence of 500 nM DAPT and/or the 10% vitreous. These studies demonstrate that DAPT does inhibit adenoviral transduction. Furthermore, transduction was inhibited even in the absence of vitreous. This suggests that intra-membraneous CD44 degradation by the γ-secretase/PSl complex is involved in the vitreous enhancement of Ad5-Luc transduction. Furthermore, taken together these results suggest that the sequential degradation of CD44 after dimerization and binding of hyaluronan by the metalloproteinases and the γ-secretase/PSl are steps required for the vitreous enhancement of Ad5-Luc transduction and adenoviral infection in general (FIG. 3). Example 4

Anti-Human CD44 Antibodies Reduce Adenoviral Infection

Studies were undertaken to study the effects of antibodies that bind to CD44 on adenovirus infection. HEK 293 cells were transduced with a helper dependent (HD) adenovirus comprising a CMV/β-Gal expression cassette. Transductions were performed with or with-out 5% vitreous alone or in the presence of control IgG (ROSl) or an anti-CD44 IgG (BRIC222 or BRIC235). Following transduction cells were harvested and β-Gal activity was assessed. Only the BRIC235 (an antibody that binds to the CD44 hyaluronan binding region) antibody inhibited HD-CMV/β-Gal either in the presence or absence of vitreous, whereas none of the other antibodies affected transduction in either condition. This result indicates that infection of HEK293 cells requires the interaction of the CD44 hyaluronan binding domain with hyaluronan from the vitreous. Thus, antibodies that bind to human CD44 at the hyaluronan binding region act as inhibitors of virus infection (FIG. 4).

Example 5 Knock-down of MMPl 5 in Y79 Cells Decreases Vitreous Enhancement of Ad- Mediated Transgene Expression

The membrane type metalloprotease MMP 15 was found to be highly expressed in the retinoblastoma tumors (data not shown). To determine if this MMP is associated with the TAPI-I /TAPI-O sensitive vitreous enhancement of Ad-mediated transgene expression observed in the retinoblastoma cell line Y79, its expression was knock-down by using a MMP 15 shRNAmir (data not shown). As a control, Y79 cells were also engineered with a non-silencing shRNAmir. Cells were plated at 2x10^Λ4 cells/well in a 96-well plate and transduced with an Ad5/CMV-Luc (MOI=250 pfu/cell) in the presence or absence of 5% vitreous. A dramatic enhancement of Ad- mediated transgene expression was observed in both the parental and non-silencing containing Y79 cells. However, this vitreous enhancement is significantly decreased in MMP15shRNA containing Y79 cells when compared to either the parental or non- silencing containing Y79 cells (n=5) (FIG. 5).

Example 6

Effect of CD44 Antagonists on Ad-Mediated Transgene Expression on Human Conjunctiva Explants The human adenovirus is a well known pathogen of the human conjunctiva among other tissues. In this experiment human conjunctiva explants were plated in a 96-well plate with DMEM culture media supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin antibiotics. Samples were transduced with an Ad5/CMV-Luc vector (1.25xlO^Λ7 pfu/sample) in the presence or absence of BRIC235 (a human CD44 blocking antibody), TAPI-O or TAPI-I (both are nonspecific MMP inhibitors), and DAPT (a potent γ-Secretase inhibitor). Tissue cells were lysed and luciferase activity determined and standardized to the amount of protein present in the lysate. The inhibition of luciferase activity was determined by using the following mathematical formula: %Inhibition = [(Activity in Media - Activity in Antagonist)/ Activity in Media] x 100. Graphed results represent average values from two independent experiments (FIG. 6).

Example 7

CD44 Degradation Inhibitors Modify Adenovector-Mediated Transgene

Expression 1. Materials and Methods a. Cell lines

HeLa cells (cervical epithelial carcinoma), COS-7 cells (African green monkey kidney), and Y79 cells (human retinoblastoma) were maintained in culture using Dulbecco's modified eagle medium (Gibco/BRL, Life Technologies, NY); Jurkat cells (T-cell lymphoma) were maintained in culture using RPMI- 1640 (Gibco/BRL, Life Technologies, NY). Both media were supplemented with 5% fetal bovine serum (HyClone, Logan UT), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco/BRL, Life Technologies, NY). Cells were incubated at 37°C in 5% carbon dioxide supplemented humidified air. i. Human conjunctiva explants

Human eyes were obtained from The Lions Eye Bank of Texas at Baylor College of Medicine. Tissues were screened for HIV 1-2, HCV, HBsAg, and

RPR/TP/STS before release for research purposes. Information other than age and cause of death was not obtained from the tissue bank. Conjunctiva isolation was performed using sterile techniques maintaining tissues in Dulbecco's modified eagle medium (Gibco/BRL, Life Technologies, NY) supplemented with 5% fetal bovine serum (HyClone, Logan UT), 100 units/ml penicillin and 100 μg/ml streptomycin

(Gibco/BRL, Life Technologies, NY). Explants were divided and transduced with adenoviral vectors immediately after isolation. ii. Reagents

Vitreous was harvested from fresh or frozen bovine eyes (Ladpak Slaughterhouse, Needville, TX). To reduce viscosity, the vitreous was sheared using a 19-gauge needle and clarified by centrifugation before dilution in serum- free culture medium. Expressed luciferase activity was quantified using the Luciferase Assay

System (Promega, Madison, WI). Protein concentrations were determined using the method of Bradford (BioRad, Hercules, CA), Bradford, (1976). The adenoviral construct containing the firefly luciferase (Ad5/CMV-luc, 22 vp/iu) reporter gene was provided by the Center for Cell and Gene Therapy, Baylor College of Medicine,

Houston, TX. Viral concentrations were determined spectroscopically (Nyberg-

Hoffman, 1997) and infectious units were determined by plaque assays using

HEK293 cells. The metalloproteinase inhibitors TAPI-O (IC₅₀=IOO nM) and TAPI-I (IC₅₀=920 nM), and the γ-secretase/PSl inhibitor DAPT (IC₅₀=20 nM) were purchased from Calbiochem (San Diego, CA) and Sigma (St. Louis, MO) respectively. iii. Statistical Analysis

Results are reported as average ± standard deviation. Differences between two groups were determined by the Student-t test. For multiple comparisons, an analysis of variance was performed followed by the Student-Newman-Keuls test. Statistical difference was considered significant when the p-value < 0.05. All tests were performed using the GraphPad Prism software version 3.02.

2. Results a. Incubation with a metalloproteinase inhibitor blocks CD44-ligand enhanced adenovector-mediated transgene expression.

Y79 cells were incubated with the indicated concentrations of vitreous, which contains physiological concentrations of the CD44 ligand hyaluronan (Sebag, 1992) in the presence or absence of the metalloproteinase inhibitor TAPI-O (1 μM) and transduced with Ad5/CMV-luc(250 pfu/cell). The cultures were incubated for 24 hours and luciferase activity was measured. The CD44-mediated enhancement of luciferase activity was inhibited in the cultures containing TAPI-O (P<0.05, n=3) (Fig. 7a). The effect of TAPI-O in the absence of vitreous and/or CD44 was determined by transducing Y79 cells or the CD44 negative cell lines Jurkat (JK) and COS-7 cells with Ad5/CMV-luc in the presence or absence of the metalloproteinase inhibitor TAPI-O (1 μM). Y79 cells without vitreous were transduced with Ad5/CMV-luc (250 pfu/cell) in either the presence or absence of the metalloproteinase inhibitor TAPI-O (1 μM). Adenovector-mediated transgene expression was not significantly inhibited in the presence of TAPI-O (P>0.05, n=4) (FIG. 7b). Jurkat (JK) cells were transduced with vitreous (5%) and Ad5/CMV-luc (1,000 pfu/cell) in either the presence or absence of the metalloproteinase inhibitor TAPI-O (1 μM). Adenovector-mediated transgene expression was not significantly inhibited in the presence of TAPI-O (P>0.05, n=5) (FIG. 7c). COS-7 cells were transduced with vitreous (5%) and Ad5/CMV-luc (50 pfu/cell) in either the presence or absence of the metalloproteinase inhibitor TAPI-O (1 μM). Adenovector-mediated transgene expression was not significantly inhibited in the presence of TAPI-O (P>0.05, n=5) (FIG. 7d). TAPI-O did not have any significant effect in the absence of vitreous (data not shown).

b. The γ-secretase/PSl inhibitor DAPT inhibits CD44-ligand enhanced adenovector-mediated transgene expression.

Y79 cells were transduced with Ad5/CMV-luc (250 pfu/cell) either in the presence or absence of the γ-secretase/PSl inhibitor DAPT (500 nM) and 10% vitreous. Vitreous enhancement of adenovector-mediated transgene expression was inhibited in the presence of DAPT (P<0.05, n=3). (FIG. 8a). Similarly, incubation with this inhibitor reduced transgene expression in the absence of vitreous. Y79 cells were transduced with Ad5/CMV-luc (250 pfu/cell) either in the presence or absence of the γ-secretase/PSl inhibitor DAPT (500 nM). Adenovector-mediated transgene expression was inhibited in the presence of DAPT (P<0.0002, n=5) (FIG. 8b). The effect of DAPT in cells that do not contain CD44 was determined by transducing JK cells or COS-7 cells with Ad5/CMV-luc in either the presence or absence of the γ- secretase/PSl inhibitor DAPT (500 nM). JK cells were transduced with Ad5/CMV- luc (1,000 pfu/cell) in either the presence or absence of the γ-secretase/PSl inhibitor DAPT (500 nM). Adenovector-mediated transgene expression was not significantly inhibited in the presence of DAPT (P>0.05, n=5). d) COS-7 cells were transduced with Ad5/CMV-luc (50 pfu/cell) in either the presence or absence of the γ- secretase/PSl inhibitor DAPT (500 nM). Adenovector-mediated transgene expression was not significantly inhibited in the presence of DAPT (P>0.05, n=5) (FIG. 8c, d). Luciferase activity was not significantly inhibited in the presence of DAPT in either of these cell lines. c. The inhibitors TAPI-I and DAPT can inhibit adenovector- mediated transgene expression in human conjunctiva explants.

Naturally occurring adenovirus infection is a human disease, and conventional animal models are poorly representative of naturally occurring infection. To mimic actual infection, human conjunctiva, a natural target of infection, was transduced by an adenoviral vector in situ. Conjunctiva was harvested from two human eyes using sterile techniques and kept in supplemented DMEM culture media. Samples were divided into small pieces and placed in the wells of a 96-well plate with fresh culture media (100 μL media per well). Explants were transduced with Ad5/CMV-luc (1.25xlO⁷ pfu) in the presence or absence of TAPI-I (10 μM), DAPT (500 nM), or DMSO (0.1%), or culture media only. TAPI-I inhibited adenovector-mediated transgene expression when compared to either samples transduced in the presence of media (P<0.05, n=3) or DMSO (P<0.05, n=3). DAPT inhibited adenovector- mediated transgene expression when compared to either samples transduced in the presence of media (P<0.05, n=3) or DMSO (P<0.05, n=3). There was not a significant difference in adenovector-mediated transgene expression between the inhibitors (P>0.05, n=3) (FIG. 9). It was demonstrated that the metalloproteinase inhibitor TAPI-O can block vitreous enhancement of transgene expression in Y79 cells. The γ-secretase/PSl inhibitor DAPT blocked both the vitreous enhancement of transgene expression and transgene expression in the absence of vitreous. TAPI-O and DAPT did not affect transgene expression in CD44 negative cells. Furthermore, these inhibitors not only have an effect in vitro but also play a role in situ.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

1. A method for treating a subject with an adenoviral infection comprising administering to the subject having the adenoviral infection an effective amount of a CD44 pathway antagonist selected from the group consisting of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor and an antibody that binds to human CD44.

2. The method of claim 1 , wherein the subject is a human.

3. The method of claim 2, wherein the subject is immune compromised.

4. The method claim 3, wherein the subject is a bone marrow transplant patient.

5. The method claim 1, wherein the adenoviral infection is conjunctivitis, acute febrile pharyngitis, pharyngoconjunctival fever, acute respiratory disease, pneumonia, epidemic keratoconjunctivitis, pertussis-like syndrome, acute hemorrhagic cystitis, gastroenteritis, meningoencephalitis, hepatitis or myocarditis.

6. The method claim 1, wherein the adenoviral infection is a systemic or persistent adenoviral infection.

7. The method of claim 1, wherein the CD44 pathway antagonist a MMP inhibitor.

8. The method of claim 7, wherein the MMP is MMP-I (interstitial collagenase), MMP-2 (Gelatinase A), MMP-3 Stomelysin-1), MMP-7 (Matrilysin), MMP-8 (Neutraphil collagenase), MMP-9 (Gelatinase B), MMP-IO (Stromelysin-2), MMP-I l (Stromelysin-3), MMP-12 (Metalloelsastase), MMP-13 (Collagenase-3), MMP-14 (MTl-MMP), MMP-15 (MT2-MMP), MMP-16 (MT3-MMP), MMP-17 (MT4- MMP), MMP-18 (Collagenase-4), MMP-19, MMP-20 (Enamelysin), MMP-21 (MT5- MMP), MMP-23 or MMP-24 inhibitor.

9. The method of claim 7, wherein the MMP inhibitor is a small molecule, a peptide, a polypeptide, a protein, an antibody, an antibody fragment, a DNA, or a RNA.

10. The method claim 9, wherein the MMP inhibitor is a small molecule.

11. The method of claim 10, wherein the MMP inhibitor is doxycycline, tetracycline, CMT-I CMT-8, Marmastat, Batimastat (BB-94), AG3340 (prinomastat), BAY 12-9566, MMI270, COL-3 (metastat), BMS-275291, CP-471,358, AE-94 (neovastat) or TAPI-O.

12. The method of claim 11 , wherein the MMP inhibitor is TAPI-O.

13. The method of claim 12, wherein the MMP inhibitor is tetracycline or doxycycline.

14. The method of claim 7, wherein the MMP inhibitor is a selective MMP inhibitor.

15. The method claim 1, wherein the CD44 pathway antagonist is a gamma- secretase inhibitor.

16. The method of claim 15, wherein the gamma-secretase inhibitor is a small molecule, a peptide, a polypeptide, a protein, an antibody, an antibody fragment, a DNA, or a RNA.

17. The method of claim 16, wherein the gamma-secretase inhibitor is a peptide.

18. The method claim 17, the peptide is a modified aldehyde dipeptide or tripeptide.

19. The method of claim 16, wherein the gamma-secretase inhibitor is a small molecule.

20. The method of claim 19, wherein the small molecule is DAPT, LY450139 dihydrate, LY-411,575, MRK-560, or L-685,458.

21. The method of claim 20, wherein the gamma-secretase inhibitor is DAPT.

22. The method of claim 1, wherein the CD44 pathway antagonist is an antibody that binds to human CD44.

23. The method of claim 22, wherein the antibody binds to the same CD44 epitope as a BRIC-235 antibody.

24. The method of claim 22, wherein the antibody comprises the VL and VH domains of a BRIC-235 antibody.

25. The method of claim 18, wherein the antibody is the BRIC-235 antibody or a BRIC-235 antibody fragment.

26. The method of claim 15, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a single chain antibody, a humanized antibody, a Fab fragment, F(ab')2 fragment, single domain antibodies or a antibody paratope peptide.

27. The method of claim 26, wherein the antibody is a monoclonal antibody.

28. The method of claim 26, wherein the antibody is a humanized antibody.

29. The method claim 1, further comprising administering an effective amount of two or more the CD44 pathway antagonist selected from the group consisting of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor and an antibody that binds to human CD44.

30. The method claim 1, wherein the CD44 pathway antagonist is administered locally.

31. The method claim 1, wherein the CD44 pathway antagonist is administered systemically.

32. The method claim 1, wherein the CD44 pathway antagonist is administered topically, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage.

33. The method claim 30, wherein the CD44 pathway antagonist is administered to the eye.

34. The method claim 33, wherein administration to the eye is by topical, subconjunctival, periocular, retrobulbar, subtenon, intracameral, intravitreal, intraocular, subretinal, posterior juxtascleral or suprachoroidal administration.

35. The method claim 33, wherein the CD44 pathway antagonist is administered as an eye drop.

36. The method of claim 1, further comprising a administering an additional adenoviral therapy.

37. The method of claim 36, wherein the additional adenoviral therapy is low molecular weight hyaluronhyaluronan, HPMPA, cidofovir or ribavirin.

38. An eye dropper comprising:

a) a CD44 pathway antagonist selected from the group consisting of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor and an antibody that binds to human CD44; and

b) a sterile and pharmaceutically acceptable aqueous carrier;

wherein the composition is comprised in a bottle comprising an exit portal that enables drop-wise administration.

39. A nasal spray comprising:

a) a CD44 pathway antagonist selected from the group consisting of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor and an antibody that binds to human CD44; and

b) a pharmaceutically acceptable carrier;

wherein the composition is comprised in a bottle comprising an exit portal that disperses the composition into a mist or aerosol.

40. A pharmaceutical composition comprising:

a) a combination of at least two CD44 pathway antagonist selected from the group consisting of a matrix metalloprotease (MMP) inhibitor, a gamma-secretase inhibitor and an antibody that binds to human CD44; and b) a pharmaceutically acceptable carrier.

41. A method of increasing the expression of a transgene at a site in a subject comprising administering to the subject an adenoviral vector comprising a transgene encoding a therapeutic polypeptide and a pharmaceutically effective amount of an agent that increases the expression and/or activity of:

a) a MMP or

b) gamma secretase;

wherein expression of the transgene is increased at the site in the subject.

42. The method of claim 41, wherein the subject is a patient with cancer, and the site is a cancer in the subject.

43. The method of claim 41 , wherein the subject is a human.

44. The method of claim 41, wherein the subject is administered an agent that increases the expression of a MMP or gamma secretase.

45. The method of claim 44, wherein the agent that increases the expression of a MMP or gamma secretase is a small molecule, a peptide, a polypeptide, a protein, an antibody, an antibody fragment, a DNA, or a RNA.

46. The method of claim 45, wherein the agent that increases the expression of a MMP or gamma secretase is a siRNA or shRNA.

47. The method of claim 41, wherein the subject is administered an agent that increases the activity of a MMP or a gamma secretase.

48. The method of claim 41 , wherein the MMP is MMP- 15.

49. The method of claim 42, wherein the cancer is wherein the cancer is breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colorectal cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia.

50. The method of claim 49, wherein the cancer is cancer of the eye.

1. The method of claim 50, wherein the cancer of the eye is retinoblastoma.