Patent Grant
9200041
Human adenoviruses (Ads) have been classified into six species (A to F), currently containing 51 serotypes. Most Ad serotypes utilize the coxsackie-adenovirus receptor (CAR) as a primary attachment receptor (Bergelson et al., 1997). This is, however, not the case for species B Ad serotypes. Recently, we have suggested a new grouping of species B Ads based on their receptor usage (Tuve et al., 2006). Group 1 (Ad16, 21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3, Ad7, 14) share a common, unidentified receptor/s, which is not CD46 and which was tentatively named receptor X; Group 3 (Ad11) preferentially interacts with CD46, but also utilizes receptor X if CD46 is blocked.
Species B Ads are common human pathogens. Since 2005, a simultaneous emergence of diverse species B serotypes at the majority of US military training facilities was observed. This included serotypes Ad3, Ad7, and Ad14 (Metzgar et al., 2007). In 2007 a new, highly pathogenic strain and possibly more virulent strain of Ad14, Ad14a, has been discovered at several sites in the US and in Asia (Louie et al., 2008; Tate et al., 2009). We recently demonstrated that Ad14a belongs to species B group 2 Ads with regards to their receptor usage (Wang et al., 2009). Collectively, all receptor X utilizing serotypes (Ad3, Ad7, Ad14, Ad14a, and Ad11) are referred to herein as AdB-2/3.
AdB-2/3 have great relevance as gene transfer vectors, particularly with regard to tumors of epithelial origin, representing most solid tumors (Yamamoto and Curiel, 2010). Epithelial cells maintain several intercellular junctions and an apical-basal polarity. Key features of epithelial cells are conserved in epithelial cancers in situ and in cancer cell lines (Turley et al., 2008). Both CAR and CD46 are often trapped in tight and adherence junctions of epithelial cancer cells and are not accessible to Ads that use these attachment receptors (Coyne and Bergelson, 2005; Strauss et al., 2009). In contrast, AdB-2/3 efficiently infect epithelial cancer cells, which is accomplished in part through induction of processes that are reminiscent of Epithelial-to-Mesenchymal Transition (EMT) (Strauss et al., 2009). Another distinctive feature of AdB-2/3 is their ability to produce subviral dodecahedral particles during their replication, consisting of Ad fiber and penton base (Norrby et al., 1967). Penton-Dodecahedra (PtDd) cannot assemble from full-length penton base protein, but require spontaneous N-terminal truncation by proteolysis between residues 37 and 38 (Fuschiotti et al., 2006). This cleaved site is conserved in Ad3, Ad7, Ad11, and Ad14 but is not present in Ad2 and Ad5. In the case of Ad3 the PtDd are formed at a massive excess of 5.5×106 PtDd per infectious virus (Fender et al., 2005), and it has been suggested that PtDd enhance Ad3 infectivity by disturbing intercellular junctions, thus favoring virus spreading (Walters et al., 2002).
The first attempts to identify receptor X date back to 1995. Recently, several candidates for receptor X such as CD46, CD80 and/or CD86 were suggested (Fleischli et al., 2007; Short et al., 2004; Short et al., 2006; Sirena et al., 2004). However, no one thus far has been able to verify that these proteins can serve as the high affinity receptor for AdB-2/3 (Gaggar et al., 2003b; Gustafsson et al., 2006; Marttila et al., 2005; Persson et al., 2008; Segerman et al., 2003; Tuve et al., 2006).
In a first aspect, the present invention provides methods for enhancing therapeutic treatment, or diagnosis of a disorder associated with epithelial tissue, and/or imaging epithelial tissues, comprising administering to a subject in need thereof:
The method of the invention can be used to treat and/or diagnose disorders associated with epithelial tissue, and/or image epithelial tissues, such as epithelial tissues in which such a disorder is believed present, where such disorders include but are not limited to solid tumors, irritable bowel syndrome, inflammatory bowel disorder, Crohn's disease, ulcerative colitis, constipation, gatroesophageal reflux disease, chronic obstructive pulmonary disease, asthma, bronchitis, pulmonary emphysema, cystic fibrosis, interstitial lung disease, pneumonia, primary pulmonary hypertension, pulmonary embolism, pulmonary sarcoidosis, tuberculosis, pancreatitis, pancreatic duct disorders, brain disorders (ie: any brain disorder that could benefit from improved transport of drugs through the epithelial blood-brain barrier), bile duct obstruction, cholecystitis, choledocholithiasis, infections (including but not limited to cellulitis, pneumonia, and pyelonephritis), and gallstones. In a preferred embodiment, the disorder is a solid tumor, including, but not limited to, breast tumors, lung tumors, colon tumors, rectal tumors, stomach tumors, prostate tumors, ovarian tumors, uterine tumors, cervical tumors, kidney tumors, skin tumors, melanomas, pancreatic tumors, liver tumors, endocrine tumors, brain tumors, head and neck tumors, nasopharyngeal tumors, gastric tumors, squamous cell carcinomas, adenocarcinomas, bladder tumors, and esophageal tumors.
The methods of any embodiment of the invention can utilize, for example, AdB-2/3 fiber multimers selected from the group consisting of an Ad3 fiber multimer, and Ad7 fiber multimer, an Ad11 fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer, combinations thereof, and functional equivalents thereof. In a preferred embodiment, the AdB-2/3 fiber multimer is an Ad3 fiber multimer, or functional equivalents thereof. In further embodiments, the AdB-2/3 fiber multimer can comprise AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd), recombinant AdB-2/3 fiber multimers, and functional equivalents thereof. In preferred embodiments, the AdB-2/3 fiber multimer comprises an Ad3 PtDd or junction opener 1 (JO-1) (SEQ ID NO:20)
Any suitable therapeutic, diagnostic, or imaging agent can be used in these methods. In embodiments where therapeutics are used, such therapeutics may include but are not limited to antibodies, immunoconjugates, vaccines, radioactive particle/radiation therapy (“radiation”), chemotherapeutics, cellular immunotherapy including adoptive T-cell therapy and dendritic cell therapy, nanoparticles, inhaled therapeutics, gene therapy constructs, and nucleic acid therapeutics or combinations thereof. In preferred embodiments, the therapeutic comprises a chemotherapeutic or an anti-tumor monoclonal antibody. Exemplary anti-tumor monoclonal antibodies that can be used in the methods of the invention include, but are not limited to trastuzumab, cetumiximab, petuzumab, apomab, conatumumab, lexatumumab, bevacizumab, bevacizumab, denosumab, zanolimumab, lintuzumab, edrecolomab, rituximab, ticilimumab, tositumomab, alemtuzumab, epratuzumab, mitumomab, gemtuzumab ozogamicin, oregovomab, pemtumomab daclizumab, panitumumab, catumaxomab, ofatumumab, and ibritumomab.
In one preferred embodiment, the disorder associated with epithelial tissue comprises a Her-2 positive tumor. Exemplary Her-2 positive tumors that can be treated under this embodiment include, but are not limited to, breast tumors, gastric tumors, colon tumors, and ovarian tumors. In this embodiment, it is further preferred that the therapeutic comprises trastuzumab; in a further preferred embodiment, the therapeutic further comprises one or more chemotherapeutics and/or radiation. In this embodiment, it is further preferred that the AdB-2/3 fiber multimer comprises an Ad3 PtDd, JO-1 (SEQ ID NO:20), or functional equivalents thereof. In another preferred embodiment, the subject to be treated has not responded to trastuzumab therapy.
In another preferred embodiment, the disorder associated with epithelial tissue comprises an EGFR-positive tumor. Exemplary EGFR-positive tumors include, but are not limited to lung tumors, colon tumors, breast tumors, rectal tumors, head and neck tumors, and pancreatic tumors. In this embodiment, it is preferred that the therapeutic comprises cetuximab; in a further preferred embodiment, the therapeutic further comprises one or more chemotherapeutics and/or radiation. In this embodiment, it is further preferred that the AdB-2/3 fiber multimer comprises an Ad3 PtDd, JO-1 (SEQ ID NO:20), or functional equivalents thereof. In another preferred embodiment, the subject to be treated has not responded to cetuximab therapy.
In another embodiment, the disorder associated with epithelial tissue comprises a solid tumor and therapeutic comprises a vascular endothelial growth factor (VEGF) inhibitor.
In a second aspect, the present invention provides methods for treating a disorder associated with epithelial tissue, comprising administering to a subject in need thereof an amount of AdB-2/3 fiber multimer or functional equivalent thereof, sufficient to treat the disorder. In this embodiment, the AdB-2/3 fiber multimer is administered as a monotherapy. In one embodiment, the method is used to treat an AdB-2/3 viral infection. In another embodiment, the disorder to be treated is a solid tumor. Exemplary solid tumors that can be treated using this aspect of the invention include but are not limited to breast tumors, lung tumors, colon tumors, rectal tumors, stomach tumors, prostate tumors, ovarian tumors, uterine tumors, cervical tumors, kidney tumors, skin cancers, melanomas, pancreatic tumors, liver tumors, endocrine tumors, brain tumors, head and neck tumors, nasopharyngeal tumors, gastric tumors, squamous cell carcinomas, adenocarcinomas, bladder tumors, and esophageal tumors. In this aspect, exemplary AdB-2/3 fiber multimers for use include, but are not limited to an Ad3 fiber multimer, and Ad7 fiber multimer, an Ad11 fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer, combinations thereof, and functional equivalents thereof. In a preferred embodiment, the AdB-2/3 fiber multimer is an Ad3 fiber multimer, and functional equivalents thereof. In a further preferred embodiment, the AdB-2/3 fiber multimer is selected from the group consisting of AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd), recombinant AdB-2/3 fiber multimers, and functional equivalents thereof. In various further preferred embodiments, the AdB-2/3 fiber multimer comprises an Ad3 PtDd or junction opener 1 (JO-1) (SEQ ID NO:20),
In a third aspect, the present invention provides recombinant AdB-2/3 fiber polypeptides, comprising:
In one embodiment, the recombinant AdB-2/3 fiber polypeptide does not include an AdB-2/3 tail domain. In another embodiment, each shaft domain is selected from the group consisting of an Ad3 shaft domain, an Ad7 shaft domain, an Ad11 shaft domain, an Ad 14 shaft domain, an Ad14a shaft domain, combinations thereof, and functional equivalents thereof. In a further embodiment, the one or more shaft domains comprise 1-22 shaft domains. In various further embodiments, each shaft domain comprises or consists of an amino acid sequence according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11 or SEQ ID NO:12. In a further embodiment, the knob domain is selected from the group consisting of an Ad3 knob domain, an Ad7 knob domain, an Ad11 knob domain, an Ad 14 knob domain, an Ad14a knob domain, and functional equivalents thereof. In various further embodiments, the knob domain comprises or consists of an amino acid sequence according to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13 or SEQ ID NO:14. In a further embodiment, the dimerization domain comprises an amino acid sequence selected from the group consisting of EVSALEK (SEQ ID NO: 22) and/or KVSALKE (SEQ ID NO: 23). In a preferred embodiment, the recombinant AdB-2/3 polypeptide comprises: (a) one or more shaft domains that each comprise or consist of an Ad3 shaft domain (SEQ ID NO: 1); and (b) a knob domain comprises or consists of an Ad3 knob domain (SEQ ID NO:6).
In a further preferred embodiment, the recombinant AdB-2/3 fiber polypeptide comprises or consists of the amino acid sequence of JO-1 (SEQ ID NO:20), In a further preferred embodiment, the AdB-2/3 fiber polypeptide is multimerized; most preferably dimerized.
In other aspects, the present invention provides isolated nucleic acids encoding the recombinant AdB-2/3 fiber polypeptides of the invention; recombinant expression vectors comprising the isolated nucleic acids of the invention; host cells comprising the recombinant expression vectors of the invention; and pharmaceutical compositions comprising AdB-2/3 multimers as described herein. In a further aspect, the present invention provides methods for improving delivery of a compound to an epithelial tissue, comprising contacting the epithelial tissue with (a) one or more compound to be delivered to the epithelial tissue; and (b) an amount of AdB-2/3 fiber multimer, or functional equivalent thereof, sufficient to enhance delivery of the one or more compounds to the epithelial tissue. The methods permit improved delivery of any compound that targets epithelial cells, including but not limited to diagnostic compounds and imaging compounds. In one preferred embodiment, the epithelial tissue comprises a solid tumor.
In a still further aspect, the present invention provides methods for improving delivery of a compound to tissue expressing desmoglein 2 (DSG2), comprising contacting the tissue expressing DSG2 with (a) one or more compound to be delivered to the tissue; and (b) an amount of AdB-2/3 fiber multimer, or functional equivalent thereof, sufficient to enhance delivery of the one or more compounds to the tissue. The methods of this aspect of the invention can be used to improve delivery of any compound of interest to a tissue expressing DSG2.
In a yet further aspect, the present invention provides methods for inducing an epithelial to mesenchymal transition (EMT) in a tissue, comprising contacting the epithelial tissue with an amount of AdB-2/3 fiber multimer, or functional equivalent thereof, sufficient to induce EMT.
In one preferred embodiment of each of these further aspects of the invention, the AdB-2/3 fiber multimer is selected from the group consisting of an Ad3 fiber multimer, and Ad7 fiber multimer, an Ad11 fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer, combinations thereof, and functional equivalents thereof. In various further preferred embodiments, the AdB-2/3 fiber multimer is an Ad3 fiber multimer, or a functional equivalent thereof; the AdB-2/3 fiber multimer is selected from the group consisting of AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd), recombinant AdB-2/3 fiber multimers, and functional equivalents thereof; the AdB-2/3 fiber multimer comprises an Ad3 PtDd; the AdB-2/3 fiber multimer comprises junction opener 1 (JO-1) (SEQ ID NO:20); and the AdB-2/3 fiber multimer is a dimer.
In another aspect, the present invention provides method for identifying candidate compounds for one or more of treating a disorder associated with epithelial tissue, improving delivery of a substance to an epithelial tissue, for improving delivery of a substance tissue expressing DSG2, inducing an EMT in a tissue, and/or treating an AdB-2/3 infection comprising (a) contacting an AdB-2/3 fiber multimer to DSG2 under conditions to promote multimer binding to DSG2, wherein the contacting is carried out in the presence of one or more test compounds; and (b) identifying positive test compounds that compete for binding of the AdB-2/3 fiber multimer to DSG2 compared to control; wherein the positive test compounds are candidate compounds for one or more of treating a disorder associated with epithelial tissue, improving delivery of a substance to an epithelial tissue, for improving delivery of a substance tissue expressing DSG2, inducing an EMT in a tissue, and/or treating an AdB-2/3 infection.
a) Amino acid alignment of N-termini of Ad pentons. The protease cleavage site at aa 37/38 is marked in orange. b) Scheme of viral particles and particle components used in this study. PtDd possess 12 units containing penton base and trimeric fiber. BsDd only contain penton base. c) Competition of 3H-labeled Ad14, Ad14a, Ad11 and Ad35 virus attachment to HeLa cells after pre-incubation with Ad3 BsDd, PtDd, or antiCD46 antibodies (aCD46). Attachment in PBS-treated cells was taken as 100%. n=5. Notably, the finding that PtDd partially blocks Ad35 could be due to the physical proximity of DSG2 and CD46 in HeLa cells. d) Ad3 attachment studies as in c) after preincubation with both Ad3 fiber knob and BsDd (BsDd+Ad3K). The molar concentration of Ad3K was equal that of fiber knob within PtDd used for competition. e) Scheme of Ad3-GFP vector. The vector is based on wild-type Ad deleted for nt 29,892-30,947 to accommodate the CMV-GFP-polyA expression cassette. The lower panel shows restriction enzyme analyses of wt Ad3 and Ad3-GFP with the expected fragments. M: DNA size marker. f and g) Validation of polyclonal rabbit antibody against recombinant Ad3 fiber knob. f) Western blot. Ad knobs (Ad3K, Ad5K, 14K, 14aK, Ad11K, Ad35K) or PtDd were separated in PAG cells with (B) and without (UB) sample denaturation. Filters were incubated with antiAd3K antiserum and anti-rabbit-HRP antibodies. g) Inhibition of Ad attachment: 3H-labeled Ad3, Ad14 and Ad5 virus were incubated with PBS (white bars) or rabbit anti-Ad3K serum (grey bars) for one hour on ice, then added to HeLa cells for attachment studies.
a) Competition of 3H-labeled Ad3 and Ad5 virus attachment to HeLa cells after pre-incubation with Ad3 BsDd, PtDd, or Ad fiber knobs. Attachment in PBS-treated cells was taken as 100%. n=5. Data are represented as mean+/−SEM). Ad3-PtDd vs. Ad3 knobP=0.0033. b) Competition of Ad3-GFP and Ad35-GFP virus infection. HeLa cells were pretreated with Ad3 fiber knob or PtDd at increasing concentrations and then exposed to Ad3-GFP (left panel) or Ad35-GFP virus (right panel) at an MOI of 100 pfu/cell. GFP expression was measured 18 hours later by flow cytometry. Data are represented as mean. Standard deviation was less than 10% for all data points. c) Attachment of 3H-labeled Ad3 and Ad35 viruses to human and non-human cell lines. Y79 and Ramos are human retinoblastoma and lymphoma cells, respectively. CHO cells are Chinese Hamster ovary cells. MMC and TC1 cells are mouse mammary carcinoma and lung carcinoma cells, respectively. TC1-CD46 cells express human CD46. Shown are the average number of viral particels attached per cells. n=5. d and e) Identification of receptor X by affinity capture and MS/MS. Membrane protein fractions were prepared from HeLa and Ramos cells. Protein blots were hybridized with Ad5/35++ virions (d) and Ad3 virions or Ad3 PtDd (e). Binding was visualized with polyclonal antibodies against Ad35++ knob (d) or Ad3 knob (e) (see also
a) Competition of 3H-labeled Ad binding by recombinant DSG2. 3H-Ad3, Ad7, Ad14, Ad14a, Ad11, Ad5 and Ad35 virus were pre-incubated with 6 μg ml−1 recombinant human DSG2 protein. Attachment of virus particles incubated with PBS was taken as 100%. For analysis of Ad11 attachment, cells were also incubated with 50 μml−1 of Ad35K on ice for one hour before adding of Ad11 virus to block CD46. b) Competition of Ad transduction by recombinant DSG1, DSG2 or DSC2 proteins. c) Competition of 3H-Ad binding by DSG2-specific antibodies. n=5. PBS vs. 6D8: P=0.013; PBS vs. 8E5: P=0.0014. The specificity of mAbs to different DSG2 domains is as follows (for a scheme of DSG2, see
a) Transduction of human cell lines that express different DSG2 levels. Human erythromyeloblastoid leukemia K562 cells and Burkitt's B-cell lymphoma BJAB and Ramos cells were infected with Ad3-GFP and Ad5/35-GFP at increasing MOIs and GFP expression was measured 18 hours later. N=3. Standard deviation was less than 10% for all data points. b) Ectopic DSG2 expression. Human histiocytic lymphoma U937 cells were infected with a lentivirus vector carrying the DSG2 cDNA under the control of the EF1a promoter. Stable DSG2 expression was detected in >98% of lentivirus transduced cells by flow cytometry. c) Attachment of 3H-Ad3 and 3H-Ad35 to Raji, U937 and DSG2-expressing U937 (U937-DSG2) cells. Note that Ad35 attachment is mediated through CD46 and can be blocked by soluble CD46 (data not shown). d) GFP expression after transduction of U937 and U937-DSG2 cells with Ad3-GFP and Ad5/35-GFP. n=3
a) Immunohistochemistry studies on human colon, foreskin and ovarian cancer paraffin sections with DSG2-specific antibodies. Positive staining appears in brown. b) Confocal microscopy immunofluorescence analysis of polarized human colon cancer T84 cells for DSG2 (green) and the intercellular junction protein Claudin 7 (red). Nuclei are blue. XY and XZ planes are shown. c) Ad3 binding to DSG2. T84 cells were incubated with Cy3-labeled Ad3 particles (red) for 15 minutes, washed, and subjected to confocal microscopy. The upper XZ image is a higher magnification. Note that at least two (green) DSG2 signals are associated with one (red) Cy3-Ad3 signal. d) Confocal microscopy of normal human small airway epithelial cells (not grown in TRANSWELL™ chambers). Cells were incubated with Cy5-labelled PtDd for 15 min and then washed with PBS. The upper XZ panel shows co-localization of DSG2 (red) and E-cadherin (green). The lower XZ panel is the same image showing purple Cy5-PtDd signals co-localized with green E-cadherin signals. The XY panel shows purple (PtDd) and green E-cadherin channels. Thin arrows mark membrane localized PtDd, thick arrows label cytoplasmic DSG2. e) Confocal microscopy immunofluorescence analysis of human cervical carcinoma HeLa cells (upper XZ and XY panels) and HeLa cells incubated for 15 min with Cy3-Ad3 (lower XZ panel). Scale bars for all confocal microphotographs are 20 μm.
a-d) Phenotypic changes triggered by PtDd in breast cancer epithelial cells. 1×105 BT474 cells were incubated with 50 ng of PtDd or BsDd for the indicated time and subjected to staining with antibodies. The scale bar is 20 μm in all ZY confocal images (a, b) and 40 μm in the standard immunofluorescense studies (c, d). e) Graphic demonstration of array data for up- and down-regulated genes (PtDd vs. PBS treated cells). Each dot represents one gene. f) Western blot analysis of ERK1/2-MAPK and PI3K phosphorylation analyzed 6 hours after incubation of BT474 cells with PtDd, BsDd, DSG2-specific antibodies (10D2, 13B11, or 6D8), or control antibody (anti-GAPDH) at the indicated concentrations. For pathway inhibition, cells were treated overnight with Erk1/2 inhibitor U0126 (5 μM) or PI3K inhibitor Wortmannin (2.5 μM) before PtDd was added. The efficacy of the drugs for inhibition of the specific pathway was validated in a previous study8. GAPDH is used to demonstrate equal loading.
a) FITC-Dextran diffusion through monolayers of BT474 cells. BT474 cells cultured in transwell chamber with 0.4 μm pore size were treated with 0.5 μml−1 BsDd, PtDd or 2×108 Ad particles per ml for 2 hours and then 4 kDa FITC-dextran was added to the apical compartment. Paracellular flux was assessed in aliquots from the apical and basal chambers. BsDd vs. PtDd: P<0.001. b) Facilitation of 3H-Ad35 uptake by PtDd. Left panel: Trapping of CD46 in intercellular junctions of T84 cells. Co-localization of CD46 and the intercellular junction protein Claudin 7 results in yellow signals. Right panel: 3H-Ad35 attachment. BT474-cells were incubated with PtDd or BsDd and 3H-Ad35 for 2 hours on ice, washed, and then incubated at 37° C. for 60 min. Non-internalized Ad particles were removed by trypsin digestion and cell-associated radioactivity was measured. c) Mice carrying subcutaneous ovc316 tumors were injected intravenously with 50 μg PtDd or BsDd eight hours before intravenous injection of 1×109 pfu of Ad5/35-bGal. Sections were stained with X-gal 72 hours after injection. The scale bar is 40 μm. d) Confocal microscopy for Her2/neu and Claudin 7 in the Her2/neu-positive human breast cancer cell line BT474. These cells do not form monolayers. Note that in PBS treated cells, most Her2/neu signals (green) colocalize with Claudin 7 (red) resulting in yellow signals. Upon PtDd treatment, Claudin 7 signals decrease while more Her2/neu staining appears on the cell surface. e) Confocal microscopy of BT474 cells two hours after treatment with PBS or PtDd. f) Ad3 and PtDd enhance killing of Her2/neu positive breast cancer cells by Herceptin. Viability of PBS-treated cells was taken 100%. n=5, *P<0.05. g) Ad3 and PtDd enhancement of Herceptin therapy is mediated by DSG2 and involves ERK/MAPK and PI3K pathways. BT474 cells were transfected with control and DSG2 siRNA as described in
All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the AMBION™ 1998 Catalog (AMBION™, Austin, Tex.).
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. “And” as used herein is interchangeably used with “or” unless expressly stated otherwise.
As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
As used herein, the abbreviation “Ad” refers to an adenovirus and is typically followed by a number indicating the serotype of the adenovirus. For example, “Ad3” refers to adenovirus serotype 3.
All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.
In a first aspect, the invention provides methods for enhancing therapeutic treatment, diagnosis, or imaging of a disorder associated with epithelial tissue, comprising administering to a subject in need thereof (a) an amount of one or more therapeutics sufficient to treat the disorder, diagnostic sufficient to diagnose the disorder, and/or imaging agent sufficient to image the epithelial tissue; and (b) an amount of AdB-2/3 fiber multimer, or functional equivalent thereof, sufficient to enhance efficacy of the one or more therapeutics, diagnostics, and/or imaging agents.
The methods of this aspect of the invention can be used to enhancing therapeutic treatment, diagnosis, or imaging of a disorder associated with epithelial tissue by improving access for the therapeutic, diagnostic, and/or imaging agent to their target and dissemination in epithelial tissue. While not being bound by any mechanism, the inventors believe this occurs through complementary mechanisms: movement of the target receptor from the basolateral to the apical cell surface thus allowing better access to the epithelial tissue target by therapeutics, diagnostics, and/or imaging agents that target the receptor, such as monoclonal antibodies), and better penetration of the therapeutic through disruption of intercellular junctions. As disclosed in detail herein, the inventors have discovered that desmogelin-2 (DSG2) is the primary high affinity receptor for AdB-2/3. DSG2 is a calcium-binding transmembrane glycoprotein belonging to the cadherin protein family. In epithelial cells, DSG2 is a component of the cell-cell adhesion structure. Its cytoplasmic tail interacts with a series of proteins that are in direct contact with regulators of cell adhesion and intercellular junctions/cell morphology. It has been shown that DSG2 is overexpressed in a series of epithelial malignancies including gastric cancer, squamous cell carcinomas, melanoma, metastatic prostate cancer, and bladder cancer.
While not being bound by a specific mechanism of action, the inventors believe that the AdB-2/3 fiber multimer binding to DSG2 serves to trigger transient DSG2-mediated opening of intercellular junctions, which serves to improve access of therapeutics, diagnostics, imaging agents, or any other compound of interest that binds to a target in epithelial cells that would otherwise be trapped to at least some extent in intercellular junctions. Detailed examples of such activity are provided herein.
The methods of the invention have broad application for delivery of any therapeutic, diagnostic, imaging agent, or other compound to epithelial tissue comprising intercellular junctions where access to a target of interest can be limited, as DSG2 is widely expressed in epithelial cells. As used herein, a “disorder associated with epithelial tissue” is any disorder wherein therapeutic, diagnostic, or imaging agent administered to/across epithelial cells/epithelial tissue provides a clinical benefit to a patient, whether in improving therapeutic, diagnostic, and/or imaging efficacy. Such disorders include, but are not limited to, solid tumors (i.e.: any tumor with epithelial cell junctions), gastrointestinal disorders (including but not limited to irritable bowel syndrome, inflammatory bowel disorder, Crohn's disease, ulcerative colitis, constipation, gastroesophageal reflux disease, Barrett's esophagus, etc.), skin diseases (including but not limited to psoriasis and dermatitis), lung disorders (including but not limited to chronic obstructive pulmonary disease, asthma, bronchitis, pulmonary emphysema, cystic fibrosis, interstitial lung disease, pneumonia, pancreatic duct disorders, brain disorders (ie: any brain disorder that could benefit from improved transport of drugs through the blood-brain barrier), primary pulmonary hypertension, pulmonary embolism, pulmonary sarcoidosis, tuberculosis, etc.), renal disorders, (including but not limited to glomerulonephritis), liver diseases (including but not limited to hepatitis), endocrine disorders (including but not limited to diabetes and thyroid disorders), pancreatic duct disorders (including but not limited to pancreatitis), and bile duct disorders (including but not limited to bile duct obstruction, cholecystitis, choledocholithiasis, gallstones, etc.) and infections of epithelial tissues (including but not limited to cellulitis, pneumonia, hepatitis, and pyelonephritis). In one preferred embodiment, the disorder associated with epithelial tissue comprises a solid tumor, including but not limited to breast tumors, lung tumors, colon tumors, rectal tumors, skin tumors, endocrine tumors, stomach tumors, prostate tumors, ovarian tumors, uterine tumors, cervical tumors, kidney tumors, melanomas, pancreatic tumors, liver tumors, brain tumors, head and neck tumors, nasopharyngeal tumors, gastric tumors, squamous cell carcinomas, adenocarcinomas, bladder tumors, and esophageal tumors. As will be understood by those of skill in the art, such tumors include primary tumors, tumors that are locally invasive, as well as tumors that have metastasized.=
As used herein, “enhancing efficacy” means any increase in therapeutic, diagnostic, and/or imaging efficacy over what would be seen using the therapeutic, diagnostic, and/or imaging agen alone. For example, measurements of therapeutic efficacy will vary depending on the disorder being treated, but are readily identified by an attending physician. For example, such increases in efficacy include, but are not limited to increasing one or more of the following relative to treatment with the therapeutic alone: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s). In one non-limiting example, treating a solid tumor provides an ability to induce egress of tumor receptors from the basolateral side of epithelial cells to enable improved access and killing of the tumor.
For cancer, there are standards for defining tumor response and standard methods of measuring response. These include tumor response, which is determined by monitoring the change in tumor size or a serum marker of disease. A partial response is more than a 50% reduction in the tumor, while a complete response is defined as complete disappearance of the tumor. Methods used to measure tumors are well known to physicians and include physical examination, radiological testing such as CT scans, MRI, PET scans, X-rays as well as serum markers such as prostate specific antigen, which is used to monitor prostate cancer. Other measures of therapeutic efficacy of cancer treatment include measurements of time to progression, progression-free survival and overall survival.
Improved diagnostic efficacy includes any improvement in efficacy compared to administration of the diagnostic alone, including but not limited to, increasing specificity and/or sensitivity of the diagnostic test. Improved imaging efficacy includes any improvement in efficacy compared to administration of the imaging agent alone, including but not limited to specificity, sensitivity, reproducibility, contrast enhancement, detection of smaller sites of disease, more accurate delineation of disease, such as size and shape of diseases, such as tumors, abscesses, etc.
In various embodiments, the increase in efficacy is a 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, or greater benefit compared to efficacy with the therapeutic, diagnostic, and/or imaging agent alone across a patient population.
Any suitable subject can be treated using the methods of the invention, preferably human subjects.
As used herein, “AdB-⅔” is any adenovirus serotype that uses DSG2 as an epithelial cell receptor for viral binding. To date, Ad3, Ad7, Ad11, Ad14, and Ad14a serotypes have been identified. As other Ad serotypes are identified, those of skill in the art can readily identify those that belong to the AbD-2/3 family based on DSG2 binding assays as disclosed herein. For example, surface plasmon resonance (SPR) studies using sensors containing immobilized recombinant DSG2 can be used to determine if new Ad serotypes bind to DSG2, combined with DSG2 competition studies. Further exemplary studies, such as loss and gain of function analyses, are described in detail in Example 1.
The adenovirus virion is an icosahedron characterized by a fiber located at the base of each of the 12 vertices of the capsid. The fiber on the virion is a homotrimeric structure consisting of 3 individual fiber polypeptides. Each adenovirus fiber polypeptide is an asymmetrical structure consisting of an N-terminal tail, which interacts with the penton base protein of the capsid and contains the signals necessary for transport of the protein to the cell nucleus; a shaft, which contains a number of 15-residue repeating units; and a C-terminal knob domain that contains the determinants for receptor binding (J. S. Hong and J. A. Engler, Journal of Virology 70:7071-7078 (1996)). All adenoviruses attach to their receptors through the knob structure on the end of the fiber.
Thus, as used herein, the term AdB-2/3“fiber polypeptide” refers the full length fiber polypeptide expressed by AdB-2/3 (for example, SEQ ID NOS 15-19, with Ad3 fiber polypeptide as SEQ ID NO:15, Ad7 fiber polypeptide as SEQ ID NO:16, Ad11 polypeptide as SEQ ID NO:17, Ad14 fiber polypeptide as SEQ ID NO:18, and Ad14(a) fiber polypeptide as SEQ ID NO:19) that comprises an N-terminal tail domain, a shaft domain, and a C-terminal knob domain. The fiber polypeptides spontaneously assemble into homotrimers, referred to as “fibers,” which are located on the outside of the adenovirus virion at the base of each of the twelve vertices of the capsid.
As used herein, an “AdB-2/3 fiber multimer” is any construct comprising a multimer (dimer, trimer, etc.) of an AdB-2/3 fiber, or functional equivalent thereof. As will be understood by those of skill in the art, the AdB-2/3 fiber comprises a homotrimeric knob. The AdB-2/3 fiber multimer is a multimer, such as a dimer, of the homotrimeric AdB-2/3 fiber. As disclosed in detail in the examples that follow, AdB-2/3 fiber multimers are required for binding to DSG2 that triggers transient DSG2-mediated opening of intercellular junctions, which serves to improve access of therapeutics to therapeutic targets that would otherwise be trapped in intercellular junctions
In one embodiment, the AdB-2/3 fiber multimer is selected from the group consisting of an Ad3 fiber multimer, an Ad7 fiber multimer, an Ad11 fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer, combinations thereof, and functional equivalents thereof. In a preferred embodiment the AdB-2/3 fiber multimer is an Ad3 fiber multimer.
Exemplary constructs comprising one or more AdB-2/3 fiber multimers (or chimeras/functional equivalents thereof) include, but are not limited to, AdB-2/3 virions (such as “killed” virions, for example UV-treated virions), AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd) (subviral dodecahedral particles produced by AdB-2/3 during their replication), recombinant AdB-2/3 fiber multimers (including but not limited to those disclosed in any embodiment or combination of embodiments below), and functional equivalents thereof. In a preferred embodiment, the one or more AdB-2/3 fiber multimers comprise or consist of an AdB-2/3 PtDd, such as an Ad3 PtDd. In another preferred embodiment, the one or more AdB-2/3 fiber multimers comprise or consist of any embodiment or combination of embodiments of the compositions of the invention described below. In a further preferred embodiment, the AdB-2/3 fiber multimer comprises or consists of the polypeptide referred to herein as JO-1 (junction-opener-1), (SEQ ID NO:20), or functional equivalents thereof. As shown in the examples that follow, JO-1 (described in the examples as a self-dimerizing Ad3 fiber derivative) is a multimer of a single chain, recombinant AdB-2/3 fiber polypeptide domain, wherein the polypeptide comprises a knob domain capable of homotrimerization. Thus, JO-1 is an AdB-2/3 fiber multimer according to the present invention.
Methods for preparing large quantities of AdB-2/3 virions and capsid are well known in the art, as are methods for large scale production of PtDd. Similarly, methods for recombinant production of AdB-2/3 fiber multimers (such as JO-1) by incorporating dimerization domains in the recombinant polypeptides are well within the level of skill in the art based on the teachings herein.
The methods of the invention can thus be carried out using any AdB-2/3 fiber multimer capable of binding to DSG2 and triggering transient DSG2-mediated opening of intercellular junctions. Thus, non-naturally occurring modifications (deletions, additions, substitutions, chimeras of different Ad serotype fiber proteins and domains thereof, etc.) to the AdB-2/3 fiber multimers disclosed herein are “equivalents thereof” of the AdB-2/3 fiber multimers and are within the scope of the present invention, so long as they function to bind DSG2 and are capable of multimerizing and triggering DSG2-mediated opening of intercellular junctions. Based on the teachings herein for testing binding to DSG2 and for assessing DSG2-mediated opening of intercellular junctions, it is well within the level of skill in the art to identify such functional equivalents of the AdB-2/3 fiber multimers. In one non-limiting example, assays to assess the flux of a labeled compound (such as FITC-dextran) through confluent polarized epithelial cells, in the presence of candidate AdB-2/3 fiber multimers, are demonstrated in Examples 1 and 2 below. In another non-limiting example, assays in the presence of candidate AdB-2/3 fiber multimers to assess access to proteins not normally accessible due to epithelial cell junctions (such as CD46, Claudin 7, and ZO-1) are demonstrated in Examples 1 and 2 below. Further such assays are also disclosed herein.
AdB-2/3 fiber multimerization can be determined according to methods well known to the practitioners in the art. For example, multimerization of the recombinant AdB-2/3 fiber constructs can be assessed by criteria including sedimentation in sucrose gradients, resistance to trypsin proteolysis, and electrophoretic mobility in polyacrylamide gels (Hong and Engler, Journal of Virology 70:7071-7078 (1996)). Regarding electrophoretic mobility, the fiber multimer is a very stable complex and will run at a molecular weight consistent with that of a multimer when the sample is not boiled prior to SDS-PAGE. Upon boiling, however, the multimeric structure is disrupted and the protein subsequently runs at a size consistent with the protein monomer. Any therapeutic, diagnostic, imaging agent, or other compound that can target epithelial tissue and whose delivery to epithelial tissue can be improved by transient opening of intercellular junctions can be used in the methods of the invention. In one embodiment, the therapeutic is selected from the group consisting of antibodies, immunoconjugates, nanoparticles, nucleic acid therapeutics, and combinations thereof, chemotherapeutics, vaccines, radioactive particle/radiation therapy (“radiation”), cellular immunotherapy including adoptive T-cell therapy and dendritic cell therapy (example: intratumoral penetration of administered T-cells), inhaled therapeutics, gene therapy constructs (including but not limited to AdB-2/3 virus as a gene therapy vector, and co-administration with an Ad5-based gene therapy vector), other nucleic acid therapeutics, and combinations thereof.
In various embodiments, the therapeutic is selected from the group consisting of alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-xL, Bcl-w and Bfl-1) inhibitors, activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, biologic response modifiers, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, demethylating agents, histone deacetylase (HDAC) inhibitors, hormonal therapies, immunologicals, inhibitors of apoptosis proteins (IAPB) intercalating antibiotics, kinase inhibitors, mammalian target of rapamycin inhibitors, microRNA's mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors and the like.
Exemplary therapeutics falling within these various classes include, but are not limited to: docetaxel, doxorubicin, irinotecan, paclitaxel (Taxol®), paclitaxel albumin bound particles (Abraxane®), doxorubicin HCL liposome (Doxil®), BiTE antibodies such as adecatumumab (Micromet MT201), blinatumomab (Micromet MT103) and the like, siRNA-based therapeutics, alkylating agents including altretamine, AMD-473, AP-5280, apaziquone, bendamustine, brostallicin, busulfan, carboquone, carmustine (BCNU), chlorambucil, CLORETAZINE.® (laromustine, VNP 40101M), cyclophosphamide, dacarbazine, decitabine, 5′-azacytidine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine (CCNU), mafosfamide, melphalan, mitobronitol, mitolactol, nimustine, nitrogen mustard N-oxide, ranimustine, temozolomide, thiotepa, TREANDA® (bendamustine), treosulfan, rofosfamide and the like; angiogenesis inhibitors including endothelial-specific receptor tyrosine kinase (Tie-2) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, insulin growth factor-2 receptor (IGFR-2) inhibitors, matrix metalloproteinase-2 (MMP-2) inhibitors, matrix metalloproteinase-9 (MMP-9) inhibitors, platelet-derived growth factor receptor (PDGFR) inhibitors, thrombospondin analogs, vascular endothelial growth factor receptor tyrosine kinase (VEGFR) inhibitors and the like; antimetabolites including ALIMTA® (pemetrexed disodium, LY231514, MTA), 5-azacitidine, XELODA® (capecitabine), carmofur, LEUSTAT® (cladribine), clofarabine, cytarabine, cytarabine ocfosfate, cytosine arabinoside, decitabine, deferoxamine, doxifluridine, eflornithine, EICAR (5-ethynyl-1-.beta.-D-ribofuranosylimidazole-4-carboxamide), enocitabine, ethnylcytidine, fludarabine, 5-fluorouracil alone or in combination with leucovorin, GEMZAR® (gemcitabine), hydroxyurea, ALKERAN® (melphalan), mercaptopurine, 6-mercaptopurine riboside, methotrexate, methotrexate analogs (such as trimetrexate and pralatraxate), mycophenolic acid, nelarabine, nolatrexed, ocfosfate, pelitrexol, pentostatin, raltitrexed, Ribavirin, triapine, trimetrexate, S-1, tiazofurin, tegafur, TS-1, vidarabine, and the like; Bcl-2 protein inhibitors including AT-101 ((−)gossypol), GENASENSE® (G3139 or oblimersen (Bcl-2-targeting antisense oligonucleotide)), IPI-194, IPI-565, N-(4-(4-((4′-chloro(1,1′-biphenyl)-2-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobe-nzenesulfonamide) (ABT-737), N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)pip-erazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl-) propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide (ABT-263), GX-070 (obatoclax) and the like; Bcr-Abl kinase inhibitors include DASATINIB® (BMS-354825), GLEEVEC® (imatinib) and the like; CDK inhibitors including AZD-5438, BMI-1040, BMS-032, BMS-387, CVT-2584, flavopyridol, GPC-286199, MCS-5A, PD0332991, PHA-690509, seliciclib (CYC-202, R-roscovitine), ZK-304709 and the like; EGFR inhibitors including ABX-EGF, anti-EGFR immunoliposomes, EGF-vaccine, EMD-7200, ERBITUX® (cetuximab), HR3, IgA antibodies, IRESSA® (gefitinib), TARCEVA® (erlotinib or OSI-774), TP-38, EGFR fusion protein, TYKERB® (lapatinib) and the like; ErbB2 receptor inhibitors include CP-724-714, Cl-1033 (canertinib), HERCEPTIN® (trastuzumab), TYKERB® (lapatinib), OMNITARG® (2C4, petuzumab), TAK-165, GW-572016 (ionafarnib), GW-282974, EKB-569, PI-166, dHER2 (HER2 vaccine), APC-8024 (HER-2 vaccine), anti-HER/2neu bispecific antibody, B7.her2IgG3, AS HER2 trifunctional bispecific antibodies, mAb AR-209, mAb 2B-1 and the like; histone deacetylase inhibitors include romidepsin, LAQ-824, MS-275, trapoxin, suberoylanilide hydroxamic acid (SAHA), TSA, valproic acid and the like; HSP-90 inhibitors including 17-AAG-nab, 17-AAG, CNF-101, CNF-1010, CNF-2024, 17-DMAG, geldanamycin, IPI-504, KOS-953, MYCOGRAB® (human recombinant antibody to HSP-90), NCS-683664, PU24FC1, PU-3, radicicol, SNX-2112, STA-9090 VER49009 and the like; activators of death receptor pathways including TRAIL, antibodies or other agents that target TRAIL or death receptors (e.g., DR4 and DR5) such as Apomab, conatumumab, ETR2-ST01, GDC0145, (lexatumumab), HGS-1029, LBY-135, PRO-1762 and trastuzumab; platinum chemotherapeutics include cisplatin, ELOXATIN® (oxaliplatin) eptaplatin, lobaplatin, nedaplatin, PARAPLATIN® (carboplatin), satraplatin, picoplatin and the like; VEGFR inhibitors including AVASTIN® (bevacizumab), ABT-869, AEE-788, axitinib (AG-13736), AZD-2171, CP-547,632, IM-862, MACUGEN (pegaptamib), NEXAVAR® (sorafenib, BAY43-9006), pazopanib (GW-786034), vatalanib (PTK-787, ZK-222584), SUTENT® (sunitinib, SU-11248), VEGF trap, ZACTIMAThi (vandetanib, ZD-6474) and the like; dendritic cell therapy (sipuleucel-T, Provenge®); topoisomerase inhibitors including aclarubicin, 9-aminocamptothecin, amonafide, amsacrine, becatecarin, belotecan, BN-80915, CAMPTOSAR® (irinotecan hydrochloride), camptothecin, dexrazoxine, diflomotecan, edotecarin, ELLENCE® or PHARMORUBICIN® (epirubicin), etoposide, exatecan, abraxane, irenotecan,10-hydroxycamptothecin, gimatecan, lurtotecan, mitoxantrone, orathecin, pirarbucin, pixantrone, rubitecan, sobuzoxane, SN-38, tafluposide, topotecan and the like; antibodies including AVASTIN® (bevacizumab), CD40-specific antibodies, chTNT-1/B, denosumab, ERBITUX® (cetuximab), HUMAX-CD4® (zanolimumab), IGF I R-specific antibodies, lintuzumab, PANOREX® (edrecolomab), RENCAREX® (WX G250), RITUXAN® (rituximab), ticilimumab, trastuzimab and the like; hormonal therapies including ARIMIDEX® (anastrozole), AROMASIN® (exemestane), arzoxifene, CASODEX® jb nnb nb nbbv(bicalutamide), CETROTIDE® (cetrorelix), degarelix, deslorelin, DESOPAN® (trilostane), dexamethasone, DROGENIL® (flutamide), EVISTA® (raloxifene), AFEMA® (fadrozole), FARESTON® (toremifene), FASLODEX® (fulvestrant), FEMARA® (letrozole), formestane, glucocorticoids, HECTOROL® (doxercalciferol), RENAGEL® (sevelamer carbonate), lasofoxifene, leuprolide acetate, MEGACE® (megesterol), MIFEPREX® (mifepristone), NILANDRON® (nilutamide), NOLVADEX® (tamoxifen citrate), PLENAXIS® (abarelix), prednisone, PROPECIA® (finasteride), rilostane, SUPREFACT® (buserelin), TRELSTAR® (luteinizing hormone releasing hormone (LHRH)), VANTAS® (Histrelin implant), VETORYL® (trilostane or modrastane), ZOLADEX® (fosrelin, goserelin) and the like; immunologicals including interferon alpha, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon gamma-1a, ACTIMMUNE® (interferon gamma-1b) or interferon gamma-n1, combinations thereof and the like. Other agents include ALFAFERONE® (IFN-alpha), BAM-002 (oxidized glutathione), BEROMUN® (tasonermin), BEXXAR® (tositumomab), CAM PATH®(alemtuzumab), CTLA4 (cytotoxic lymphocyte antigen 4), decarbazine, denileukin, epratuzumab, GRANOCYTE® (lenograstim), lentinan, leukocyte alpha interferon, imiquimod, MDX-010 (anti-CTLA-4), melanoma vaccine, mitumomab, molgramostim, MYLOTARG™ (gemtuzumab ozogamicin), NEUPOGEN® (filgrastim), OncoVAC-CL, OVAREX® (oregovomab), pemtumomab (Y-muHMFG1), PROVENGE® (sipuleucel-T), sargaramostim, sizofilan, teceleukin, THERACYS® (Bacillus Calmette-Guerin), ubenimex, VIRULIZIN® (immunotherapeutic, Lorus Pharmaceuticals), Z-100 (Specific Substance of Maruyama (SSM)), WF-10 (Tetrachlorodecaoxide (TCDO)), PROLEUKIN® (aldesleukin), ZADAXIN® (thymalfasin), ZENAPAX® (daclizumab), ZEVALIN®. (90Y-Ibritumomab tiuxetan) and the like; ofatumumab; biological response modifiers agents including krestin, lentinan, sizofuran, picibanil PF-3512676 (CpG-8954), ubenimex and the like; pyrimidine analogs include cytarabine (ara C or Arabinoside C), cytosine arabinoside, doxifluridine, FLUDARA® (fludarabine), 5-FU (5-fluorouracil), floxuridine, GEMZAR® (gemcitabine), TOMUDEX® (ratitrexed), TROXATYL® (triacetyluridine troxacitabine) and the like; purine analogs including LANVIS® (thioguanine) and PURI-NETHOL® (mercaptopurine).; antimitotic agents including batabulin, epothilone D (KOS-862), N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide, ixabepilone (BMS 247550), paclitaxel, TAXOTERE® (docetaxel), PNU100940 (109881), patupilone, XRP-9881 (larotaxel), vinflunine, ZK-EPO (synthetic epothilone) and the like; and other chemotherapeutic agents such as ABRAXANE® (ABI-007), ABT-100 (farnesyl transferase inhibitor), ADVEXIN® (Ad5CMV-p53 vaccine), ALTOCOR® or MEVACOR® (lovastatin), AMPLIGE®. (poly I:poly C12U, a synthetic RNA), APTOSYN® (exisulind), AREDIA® (pamidronic acid), arglabin, L-asparaginase, atamestane (1-methyl-3,17-dione-androsta-1,4-diene), AVAGE® (tazarotene), AVE-8062 (combreastatin derivative) BEC2 (mitumomab), cachectin or cachexin (tumor necrosis factor), canvaxin (vaccine), CEAVAC® (cancer vaccine), CELEUK® (celmoleukin), CEPLENE® (histamine dihydrochloride), CERVARIX® (human papillomavirus vaccine), CHOP® (C: CYTOXAN® (cyclophosphamide); H: ADRIAMYCIN® (hydroxydoxorubicin); 0: Vincristine (ONCOVIN®); P: prednisone), CYPAT® (cyproterone acetate), combrestatin A4P, DAB(389)EGF (catalytic and translocation domains of diphtheria toxin fused via a His-Ala linker to human epidermal growth factor) or TransMlD-107R® (diphtheria toxins), dacarbazine, dactinomycin, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), eniluracil, EVIZON™ (squalamine lactate), DIMERICINE® (T4N5 liposome lotion), discodermolide, DX-8951f (exatecan mesylate), enzastaurin, EP0906 (epithilone B), GARDASIL® (quadrivalent human papillomavirus (Types 6, 11, 16, 18) recombinant vaccine), GASTRIMMUNE®, GENASENSE®, GMK (ganglioside conjugate vaccine), GVAX® (prostate cancer vaccine), halofuginone, histerelin, hydroxycarbamide, ibandronic acid, IGN-101, IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox), IL-13-pseudomonas exotoxin, interferon-.alpha., interferon-.gamma., JUNOVAN® or MEPACT® (mifamurtide), lonafarnib, 5,10-methylenetetrahydrofolate, miltefosine (hexadecylphosphocholine), NEOVASTAT® (AE-941), NEUTREXIN® (trimetrexate glucuronate), NIPENT® (pentostatin), ONCONASE® (a ribonuclease enzyme), ONCOPHAGE® (melanoma vaccine treatment), ONCOVAX® (IL-2 Vaccine), ORATHECIN® (rubitecan), OSIDEM® (antibody-based cell drug), OVAREX® MAb (murine monoclonal antibody), paclitaxel, PANDIMEX® (aglycone saponins from ginseng comprising 20(S)protopanaxadiol (aPPD) and 20(S)protopanaxatriol (aPPT)), panitumumab, PANVAC®-VF (investigational cancer vaccine), pegaspargase, PEG Interferon A, phenoxodiol, procarbazine, rebimastat, REMOVAB® (catumaxomab), REVLIMID® (lenalidomide), RSR13 (efaproxiral), SOMATULINE® LA (lanreotide), SORIATANE® (acitretin), staurosporine (Streptomyces staurospores), talabostat (PT100), TARGRETIN® (bexarotene), TAXOPREXIN® (DHA-paclitaxel), TELCYTA® (canfosfamide, TLK286), temilifene, TEMODAR® (temozolomide), tesmilifene, thalidomide, THERATOPE® (STn-KLH), thymitaq (2-amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline dihydrochloride), TNFERADE® (adenovector: DNA carrier containing the gene for tumor necrosis factor-.alpha.), TRACLEER® or ZAVESCA® (bosentan), tretinoin (Retin-A), tetrandrine, TRISENOX®. (arsenic trioxide), VIRULIZIN®, ukrain (derivative of alkaloids from the greater celandine plant), vitaxin (anti-alphavbeta3 antibody), XCYTRIN® (motexafin gadolinium), XINLAY® (atrasentan), XYOTAX® (paclitaxel poliglumex), YONDELIS® (trabectedin), ZD-6126, ZINECARD® (dexrazoxane), ZOMETA® (zolendronic acid), crizotinib, zorubicin and the like.
In another preferred embodiment, the therapeutic comprises a compound that binds to desmoglein-2; preferably a compound that binds to DSG2 and opens up tight junctions.
In other embodiments, the therapeutic comprises radioactive particles/radiation therapy. Any suitable radioactive therapy or particle can be used as deemed appropriate by an attending physician, including but not limited to cobalt-60, iodine-131, iridium-192, strontium-89, samarium 153, rhenium-186 and lead-212.
In a preferred embodiment, the therapeutic is an anti-tumor therapeutic and comprises a chemotherapeutic or anti-tumor monoclonal antibody as described herein. In a further preferred embodiment, the anti-tumor therapeutic comprises an antibody selected from the group consisting of trastuzumab, cetumiximab, petuzumab, apomab, conatumumab, lexatumumab, bevacizumab, bevacizumab, denosumab, zanolimumab, lintuzumab, edrecolomab, rituximab, ticilimumab, tositumomab, alemtuzumab, epratuzumab, mitumomab, gemtuzumab ozogamicin, oregovomab, pemtumomab daclizumab, panitumumab, catumaxomab, ofatumumab, and ibritumomab. Non-limiting examples of useful anti-tumor mAb and their specific uses are listed in Table 1, and as further described in Campoli, M., et al., Principles & Practice of Oncology 23(1&2):1-19 (2009), incorporated herein by reference.
The monoclonal antibody therapeutics can be any type of monoclonal antibody, including but not limited to standard monoclonal antibodies, humanized monoclonals, fully human antibodies generated from mice or other sources, chimeric monoclonals, and fragments thereof. “Humanized monoclonal antibodies” refers to monoclonal antibodies derived from a non-human monoclonal antibody, such as a mouse monoclonal antibody. Alternatively, humanized monoclonal antibodies can be derived from chimeric antibodies that retain, or substantially retain, the antigen-binding properties of the parental, non-human, monoclonal antibodies but which exhibit diminished immunogenicity as compared to the parental monoclonal antibody when administered to humans. For example, chimeric monoclonal antibodies can comprise human and murine antibody fragments, generally human constant and mouse variable regions. Humanized monoclonal antibodies can be prepared using a variety of methods known in the art, including but not limited to (1) grafting complementarity determining regions from a non-human monoclonal antibody onto a human framework and constant region (“humanizing”), and (2) transplanting the non-human monoclonal antibody variable domains, but “cloaking” them with a human-like surface by replacement of surface residues (“veneering”). These methods are disclosed, for example, in, e.g., Jones et al., Nature 321:522-525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immunol. 31(3):169-217 (1994); and Kettleborough, C. A. et al., Protein Eng. 4(7):773-83 (1991). Monoclonal antibodies can be fragmented using conventional techniques, and the fragments screened for utility in the same manner as for whole antibodies. For example, F(ab′)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments. Fab fragments can be obtained by treating an IgG antibody with papain; F(ab′) fragments can be obtained with pepsin digestion of IgG antibody. A F(ab′) fragment also can be produced by binding Fab′ described below via a thioether bond or a disulfide bond. A Fab′ fragment is an antibody fragment obtained by cutting a disulfide bond of the hinge region of the F(ab′)2. A Fab′ fragment can be obtained by treating a F(ab′)2 fragment with a reducing agent, such as dithiothreitol. Antibody fragment peptides can also be generated by expression of nucleic acids encoding such peptides in recombinant cells (see, e.g., Evans et al., J. Immunol. Meth. 184: 123-38 (1995)). For example, a chimeric gene encoding a portion of a F(ab′)2 fragment can include DNA sequences encoding the CH I domain and hinge region of the H chain, followed by a translational stop codon to yield such a truncated antibody fragment molecule. Non-limiting examples of monoclonal antibody fragments include (i) a Fab fragment, a monovalent fragment consisting essentially of the VL, VH, CL and CH I domains; (ii) F(ab)2 and F(ab′)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists essentially of a VH domain; and (vi) one or more isolated CDRs or a functional paratope.
In one preferred embodiment that can be combined with any embodiment or combination of embodiments of the invention, the disorder comprises a Her-2 positive tumor, and the method comprises co-administering the AdB-2/3 fiber multimer together with suitable monoclonal antibody therapy, alone or in combination with a chemotherapeutic, radiation, or combinations thereof. In a further preferred embodiment, the monoclonal antibody is trastuzumab. In a further preferred embodiment that can be combined with any of these embodiments, the Her-2 positive tumor is selected from the group consisting of a breast tumor, a gastric tumor, a colon tumor, and an ovarian tumor, In a most preferred embodiment, the AdB-2/3 fiber multimer comprises an Ad3 PtDd, JO-1 multimers (SEQ ID NO:20), or functional equivalents thereof. As shown in the examples that follow, AdB-2/3 fiber multimer co-administration with trastuzumab leads to improved trastuzumab access to Her-2 receptors in a breast tumor model, resulting in greatly improved trastuzumab therapeutic efficacy. In a further preferred embodiment, the method is carried out on patients who have not responded adequately to trastuzumab, such as by lack of tumor remission, by tumor relapse, or by development of resistance to trastuzumab. The methods of these embodiments can also be used to help reduce the dosage of trastuzumab required to obtain therapeutic efficacy, and can thus serve to limit side effects (such as trastuzumab-associated cardiotoxicity).
In another preferred embodiment that can be combined with any embodiment or combination of embodiments of the invention, the disorder comprises an EGFR-positive tumor, and the method comprises co-administering the AdB-2/3 fiber multimer together with suitable monoclonal antibody therapy, alone or in combination with a chemotherapeutic, radiation, or combinations thereof. In a further preferred embodiment, the monoclonal antibody is cetuximab. In a further preferred embodiment that can be combined with any of these embodiments, the EGFR-positive tumor is selected from the group consisting of a lung tumor, a colon tumor, a breast tumor, a rectal tumor, a head and neck tumor, and a pancreatic tumor. In a most preferred embodiment, the AdB-2/3 fiber multimer comprises an Ad3 PtDd, JO-1 multimers (SEQ ID NO:20), or functional equivalents thereof. As shown in the examples that follow, AdB-2/3 fiber multimer co-administration with cetuximab leads to improved cetuximab access to EGFR receptors in a lung tumor model, resulting in greatly improved cetuximab therapeutic efficacy. In a further preferred embodiment, the method is carried out on patients who have not responded adequately to cetuximab, such as by lack of tumor remission, by tumor relapse, or by development of resistance to cetuximab. The methods of these embodiments can also be used to help reduce the dosage of cetuximab required to obtain therapeutic efficacy, and can thus serve to limit side effects (such as acne-like rashes that often occur during cetuximab therapy).
In one preferred embodiment that can be combined with any embodiment or combination of embodiments of the invention, the disorder comprises an epithelial tumor, and the method comprises co-administering the AdB-2/3 fiber multimer together with a vascular endothelial growth factor (VEGF) inhibitor, alone or in combination with other chemotherapeutic, radiation, or combinations thereof. Any suitable VEGF inhibitor can be used, including but not limited to bevacizumab.
In a further embodiment that can be combined with any embodiment or combination of embodiments herein, the methods involving solid tumors further comprise administering a compound capable of degrading tumor stroma proteins. As shown in the examples that follow, such an approach (combination of compound to degrade tumor stroma protein with JO-1 multimers (SEQ ID NO:20) and trastuzumab) resulted in complete tumor eradication in a breast cancer model. Any suitable compound for degrading tumor stroma proteins can be used, including but not limited to relaxin, collagenase, trypsin, dispase, MMP(metalloproteinase)-1, and MMP8. Delivery of such compounds can be by any suitable mechanism, including gene therapy, separate administration with the AdB-2/3 fiber multimer and the therapeutic, or administration as a conjugate with the AdB-2/3 fiber or therapeutic.
In a further embodiment that can be combined with any embodiment or combination of embodiments herein, the methods further comprise administering the AdB-2/3 multimer in combination with other junction openers. As used herein, a “junction opener” is a compound capable of transiently opening intercellular junctions. Any suitable junction openers can be used. In one non-limiting embodiment, the junction opener comprises Zona occludens toxin (Zot), a Vibrio cholerae (V. cholerae)-produced toxin that possess the ability to reversibly alter intestinal epithelial junctions, allowing the passage of macromolecules through mucosal barriers (Fasano et al. (1991) Proc Natl Acad Sci USA 88: 5242-5246)]. A Zot-derived hexapeptide (AT-1001) has been developed,) In another embodiment, Clostridium perfringens enterotoxin removes claudins-3 and -4 from the tight junctions to facilitate bacterial invasion (Sonoda N, et al. (1999) J Cell Biol 147: 195-204.]. In a further embodiment, oncoproteins encoded by human Ad, HPV, HTLV-1 can transiently open epithelial junctions by mislocalizing the junction protein ZO-1 (Latorre L I, et al. (2005) J Cell Sci 118: 4283-4293). In other embodiments, several human viruses engage tight junction or other cell junction molecules to achieve entry into epithelial cells. Among these viruses are hepatitis C virus (Evans M J, et al. (2007) Nature 446: 801-805), reovirus (Barton E S, et al. (2001) Cell 104: 441-451), and herpes simplex virus Geraghty R J, et al. (1998) Science 280: 1618-1620).
In another embodiment, the therapeutic is an inhaled therapeutic. Any suitable inhaled therapeutic can be used in the methods of the invention. In various non-limiting embodiments, the inhaled therapeutic is selected from the group consisting of corticosteroids, bronchodilators, beta agonists, anticholinergics, albuterol (PROVENTIL®; VENOLIN®; ACCUNEB®; PROAIR®), levalbuterol (XOPENEX®), pirbutrol (MAXAIR®), ipratropium bromide (ATROVENT®), beclomethasone, budesonide, flunisolide (AEROBID®), fluticasone, triamcinolone acetonide, fluticasone (a corticosteroid) and salmeterol (ADVAIR®), formtorol (a long-acting, beta-agonist bronchodilator) and budesonide (a corticosteroid) (SYMICORT®), albuterol (a beta agonist) and ipratropium (COMBIVENT®; an anticholinergic) (budesonide (PULMICORT RESPULES®), and tiopropium (SPIRIVA®; an anticholinergic bronchodilator).
In another embodiment, the compound comprises a diagnostic or imaging agent. The methods of the invention have broad application for delivery of any diagnostic, imaging agent, or other compound to epithelial tissue comprising intercellular junctions where access to a target of interest can be limited. In various non-limiting embodiments, the imaging agents can include any chemical compound that can produce a detectable signal, either directly or indirectly. Many such imaging agents are known to those of skill in the art. Examples of imaging agents suitable for use in the disclosed methods and compositions are radioactive isotopes, fluorescent molecules, magnetic particles (including nanoparticles), metal particles (including nanoparticles), phosphorescent molecules, enzymes, antibodies, ligands, and combinations thereof, while diagnostic agents may comprise a compound that is a diagnostic marker for a particular epithelial disorder bound to the such an imaging agent. Methods for detecting and measuring signals generated by imaging agents are also known to those of skill in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary detection label coupled to the antibody. In one preferred embodiment, the imaging agent and/or diagnostic is one that can be used to detect a tumor, whether by direct tumor binding, or by coupling of the imaging or diagnostic agent with a compound that can bind the tumor.
In one example, the imaging agents can comprise a fluorescent imaging agent, while diagnostic agents may comprise a compound that is a diagnostic marker for a particular epithelial disorder bound to the fluorescent imaging agent. A fluorescent imaging agent is any chemical moiety that has a detectable fluorescence signal. This imaging agent can be used alone or in combination with other imaging agents. Examples of suitable fluorescent agents that can be used in the compositions and methods disclosed herein include, but are not limited to, fluorescein (FITC), 5-carboxyfluorescein-N-hydroxysuccinimide ester, 5,6-carboxymethyl fluorescein, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), fluorescamine, OPA, NDA, indocyanine green dye, the cyanine dyes (e.g., Cy3, Cy3.5, Cy5, Cy5.5 and Cy7), 4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid, acridine, acridine isothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenylinaphthalimide-3,5 disulfonate, N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BODIPY, Brilliant Yellow, coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151), cyanosine, 4′,6-diaminidino-2-phenylindole (DAPI), 5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red), 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin diethylenetriamine pentaacetate, 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), eosin, eosin isothiocyanate, erythrosin B, erythrosine, isothiocyanate, ethidium bromide, ethidium, 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate, IR144, IR1446, Malachite Green isothiocyanate, 4-methylumbelliferone, ortho cresolphthalein, nitrotyrosine, pararosaniline, Phenol Red, B-phycoerythrin, o-phthaldialdehyde, pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron[R] Brilliant Red 3B-A), 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), 5,6-tetramethyl rhodamine, rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, coumarin-6, and the like, including combinations thereof. These fluorescent imaging moieties can be obtained from a variety of commercial sources, including MOLECULAR PROBES™, Eugene, Oreg. and RESEARCH ORGANICS™ Cleveland, Ohio, or can be synthesized by those of ordinary skill in the art.
In another example, the imaging agents can comprise a Magnetic Resonance Imaging (MRI) agent, while diagnostic agents may comprise a compound that is a diagnostic marker for a particular epithelial disorder bound to the MRI agent. A MRI agent is any chemical moiety that has a detectable magnetic resonance signal or that can influence (e.g., increase or shift) the magnetic resonance signal of another agent. This type of imaging agent can be used alone or in combination with other imaging agent. In still another example, a gadolinium-based MRI agent can serve as an imaging agent. An example of a suitable MRI agent that can be incorporated into the disclosed imaging agents is para-amino-benzyl diethylenetriaminepentaacetic acid (p-NH2-Bz-DTPA, Compound 7), a conjugable form of diethylenetriaminepentaacetic acid (DTPA), which is known to strongly bind gadolinium and is approved for clinical use as a magnetic resonance contrast agent. Incorporation of an MRI agent on a large macromolecule such as a dendrimeric substrate as disclosed herein can allow large T1 relaxation (high contrast) and multiple copies of agent on a single molecule, which can increase signal. By combining an MRI imaging agent and, for example, a fluorescent imaging agent, the resulting agent can be detected, imaged, and followed in real-time via MR I. Other imaging agents include PET agents that can be prepared by incorporating an 18F or a chelator for 64Cu or 68Ga. Also, addition of a radionuclide can be used to facilitate SPECT imaging or delivery of a radiation dose, while diagnostic agents may comprise a compound that is a diagnostic marker for a particular epithelial disorder bound to the PET agent.
In some embodiments, the diagnostic agent is a diagnostic imaging agent, including but not limited to position emission tomography (PET) agents, computerized tomography (CT) agents, magnetic resonance imaging (MRI) agents, nuclear magnetic imaging agents (NMI), fluoroscopy agents and ultrasound contrast agents. Such diagnostic agents include radioisotopes of such elements as iodine (I), including 123I, 125I, 131I, etc. barium (Ba), gadolinium (Gd), technetium (Tc), including 99Tc, phosphorus (P), including 31P, iron (Fe), manganese (Mn), thallium (TI), chromium (Cr), including 51Cr, carbon (C), including 14C, or the like, fluorescently labeled compounds, or their complexes, chelates, adducts and conjugates. Any suitable PET agents can be used, including but not limited to carbon-11, nitrogen-13, oxygen-15, fluorine-18,11C-metomidate, and glucose analogues thereof, including but not limited to fludeoxyglucose (a glucose analog labeled with fluorine-18.
In other embodiments, the diagnostic agent is a marker gene that encode proteins that are readily detectable when expressed in a cell (including, but not limited to, beta-galactosidase, green fluorescent protein, luciferase, and the like) and labeled nucleic acid probes (e.g., radiolabeled or fluorescently labeled probes). In some embodiments, covalent conjugation of diagnostics agents to the AdB-2/3 multimers provided herein is achieved according to a variety of conjugation processes. In other embodiments, the diagnostic agent is non-covalently associated with AdB-2/3 multimers provided
In a further aspect, the present invention provides methods for improving delivery of a substance to an epithelial tissue, comprising contacting the epithelial tissue with (a) one or more compound to be delivered to the epithelial tissue; and (b) an amount of AdB-2/3 fiber multimer, or functional equivalent thereof, sufficient to enhance delivery of the one or more compounds to the epithelial tissue. In this aspect, the compounds may be any suitable compound such as those described in detail above. In a preferred embodiment, the one or more compounds comprise an imaging agent. In a further preferred embodiment the epithelial tissue comprises a solid tumor, including any of those disclosed in the present application. In various non-limiting embodiments, the solid tumor is selected from the group consisting of breast tumors, lung tumors, colon tumors, rectal tumors, stomach tumors, prostate tumors, ovarian tumors, uterine tumors, skin tumors, endocrine tumors, cervical tumors, kidney tumors, melanomas, pancreatic tumors, liver tumors, brain tumors, head and neck tumors, nasopharyngeal tumors, gastric tumors, squamous cell carcinomas, adenocarcinomas, bladder tumors, and esophageal tumors.
In a still further aspect, the present invention provides methods for improving delivery of a substance cell or tissue expressing desmoglein 2 (DSG2), comprising contacting the cell or tissue expressing DSG2 with (a) one or more compound to be delivered to the cell or tissue; and (b) an amount of AdB-2/3 fiber multimer, or functional equivalent thereof, sufficient to enhance delivery of the one or more compounds to the tissue. Exemplary tissue types expressing DSG2 include, but are not limited to epithelial cells/tissue (such as those disclosed herein), human platelets and granulocytes. As shown in the examples that follow, DSG2 also acts as receptor in non-polarized cells. Thus, these methods find application not only in epithelial cells and tissue, but also are relevant, for example, in AdB-2/3 pathogenesis and the intravascular application of AdB-2/3 vectors for gene therapy purposes.
In a still further aspect, the present invention provides methods for inducing an epithelial to mesenchymal transition (EMT) in a tissue, comprising contacting the epithelial tissue with an amount of AdB-2/3 fiber multimer, or functional equivalent thereof, sufficient to induce EMT. EMT is a cellular transdifferentiation program where epithelial cells lose characteristics such as intercellular junctions and gain properties of mesenchymal cells. EMT is characterized by increased expression of mesenchymal markers, increased expression of extracellular matrix compounds, decreased expression of epithelial markers, altered location of transcription factors, and activation of kinases, and disassociation of intercellular junctions.
In each of these further aspects, any embodiment of the compounds and AdB-2/3 fiber multimers described herein can be used. In one non-limiting embodiment, the AdB-2/3 fiber multimer is selected from the group consisting of an Ad3 fiber multimer, an Ad7 fiber multimer, an Ad11 fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer, combinations thereof, and functional equivalents thereof. In another embodiment, the AdB-2/3 fiber multimer is an Ad3 fiber multimer, or a functional equivalent thereof. In another embodiment, the AdB-2/3 fiber multimer is selected from the group consisting of AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd), recombinant AdB-2/3 fiber multimers, and functional equivalents thereof. In various preferred embodiments, the AdB-2/3 fiber multimer comprises an Ad3 PtDd or a junction opener 1 (JO-1) (SEQ ID NO:20) multimer (such as a dimer), or functional equivalent thereof. In a further preferred embodiment that can be combined with each of these embodiments, the AdB-2/3 fiber multimer is a dimer. While not being bound by any specific mechanism of action, it is believed that these further aspects each take advantage of the AdB-2/3 fiber multimer serving to disrupt intercellular junctions.
In all of the aspects and embodiments of the methods of the invention, the therapeutic, diagnostic, and/or imaging agent can be administered together with the AdB-2/3 multimer or may be administered together. In one embodiment, the therapeutic and AdB-2/3 multimer are attached, via any suitable covalent or non-covalent binding. In one non-limiting embodiment, an AbB-2/3 multimer can attached to a toxin or other drug to kill solid tumor cells.
The AdB-2/3 fiber multimer and/or therapeutic can be administered in any way deemed suitable by an attending physician, depending on whether a local or systemic mode of administration is most appropriate for the condition being treated. As used herein, the terms “systemic delivery” and “systemic administration” are intended to include, but are not limited to, oral and parenteral routes including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalational, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal, and other routes of administration that effectively result in dispersement of the delivered agent to a single or multiple sites of intended therapeutic action. Preferred routes of systemic delivery for the present compositions include intravenous, intramuscular, subcutaneous, and inhalational. In one preferred embodiment, intravenous administration is used, such as for treatment of disseminated tumors (and for monoclonal antibody delivery). In another embodiment, oral delivery may be preferred, for example, for treating gastrointestinal (GI) epithelial disorders. In another embodiment, nasal or aerosol delivery may be preferred for delivery to the lungs, such as for lung epithelial disorders.
The AdB-2/3 fiber multimer may be introduced in association with another molecule, such as a lipid or liposome to protect the polypeptides from enzymatic degradation. For example, the covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thus prolong half-life.
The AdB-2/3 fiber multimer and/or therapeutic may be systemically administered on a periodic basis at intervals determined to maintain a desired level of therapeutic effect. For example, administration by intravenous injection may be once per day, once per week, every two to four weeks or at less frequent intervals. The dosage regimen will be determined by the physician considering various factors that may influence the action of the combination of agents. These factors will include the extent of progress of the condition being treated, the patient's age, sex and weight, and other clinical factors. The dosage for AdB-2/3 fiber multimer and/or therapeutic will vary as a function of the multimer and/or therapeutic being administered, as well as the presence and nature of any drug delivery vehicle (e.g., a sustained release delivery vehicle). In addition, the dosage quantity may be adjusted to account for variation in the frequency of administration and the pharmacokinetic behavior of the delivered agent(s). Dosage ranges of AdB-2/3 fiber multimers will generally range between 0.01 and 250 mg/kg, preferably between 0.1 and 10 mg/kg, and more preferably between 0.10 to 0.5 mg/kg. Dosages of approved therapeutics are readily identifiable by medical practitioners. The therapeutic may also be able to be administered at a reduced dose due to enhanced penetration into epithelial tissues, such as cancers.
The AdB-2/3 fiber multimer may be administered to the subject before, simultaneously, or after administration of the therapeutic. In a preferred embodiment, administration of the therapeutic and the AdB-2/3 fiber multimer are carried out at the same time. The timing of administrations of the therapeutic relative to the AdB-2/3 fiber multimer can be varied to achieve the greatest therapeutic effect. Preferably, the therapeutic is administered at a time to ensure its contact with the transient opening of the intercellular junction caused by AdB-2/3 fiber multimer binding to DSG2. For example, the therapeutic can be administered prior to, simultaneously with, after each administration of the AdB-2/3 fiber multimer. In other preferred embodiments, the therapeutic can be administered after the administration of the AdB-2/3 fiber multimer, for example up to 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 40 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, or even up to 96 hours after the administration of the AdB-2/3 fiber multimer.
In another aspect, the present invention provides methods for treating a disorder associated with epithelial tissue, comprising administering to a subject in need thereof an amount of AdB-2/3 fiber multimer, or functional equivalent thereof, sufficient to treat the disorder. In this embodiment, no other therapeutic is delivered. In non-limiting embodiments, the monotherapy is used to treat a disorder selected from the group consisting of an AdB-2/3 viral infection, a solid tumor, or a disorder that can be treated using an AdB-2/3-based gene delivery vector. For example, in treating solid tumors, the method comprises improving access of immune system cells to the site of the disorder, such as by penetration (such as intratumoral penetration of pre-existing natural killer cells, T-cells or dendritic cells). The method can also be used to treat any of the disorders associated with epithelial cells discussed above that can benefit from improved access of cells of the immune system to the target epithelial cells. In one preferred embodiment, the disorder is a solid tumor, and the method comprises improving immune system attack of the tumor. The method can be used with any of the solid tumors discussed above. All embodiments and combinations of embodiments of the first aspect of the invention can be used in this second aspect as well, unless the context clearly dictates otherwise.
Thus, in various embodiments of this second aspect, the AdB-2/3 fiber multimer is selected from the group consisting of an Ad3 fiber multimer, an Ad7 fiber multimer, an Ad11 fiber multimer, an Ad14 fiber multimer, and an Ad14a fiber multimer. Similarly, exemplary constructs comprising one or more AdB-2/3 fiber multimers (or chimeras/functional equivalents thereof) for use in this aspect of the invention include, but are not limited to, AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd) (subviral dodecahedral particles produced by AdB-2/3 during their replication), recombinant AdB-2/3 fiber multimers (including but not limited to those disclosed in any embodiment or combination of embodiments below, such as a JO-1 multimer, such as a JO-1 dimer), and functional equivalents thereof. In a preferred embodiment, the one or more AdB-2/3 fiber multimers comprise or consist of an AdB-2/3 PtDd, such as an Ad3 PtDd. In another preferred embodiment, the one or more AdB-2/3 fiber multimers comprise or consist of any embodiment or combination of embodiments of the compositions of the invention described below, such as a JO-1 dimer. Further, the methods of this aspect of the invention can be carried out using any AdB-2/3 fiber multimer capable of binding to DSG2 and triggering transient DSG2-mediated opening of intercellular junctions. Thus, non-naturally occurring modifications (deletions, additions, substitutions, chimeras of different Ad serotype fiber proteins and domains thereof, etc.) to the AdB-2/3 fiber multimers disclosed herein are within the scope of the present invention, so long as they function to bind DSG2 and trigger DSG2-mediated opening of intercellular junctions. Based on the teachings herein for testing binding to DSG2 and for assessing DSG2-mediated opening of intercellular junctions, it is well within the level of skill in the art to identify such functional equivalents of the AdB-2/3 fiber multimers.
In another aspect, the present invention provides recombinant AdB-2/3 fiber polypeptides, comprising: (a) one or more AdB-2/3 fiber polypeptide shaft domains, or functional equivalents thereof; (b) an AdB-2/3 fiber polypeptide knob domain, or functional equivalent thereof, operatively linked to and located C-terminal to the one or more AdB-2/3 fiber protein shaft domains; and (c) one or more non-AdB-2/3-derived dimerization domains operatively linked to and located N-terminal to the one or more AdB-2/3 fiber polypeptide shaft domains.
In a preferred embodiment, the recombinant polypeptides do not include a tail domain from an Ad fiber polypeptide. As is disclosed in detail below, the inventors have localized the required sites for fiber polypeptide multimerization and binding to DSG2 to the shaft and knob domains of the AdB-2/3 proteins. The polypeptides of this aspect of the invention can thus be used, for example, to form AdB-2/3 fiber multimers for use in the various methods of the invention discussed above. In this aspect, the recombinant polypeptides can include shaft and knob domains from any AdB-2/3 virus, or any mutants (substitutions, additions, deletions, chimeras, etc.) to such shaft and knob domains that retain or improve binding affinity to DSG2, and are capable of forming multimers (such as dimers) via the dimerization domain (functional equivalents). It is well within the level of skill in the art to generate such mutants, and testing of such mutants for DSG2 binding and resulting suitability for use in the recombinant polypeptides of the invention is also within the level of skill in the art based on the teachings herein. For example, surface plasmon resonance (SPR) studies using sensors containing immobilized recombinant DSG2 can be used to determine if recombinant polypeptides being assessed bind to DSG2, combined with DSG2 competition studies. In one non-limiting embodiment, the polypeptide may be modified or mutated to reduce its immunogenicity by changing its amino acid sequence using techniques well known to those skilled in the art. Further exemplary studies, such as loss and gain of function analyses, are described in detail in Example 1.
As used throughout the present application, the term “polypeptide” is used in its broadest sense to refer to a sequence of subunit amino acids. The polypeptides of the invention may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids. The polypeptides described herein may be chemically synthesized or recombinantly expressed. The polypeptides may be linked to other compounds to promote an increased half-life in vivo, such as by PEGylation, HESylation, PASylation, glycosylation, or may be produced as an Fc-fusion or in deimmunized variants. Such linkage can be covalent or non-covalent as is understood by those of skill in the art.
As used herein, the term “operatively linked” refers to an arrangement of elements wherein the domains are configured so that they function as a unit for their intended purpose. The term does not require that the domains are immediately adjacent on the polypeptide, as spacer/linker sequences may be present between the domains, the lengths of which can be quite variable. In one non-limiting embodiment, the spacer length between any two domains of the recombinant AdB-2/3 fiber polypeptides can be between about 0 amino acids and about 20 amino acids. In various other non-limiting embodiments, the spacer length can be 0-20, 0-19, 0-18, 0-17, 0-16, 0-15, 0-14, 0-13, 0-12, 0-11, 0-10, 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, 0-3, 0-2, 0-1, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6- 17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, 19-20, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acids in length.
As used herein, “recombinant polypeptide” means a non-naturally occurring protein product, wherein the domains of the recombinant polypeptide are derived from one or more other proteins or artificially derived sequences. For example, each domain can be derived from a different naturally occurring protein sequence (ex: shaft sequence from one AdB-2/3 serotype; knob domain from a different AdB-2/3 serotype; etc.). The recombinant polypeptide may be constructed by a variety of mechanisms including, but not limited to, standard DNA manipulation techniques and chemical assembly via subunit parts of the recombinant polypeptide. The chemical assembly may lead to an equivalent form as the molecular genetic form or alternative associations with equivalent function. In a preferred embodiment, the recombinant polypeptide is produced by standard recombinant DNA techniques. Techniques for such recombinant production and isolation of the recombinant polypeptides of the invention are well within the level of skill in the art based on the teaching herein.
In one embodiment, each shaft domain is selected from the group consisting of an Ad3 shaft domain, an Ad7 shaft domain, an Ad11 shaft domain, an Ad 14 shaft domain, an Ad14a shaft domain, combinations thereof, and functional equivalents thereof. The shaft domain is required for fiber knob dimerization, which is required for binding to DSG2 and resulting transient opening of intercellular junctions. Thus, functional equivalents of the shaft domains of these Ad virus serotypes can be readily determined by those of skill in the art, based on the examples provided below. For example, surface plasmon resonance (SPR) studies using sensors containing immobilized recombinant DSG2 can be used to determine if recombinant polypeptides being assessed bind to DSG2, combined with DSG2 competition studies. Further exemplary studies, such as loss and gain of function analyses, are described in detail in Example 1.
The recombinant polypeptides may comprise between 1 and 22 AdB-2/3 fiber polypeptide shaft domains. Thus, in various embodiments to polypeptides comprise 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5- 16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6- 14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-22, 7-21, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-22, 9-21, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-22, 11-21, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-22, 12-21, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-22, 13-21, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-22, 14-21, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 15-16, 16-22, 16-21, 16-20, 16-19, 16-18, 16-17, 17-22, 17-21, 17-20, 17-19, 17-18, 18-22, 18-21, 18-20, 18-19, 19-22, 19-21, 19-20, 20-22, 20-21, 21-22, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 AdB-2/3 fiber protein shaft domains. Where more than 1 AdB-2/3 fiber protein shaft domain is present, each shaft domain can be identical, or one or more copies of the shaft domain may differ in a single recombinant polypeptide. In a preferred embodiment, the recombinant AdB-2/3 fiber polypeptide has a single shaft domain.
In another embodiment, one or more (or all) shaft domains in the recombinant polypeptide comprise or consist of an amino acid sequence according to SEQ ID NO 11:
In this sequence and other variable sequences shown herein, the variable residues are noted within parentheses, and a “-” indicates that the residue may be absent.
In another embodiment, one or more (or all) shaft domains in the recombinant polypeptide comprise or consist of an amino acid sequence according to SEQ ID NO 12:
In a still further embodiment, one or more (or all) shaft domains in the recombinant polypeptide comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:1 (Ad3), SEQ ID NO:2 (Ad7), SEQ ID NO:3 (Ad11), SEID NO:4 (Ad14), and SEQ ID NO:5 (Ad14(a). See
In another embodiment, the knob domain comprises or consists of an amino acid sequence according to SEQ ID NO 13:
In a further embodiment, the knob domain comprises or consists of an amino acid sequence according to SEQ ID NO:14:
In a still further embodiment, the knob domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:6 (Ad3), SEQ ID NO:7 (Ad7), SEQ ID NO:8 (Ad11), SEQ ID NO:9 (Ad14), and SEQ ID NO:10 (Ad14a). See
As used herein a “dimerization domain” is a peptide sequence that promotes dimerization in the recombinant polypeptide that contains it. Any suitable non-AdB-2/3-derived dimerization domain can be used in the recombinant polypeptide of the invention, so long as it permits dimerization of the recombinant polypeptide and thus binding to DSG2. The dimerization domain is non-AdB-2/3-derived, in that it is not a naturally occurring domain in an AdB-2/3 fiber polypeptide.
Non-limiting examples of the numerous dimerization domains known to those of skill in the art and suitable for use in the present invention include, but are not limited to peptide helices containing at least one helix, or a structure formed by a helix, a coil and another helix, etc., coiled coil structures, dimerization domains within, for example, many cell surface signaling receptors, Fc regions or hinge regions of an antibody, leucine zippers, the STAT protein N terminal domain, FK506 binding protein, the LexA protein C-terminal domain, nuclear receptors, the FkpA N-terminal domain, orange carotenoid protein from A. maxima, M1 matrix protein from influenza, neuraminidase from influenza virus, E. coli fuculose aldolase; and the like. (see, e.g., O'Shea, Science. 254: 539 (1991), Barahmand-Pour et al., Curr. Top. Microbiol. Immunol. 211: 121-128 (1996); Klemm et al., Annu. Rev. Immunol. 16: 569-592 (1998); Klemm et al., Annu. Rev. Immunol. 16: 569-592 (1998); Ho et al., Nature. 382: 822-826 (1996); and Pomeranz et al., Biochem. 37: 965 (1998)). Further examples include residues 325 to 410 in the bovine papillomavirus E2 protein, (Dostatni, N., et al., EMBO J. 7 (1988) 3807-3816; Haugen, T., et al. EMBO J. 7 (1988) 4245-4253; McBride, A., et al., EMBO J. 7 (1988) 533-539; McBride, A., et al., Proc Natl Acad Sci USA 86 (1989) 510-514), Type I deodinase (D1): DFLVIYIEEAHASDGW (SEQ ID NO: 24) or ADFL-YI-EAH-DGW (SEQ ID NO: 25); HIV-1 Capsid Protein: QGPKEPFRDYVDRFYKTLRA (SEQ ID NO: 26); leucine zipper dimerization motif of yeast GCN4: HMKQL D VEEL S NYHL N VARL K VGER (SEQ ID NO: 27); leucine zipper in Escherichia coli transcriptional antiterminator protein; and BgIG: GVTQLMREMLQLIKFQFSLNYQEESLSYQRLVT (SEQ ID NO: 28). In preferred embodiments, the dimerization domain comprises one or more copies of EVSALEK (SEQ ID NO:22) and/or KVSALKE (SEQ ID NO: 23).
It is well within the level of skill in the art to identify appropriate peptide sequences that can serve as dimerization domains, and mutants thereof, in the recombinant polypeptides of the present invention, based on the teachings herein, such as those disclosed below in Example 2. For example, dimerization of the recombinant AdB-2/3 fiber polypeptides can be assessed by criteria including sedimentation in sucrose gradients, resistance to trypsin proteolysis, and electrophoretic mobility in polyacrylamide gels (Hong and Engler, Journal of Virology 70:7071-7078 (1996)).
The recombinant polypeptides may comprise one or more non-AdB-2/3-derived dimerization domains. Thus, in various embodiments, the recombinant polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-AdB-2/3-derived dimerization domains. Where multiple domains are present in a polypeptide, it is preferred that each dimerization domain is the same.
In a preferred embodiment a spacer peptide is located between the dimerization domain and the one or more shaft domains. In a further preferred embodiment, the spacer peptide is a peptide with structural flexibility. Virtually any peptide with structural flexibility can be used. As an example, the flexible peptide may comprise repetitions of amino acid residues, such as Gly-Gly-Gly-Ser (SEQ ID NO: 62), or any other suitable repetition of amino acid residues. In another embodiment, the hinge region of an antibody can be used. The spacer can be any suitable length that maintains the ability of the recombinant polypeptide to dimerize and to maintain binding of the recombinant polypeptide to DSG2.
In one preferred embodiment, the recombinant AdB-2/3 polypeptide comprises:
This preferred embodiment can be used with any embodiment or combination of embodiments described herein. For example, any suitable dimerization domain can be used, including but not limited to one or more copies of EVSALEK (SEQ ID NO:22) and/or KVSALKE (SEQ ID NO: 23). Similarly any suitable spacer peptides can be used between the dimerization domain and the shaft domain and/or between the shaft domain and the knob domain. In a most preferred embodiment, the recombinant AdB-2/3 polypeptide comprises or consists of JO-1 (SEQ ID NO:20), or a multimer thereof (such as a dimer).
The recombinant polypeptides may comprise further domains, such as a domain for isolation of the polypeptide and/or a detection domain, see, for example, JO-1 with a His Tag in the examples and in SEQ ID NO:21). An isolation domain can be added to facilitate purification/isolation of the polypeptide following, for example, recombinant polypeptide production. Any suitable isolation domain can be used, including but not limited to HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, GST and MBP affinity tags. As used herein, “detection domain” means one or more amino acid sequence that can be detected. Any suitable detection domain can be used, including but not limited to, inherently fluorescent proteins (e.g. Green Fluorescent Proteins and fluorescent proteins from nonbioluminescent Anthozoa species), cofactor-requiring fluorescent or luminescent proteins (e.g. phycobiliproteins or luciferases), and epitopes recognizable by specific antibodies or other specific natural or unnatural binding probes, including, but not limited to, dyes, enzyme cofactors and engineered binding molecules, which are fluorescently or luminescently labeled.
In another embodiment, the recombinant polypeptides are in a multimeric form, such as a dimer, trimer, etc. In a preferred embodiment, a JO-1 (SEQ ID NO:20) multimer comprises a JO-1 dimer formed by dimerization through the dimerization domains in each JO-1 homotrimer (ie: a JO-1 polypeptide is a homotrimer through trimerization of the knob domain) In multimeric form (such as a dimer), the recombinant polypeptides comprise AdB-2/3 fiber multimers, and can be used in the various methods of the invention discussed above. As will be understood by those of skill in the art, such multimers may comprise multimers of identical recombinant polypeptide of the invention, or may comprise multimers of different recombinant polypeptides of the invention. In one embodiment, the dimerization domains are the same in each recombinant polypeptide forming part of the multimer. In another embodiment, the dimerization domains are different in each recombinant polypeptide forming part of the multimer. In another embodiment, the shaft and/or knob domains are the same in each recombinant polypeptide forming part of the multimer. In another embodiment, the shaft and/or knob domains are different in each recombinant polypeptide forming part of the multimer.
AdB-2/3 fiber multimerization can be determined according to methods well known to the practitioners in the art. For example, multimerization of the recombinant AdB-2/3 fiber constructs can be assessed by criteria including sedimentation in sucrose gradients, resistance to trypsin proteolysis, and electrophoretic mobility in polyacrylamide gels (Hong and Engler, Journal of Virology 70:7071-7078 (1996)). Regarding electrophoretic mobility, the fiber multimer is a very stable complex and will run at a molecular weight consistent with that of a multimer when the sample is not boiled prior to SDS-PAGE. Upon boiling, however, the multimeric structure is disrupted and the protein subsequently runs at a size consistent with the protein monomer.
The recombinant polypeptides, or multimeric versions thereof, may be stored in solution or frozen.
In another embodiment, the recombinant polypeptides of the invention are combined with (such as conjugated to) one or more therapeutics for a disorder associated with epithelial tissue. Such conjugates can be used, for example, in the therapeutic methods of the invention. Methods for conjugating the polypeptides of the invention to a therapeutic of interest, such as by covalent binding or chemical cross-linking, are well known to those of skill in the art. Any suitable therapeutic can be used to form a conjugate according to this embodiment of the invention, including but not limited to those disclosed above, as well as tumor stroma degrading compounds, such as relaxin. In a preferred embodiment, the therapeutic is an anti-tumor therapeutic and comprises a chemotherapeutic or anti-tumor monoclonal antibody as described herein. In a further preferred embodiment, the anti-tumor therapeutic comprises an antibody selected from the group consisting of trastuzumab, cetumiximab, petuzumab, Apomab, conatumumab, lexatumumab, bevacizumab, bevacizumab, denosumab, zanolimumab, lintuzumab, edrecolomab, rituximab, ticilimumab, tositumomab, alemtuzumab, epratuzumab, mitumomab, gemtuzumab ozogamicin, oregovomab, pemtumomab daclizumab, panitumumab, catumaxomab, ofatumumab, and ibritumomab.
In another aspect, the present invention provides nucleic acids encoding the polypeptide or any embodiment of the invention. The nucleic acids may comprise RNA or DNA, and can be prepared and isolated using standard molecular biological techniques, based on the teachings herein. The nucleic acids may comprise additional domains useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals.
In a further aspect, the present invention provides recombinant expression vectors comprising the nucleic acid of any aspect of the invention operatively linked to a promoter. “Recombinant expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any promoter capable of effecting expression of the gene product. The promoter sequence used to drive expression of the disclosed nucleic acids in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The construction of expression vectors for use in transfecting prokaryotic cells is also well known in the art, and thus can be accomplished via standard techniques. (See, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the AMBION™ 1998 Catalog (AMBION™, Austin, Tex.). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA, and may comprise any other components as deemed appropriate for a given use, including but not limited to selection markers such as an antibiotic-resistance gene.
In a still further aspect, the present invention provides host cells comprising the recombinant expression vectors disclosed herein, and progeny thereof, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.). Techniques utilizing cultured cells transfected with expression vectors to produce quantities of polypeptides are well known in the art.
In another aspect, the present invention provides pharmaceutical compositions, comprising
(a) an AdB-2/3 fiber multimer, or functional equivalent thereof; and
(b) a pharmaceutically acceptable carrier.
The AdB-2/3 fiber multimer can be any such multimer as described herein according to any aspect, embodiment, or combination of embodiments of the invention. In various preferred embodiments, the AsB-2/3 fiber multimer comprises an AdB-2/3 virion, an AdB-2/3 capsid, an AdB-2/3 PtDd, or a recombinant AdB-2/3 fiber multimer of the invention, or functional equivalents thereof. In various other preferred embodiments, the AdB-2/3 fiber multimer is selected from the group consisting of an Ad3 fiber multimer, an Ad7 fiber multimer, an Ad11 fiber multimer, an Ad14 fiber multimer, an Ad14a fiber multimer, and combinations or chimeras thereof. In a preferred embodiment, the one or more AdB-2/3 fiber multimers comprise or consist of an AdB-2/3 PtDd (such as Ad3 PtDd). In another preferred embodiment, the one or more AdB-2/3 fiber multimers comprise or consist of any embodiment or combination of embodiments of the compositions of the invention described herein, such as a JO-1 (SEQ ID NO:20) dimer.
The pharmaceutical composition may further comprise one or more therapeutic for treating a disorder associated with epithelial tissue, including but not limited to those disclosed above. In a preferred embodiment, the therapeutic is an anti-tumor therapeutic and comprises a chemotherapeutic or anti-tumor monoclonal antibody as described herein. In a further preferred embodiment, the anti-tumor therapeutic comprises an antibody selected from the group consisting of trastuzumab, cetumiximab, petuzumab, Apomab, conatumumab, lexatumumab, bevacizumab, bevacizumab, denosumab, zanolimumab, lintuzumab, edrecolomab, rituximab, ticilimumab, tositumomab, alemtuzumab, epratuzumab, mitumomab, gemtuzumab ozogamicin, oregovomab, pemtumomab daclizumab, panitumumab, catumaxomab, ofatumumab, and ibritumomab.
The pharmaceutically acceptable carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the multimers (and any other therapeutic agents combined therewith). Exemplary pharmaceutically acceptable carriers for peptides are described in U.S. Pat. No. 5,211,657 to Yamada. The compositions may be formulated into preparations in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, inhalants, and injections, allowing for oral, parenteral, or surgical administration. Suitable carriers for parenteral delivery via injectable, infusion, or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid, find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve. The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay, or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability, or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting example, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles. Suitable hydrogel and micelle delivery systems include the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes disclosed in International Publication No. WO 2004/009664 A2, and the PEO and PEO/cyclodextrin complexes disclosed in U.S. Publication No. 2002/0019369 A1. Such hydrogels may be injected locally at the site of intended action, or subcutaneously or intramuscularly to form a sustained release depot.
For intrathecal (IT) or intracerebroventricular (ICV) delivery, appropriately sterile delivery systems (e.g., liquids; gels, suspensions, etc.) can be used to administer the compositions. For oral administration of non-peptidergic agents, the compositions may be carried in an inert filler or diluent such as sucrose, cornstarch, or cellulose.
The compositions of the present invention may also include biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifiers, binders, thickeners, flavoring agents (for oral administration). Exemplary formulations can be parenterally administered as injectable dosages of a solution or suspension of the multimer in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions comprising modified polypeptides. Additional components of pharmaceutical compositions include petroleum (such as of animal, vegetable, or synthetic origin), for example, soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.
The pharmaceutical composition can also be administered in the form of a depot injection or implant preparation that can be formulated in such a manner as to permit a sustained or pulsatile release of the multimers and other therapeutic (if present).
The pharmaceutical composition may comprise in addition to the polypeptide of the invention (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer. in some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic: acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan rnonopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.
The pharmaceutical composition can be packaged in any suitable manner. In one embodiment, the pharmaceutical composition is packaged as a kit containing a container (such as a vial) of the AdB-2/3 fiber multimer. In a preferred embodiment, the kit further comprises, in the same or a separate container (such as a vial), a therapeutic, diagnostic, or imaging agent to be administered to a subject, together with the AdB-2/3 fiber multimer. Any suitable AdB-2/3 fiber multimer can be used in the kits; in a most preferred embodiment AdB-2/3 fiber multimer is a mulitmer (such as a dimer) of JO-1.
In a further aspect, the present invention provides kits comprising (a) one or more recombinant polypeptides/AdB-2/3 fiber multimers, isolated nucleic acids, recombinant expression vectors, and/or host cells of the invention; and (b) instructions for its/their use in treating a disorder associated with epithelial tissue. The kits may further comprise a therapeutic for use in the methods of the present invention.
In another aspect, the present invention provides method for identifying candidate compounds for one or more of treating a disorder associated with epithelial tissue, improving delivery of a substance to an epithelial tissue, for improving delivery of a substance tissue expressing DSG2, inducing an EMT in a tissue, and/or treating an AdB-2/3 infection comprising (a) contacting an AdB-2/3 fiber multimer to DSG2 under conditions to promote multimer binding to DSG2, wherein the contacting is carried out in the presence of one or more test compounds; and (b) identifying positive test compounds that compete with the AdB-2/3 fiber multimer for binding to DSG2 compared to control; wherein the positive test compounds are candidate compounds for one or more of treating a disorder associated with epithelial tissue, improving delivery of a substance to an epithelial tissue, for improving delivery of a substance tissue expressing DSG2, inducing an EMT in a tissue, and/or treating an AdB-2/3 infection.
Positive test compounds that compete with the AdB-2/3 fiber multimer for binding to DSG2 are candidate compounds for transiently opening intracellular junctions through their interaction with DSG2. Follow-up assays to verify the ability of the compounds to transiently open intracellular junctions through their interaction with DSG2 can be carried out by any suitable methods, including but not limited to studies disclosed in the examples that follow. Compounds so identified for treating a disorder associated with epithelial tissue, improving delivery of a substance to an epithelial tissue, improving delivery of a substance tissue expressing DSG2, or inducing an EMT in a tissue, can be used as substitutes for the AdB-2/3 multimer in any of the methods of the present invention. Furthermore, AdB-2/3 represent important human pathogens causing respiratory tract infections (some sever) and pharyngoconjunctival fever. Thus compounds that can treat AdB-2/3 infection would be useful. As disclosed herein, DSG2 as the primary high-affinity receptor used by AdB-2/3, and thus compounds that can diminish AdB-2/3 binding to DSG2 are candidate compounds for treating or limiting development of AdB-2/3 infection.
In this aspect, the AdB-2/3 multimer can be any AdB-2/3 multimer disclosed in any embodiment or combination of embodiments. Thus, in various non-limiting embodiments, the AdB-2/3 multimer can be AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd) (subviral dodecahedral particles produced by AdB-2/3 during their replication), recombinant AdB-2/3 fiber multimers (including but not limited to those disclosed in any embodiment or combination of embodiments below), and functional equivalents thereof. In a preferred embodiment, the one or more AdB-2/3 fiber multimers comprise or consist of an AdB-2/3 PtDd, such as an Ad3 PtDd. In another preferred embodiment, the one or more AdB-2/3 fiber multimers comprise or consist of any embodiment or combination of embodiments of the compositions of the invention described below. In a further preferred embodiment, the AdB-2/3 fiber multimer comprises or consists of the a JO-1 (SEQ ID NO:20) dimer, or functional equivalents thereof.
Any suitable control can be used, including but not limited to an AdB-2/3 multimer binding to DSG2 in the absence of test compounds,
In one embodiment, the DSG comprises recombinant DSG2. In another embodiment, the methods use cells expressing DSG2 (endogenously or recombinantly) on the cell surface.
In one non-limiting embodiment, surface plasmon resonance (SPR) studies using sensors containing immobilized recombinant DSG2 can be used to identify candidate compounds that AdB-2/3 fiber multimer binding to DSG2, combined with DSG2 competition studies. Further exemplary studies, such as loss and gain of function analyses, are described in detail in Example 1.
In another embodiment, the identifying comprises transduction studies, where the ability of test compounds to diminish binding is detected as a decrease in the ability of functional AdB-2/3 virions to transduce DSG2-expressing epithelial cells, such as disclosed in Example 1.
In another embodiment, DSG2-expressing cell extract is electrophoretically separated and Western blotted, and labeled AdB-2/3 fiber multimer is used to probe the Western blot in the presence of the test compounds. In a further embodiment, dot-blot assays can be used, such as those described in Wang et al., J. Virology (2007) 81:12785-12792; and Wang et al. (2008) 82:10567-10579.
Further examples of techniques for identifying candidate compounds for treating an AdB-2/3 infection are provided in the examples that follow.
When the test compounds comprise polypeptide sequences, such polypeptides may be chemically synthesized or recombinantly expressed. Recombinant expression can be accomplished using standard methods in the art, as disclosed above. Such expression vectors can comprise bacterial or viral expression vectors, and such host cells can be prokaryotic or eukaryotic. Synthetic polypeptides, prepared using the well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (Na-amino protected Na-t-butyloxycarbonyl) amino acid resin with standard deprotecting, neutralization, coupling and wash protocols, or standard base-labile Na-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids. Both Fmoc and Boc Na-amino protected amino acids can be obtained from SIGMA™, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art. In addition, the polypeptides can be synthesized with other Na-protecting groups that are familiar to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, such as by using automated synthesizers.
When the test compounds comprise antibodies, such antibodies can be polyclonal or monoclonal. The antibodies can be humanized, fully human, or murine forms of the antibodies. Such antibodies can be made by well-known methods, such as described in Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988).
When the test compounds comprise nucleic acid sequences, such nucleic acids may be chemically synthesized or recombinantly expressed as well. Recombinant expression techniques are well known to those in the art (See, for example, Sambrook, et al., 1989, supra). The nucleic acids may be DNA or RNA, and may be single stranded or double. Similarly, such nucleic acids can be chemically or enzymatically synthesized by manual or automated reactions, using standard techniques in the art. If synthesized chemically or by in vitro enzymatic synthesis, the nucleic acid may be purified prior to introduction into the cell. For example, the nucleic acids can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, the nucleic acids may be used with no or a minimum of purification to avoid losses due to sample processing.
When the test compounds comprise compounds other than polypeptides, antibodies, or nucleic acids, such compounds can be made by any of the variety of methods in the art for conducting organic chemical synthesis.
Abstract
We have identified desmoglein 2 (DSG2) as the primary high-affinity receptor used by adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14. These serotypes represent important human pathogens causing respiratory tract infections. In epithelial cells, adenovirus binding to DSG2 triggers events reminiscent of epithelial-to-mesenchymal transition, leading to transient opening of intercellular junctions. This improves access to receptors, e.g. CD46 and Her2/neu, which are trapped in intercellular junctions. In addition to complete virions, dodecahedral particles (PtDd), formed by viral penton and fiber in excess during viral replication, can trigger DSG2-mediated opening of intercellular junctions as shown by studies with recombinant Ad3 PtDd. Our findings shed light on adenovirus biology and pathogenesis and have implications for cancer therapy.
Introduction
Human adenoviruses (Ads) have been classified into six species (A to F) currently containing 55 serotypes. Most Ad serotypes utilize the coxsackie-adenovirus-receptor (CAR) as a primary attachment receptor1. This is, however, not the case for species B Ad serotypes. Recently, we have suggested a new grouping of species B Ads based on their receptor usage2. Group 1 (Ad16, 21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3, Ad7, 14) share a common, unidentified receptor/s, which is not CD46 and which was tentatively named receptor X; Group 3 (Ad11) preferentially interacts with CD46, but also utilizes receptor X if CD46 is blocked.
Species B Ads are common human pathogens. Since 2005, a simultaneous emergence of diverse species B serotypes, including serotypes Ad3, Ad7, and Ad14, was observed. In 2007, a new, highly pathogenic and possibly more virulent strain of Ad14, Ad14a, has been discovered at several sites in the US and in Asia3-4. We recently demonstrated that Ad14a belongs to species B group 2 Ads with regards to their receptor usage5. Collectively, we will refer to all receptor X-utilizing serotypes (Ad3, Ad7, Ad14, Ad14a, and Ad11) as AdB-2/3.
AdB-2/3 have potential as gene transfer vectors, particularly with regard to tumors of epithelial origin6. Epithelial cells maintain several intercellular junctions (tight junctions, adherens junctions, gap junctions, and desmosomes), a feature which is often conserved in epithelial cancers in situ and in cancer cell lines7. Both CAR and CD46 are trapped in intercellular junctions of epithelial cancer cells and are not accessible to Ads that use these attachment receptors8-9. In contrast, AdB-2/3 efficiently infect epithelial cancer cells, which is accomplished in part through induction of processes that are reminiscent of Epithelial-to-Mesenchymal Transition (EMT)8, a cellular transdifferentiation program in which epithelial cells lose characteristics, such as intercellular junctions, and gain properties of mesenchymal cells10. Another distinctive feature of AdB-2/3 is their ability to produce subviral dodecahedral particles during their replication, consisting of Ad fiber and penton base11. Penton-Dodecahedra (PtDd) cannot assemble from full-length penton base protein, but require spontaneous N-terminal truncation by proteolysis between residues 37 and 3812. This cleaved site is conserved in Ad3, Ad7, Ad11, and Ad14 but is not present in Ad2 and Ad5 (
The first attempts to identify receptor X date back to 1995. These initial studies indicated the interaction of Ad3 with a ˜130 kDa HeLa cell protein14. Recently, several candidates for receptor X such as CD46, CD80 and/or CD86 were suggested15-18. However, we and others have thus far been unable to verify that these proteins can serve as the high affinity receptor for AdB-2/32,19-23.
In the present study, using Ad3 virions and recombinant Ad3 PtDd as a probe for receptor X, we identified desmoglein 2 (DSG2) as a high affinity receptor for AdB-2/3 serotypes. DSG2 is a calcium-binding transmembrane glycoprotein belonging to the cadherin protein family. In epithelial cells, DSG2 is a component of the cell-cell adhesion structure24. Its cytoplasmic tail interacts with a series of proteins that are in direct contact with regulators of cell adhesion and intercellular junctions/cell morphology25. It has been shown that DSG2 is overexpressed in a series of epithelial malignancies including gastric cancer26, squamous cell carcinomas27, melanoma28, metastatic prostate cancer29, and bladder cancer30.
Results
DSG2 is a Receptor for AdB-2/3 Viruses.
Our previous studies showed that Ad3 binds at nanomolar affinity to a high-density cellular receptor2. Ad3 binding was sensitive to trypsin and could be blocked by EDTA, implying that binding required divalent cations. First we sought to identify the Ad3 capsid protein that mediates high-affinity binding to cells, which we would later use to search for the high-affinity receptor X. Notably, high-affinity binding of Ad5 to CAR and Ad35 binding to CD46, respectively, is mediated by the corresponding fiber knob31. Our previous studies, however, revealed that a single recombinant, timeric Ad3 knob could not completely block Ad3 virus binding even when very high concentrations were used, indicating that other or additional capsid moieties are involved in Ad3 binding32. Consequently, we utilized recombinant Ad3 dodecahedra composed of Ad3 penton bases (BsDd) or Ad3 penton bases and fibers (PtDd) (
To select an optimal cell line for receptor X identification, we compared Ad3 virus binding to several human and animal cell lines (
To identify Ad receptor candidates, HeLa cell membrane proteins were solubilized, separated on polyacrylamide gels, and blotted. Blots were hybridized with viral particles and binding was visualized with virus fiber knob specific antibodies. Specific gel bands were excised and analyzed by tandem mass-spectroscopy (MS/MS). First, we tested whether this assay can detect a known Ad receptor, CD46. When filters were incubated with CD46-targeting Ad5/35++virions35, a single band was found that matched CD46 (
BIACORE™ surface plasmon resonance (SPR) studies with sensors containing immobilized recombinant human DSG2 demonstrated that Ad3, but not Ad2 or Ad5, virions interact with DSG2 (FIG. 2g). Recombinant PtDd but not BsDd particles bound to DSG2 (
Loss- and gain-of-function studies were performed on cell lines to validate DSG2 as a critical receptor for AdB-2/3 binding/infection. Recombinant DSG2 protein blocked the binding of Ad3 as well as other AdB2/3 Ads, i.e. Ad7, Ad14, Ad14a, and Ad11 to HeLa cells, but not the binding of Ad5 and Ad35 (
siRNA mediated DSG2 downregulation also decreased viral cytolysis and spread in cells that were infected at 100% confluence at an MOI of one Ad3-GFP pfu per cell. Using adjusted multiplicities of infection (MOIs) (to achieve comparable percentages of GFP expression at 16 hours post-infection), we followed viral cytolysis over time and found larger lytic plaques in control siRNA than in DSG2 siRNA-transfected cells at day 7 p.i. This is reflected in crystal violet staining of viable cells that remained attached to tissue culture wells, i.e. cells that did not develop cytopathic effects due to virus infection (
For gain-of-function studies, we selected a series of cell lines with different DSG2 expression levels and measured Ad3-GFP transduction (
DSG2 Localization in Human Cells:
As expected, we found DSG2 in cell membranes of normal epithelial tissues (foreskin and colon) and epithelial cancers (breast and ovarian cancer) (
In contrast to polarized epithelial cell lines, in non-polarized cells, such as HeLa cells, intercellular junctions (i.e. membrane-localized Claudin 7 and E-cadherin signals) were absent. DSG2 and Ad3 were found dispersed over the cell surface (
Our studies on Ad3 infection of HeLa cells (
Ad3 interaction with DSG2 triggers EMT.
Recently, we found that AdB-2/3 interaction with epithelial ovarian cancer cells triggered EMT. EMT is characterized by increased expression of mesenchymal markers, increased expression of extracellular matrix compounds, decreased expression of epithelial markers, altered location of transcription factors, and activation of kinases7. Here, we attempted to prove that Ad3 interaction with DSG2 triggers EMT-like events. To avoid potential side effects of viral gene expression on cell morphology, the studies utilized ultraviolet light (UV)-inactivated Ad particles and recombinant Ad3 PtDd. Overall, the results with both types of particles were similar. Incubation of epithelial cancer cells with PtDd (
Western blot analysis using phosphorylation-specific antibodies showed that PtDd, but not BsDd, triggered the activation of PI3K and MAPK/ERK1/2, i.e. key kinases involved in EMT (
Ad3 and PtDd Increase Access to Receptors that are Trapped in Intercellular Junctions.
To test whether Ad3 virion- or Ad3 PtDd-triggered EMT also results in opening of intercellular junctions, we studied barrier properties in monolayers of epithelial cells. First we measured the flux of 4 kDa FITC-dextran through confluent polarized BT474 cells cultured in TRANSWELL™ chambers (
In another line of experiments in breast cancer cell cultures, we found that Her2/neu, the receptor for the widely used monoclonal antibody Herceptin (trastuzumab) co-stained with the intercellular junction protein Claudin 7 (
Discussion
In this study we describe two major findings: i) DSG2 is a receptor crucial for infection of a series of human adenoviruses that are common pathogens and important biomedical tools. ii) Ad interaction with DSG2 results in the opening of intercellular junctions, thus increasing access to receptors trapped therein.
DSG2 is an Ad Attachment Receptor.
The use of complete Ad3 particles or PtDd as a receptor probe was instrumental in the identification of DSG2 as the attachment receptor for Ad3, Ad7, Ad11, and Ad14. Previous attempts with Ad3 fiber knob domains as a bait did not yield meaningful receptor candidates32. Our competition and surface plasmon resonance studies shown in
Role of Ad3 and PtDd Interaction with DSG2 in Viral Dissemination.
During replication of Ad5, excess production of fiber results in the disruption of epithelial junctions either by interfering with CAR dimerization (which is critical for maintenance of junctions) or by triggering intracellular signaling that leads to reorganization of intercellular junctions42-43. Both mechanisms could also be involved in Ad3 virion/DSG2- and Ad3 PtDd/DSG2-mediated intercellular junction opening. We have experimental support for intracellular signaling triggered by Ad3 and PtDd binding to DSG2 in epithelial cells. Immunofluorescence, PI3K/MAPK phosphorylation, mRNA expression array, and metabolic pathway inhibition data suggest that Ad3 and PtDd trigger EMT in epithelial cells resulting in transient opening of intercellular junctions. Intercellular junction opening mediated by interaction of Ad3 particles or recombinant PtDd with DSG2 is further supported by increased cell permeability and access to receptors that are trapped in intercellular junctions (CD46 and Her2/neu). Along this line, a recent study showed that antibodies against the extracellular domain of DSG2 resulted in the opening of intercellular junctions in CaCo-2 cell monolayers44.
Ad3 virion- and PtDd-triggered EMT, i.e. the dissociation of the intercellular junctions, appears to have an important biological role. We speculate that, specifically the massive overproduction of PtDd during viral infection and its interaction with DSG2, facilitate the lateral viral spread in epithelial cells and, potentially, the penetration of Ad into subepithelial cell layers and the blood stream.
Implications for Ad Pathogenesis.
Our findings that DSG2 is an attachment receptor that facilitates further viral spread, sheds light on the Ad3 infection mechanism of the respiratory tract epithelium. Furthermore, the observation that Ad3 binds to DSG2 on platelet and granulocytes has potential implications on systemic spread of this virus once it has entered the blood stream. Although mouse DSG2 shares 76% homology with human DSG245, our data, showing that mouse cells are refractory to Ad3-GFP infection, indicate that mouse DSG2 cannot function as an Ad3 receptor. To study pathogenesis of AdB-2/3 serotypes, transgenic mice that express human DSG2 under the control of adequate endogenous regulatory elements resulting in DSG2 expression in a pattern and at levels similar to humans, would be a critical tool.
Implications for Cancer Therapy.
DSG2 has been proposed as a marker for epithelial tumors46. The epithelial phenotype of cancer cells and the ability to form physical barriers represent a mechanism that restricts access of drugs, antibodies, oncolytic viruses, or immune cells to the sites of tumors, thus diminishing the efficacy of such therapeutic modalities47. We demonstrated here, in three examples (Ad5/35 vectors, Herceptin, and Erbitux), that this important problem in cancer therapy can perhaps be addressed by the use of DSG2-interacting Ad3 components.
In conclusion, we report the discovery of the high affinity receptor for a series of common human Ads. Our study contributes to the understanding of how Ads induce cellular processes in order to gain access to epithelial tissue. Our findings have implications for improving cancer therapies.
Methods
Proteins and Antibodies.
The knob domains of Ad3, Ad5, and Ad35 fibers were produced in E. coli as described elsewhere48. Recombinant Ad3 penton-dodecahedra (PtDd) and base dodecahedra (BsDd) were produced in insect cells and purified as described previously33. Polyclonal rabbit antibodies against purified recombinant Ad3 and Ad35K++ knob were produced by PickCell Laboratories B.V. (Amsterdam, The Netherlands). DSG2-specific monoclonal antibodies 20G1, 7H9, 13B11, 10D2 and 8E549 were purified from hybridoma culture supernatant.
Cell Lines.
Cells were cultured as described in the SI. BT474 is a Her2/neu-positive breast cancer cell line with epithelial cell features. To achieve cell polarization, 1.4×105 BT474, T84 and CaCo2 cells were cultured in collagen-coated 6.5 mm TRANSWELL™ inserts (0.4 μm pore size) (Costar TRANSWELL™ Clears) for 10 days until transepithelial resistance was stable.
Adenoviruses.
Wild-typeAd3 (GB strain), Ad7p (Gomen stain), Ad11p (Slobitski strain), Ad14 (DeWit strain), and Ad35 (Holden strain) were obtained from the ATCC. Ad14a is new genomic variant of Ad145. Propagation, methyl-3H thymidine labeling, purification and titering of Ads was performed as described elsewhere2. Ad5/35-GFP and Ad5-GFP are Ad5 vectors containing Ad35 and Ad5 fibers and a CMV-GFP expression cassette50. Ad3-GFP and Ad35-GFP are wild-type Ad3- and Ad35-based vectors containing a CMV-GFP expression cassette inserted into the E3 region. Construction of Ad3-GFP is described in SI. Ad35-GFP has been described previously34. For transduction studies, cells were exposed to Ad vectors at the indicated MOIs for one hour, washed, and GFP expression was measured by flow cytometry 18 hours later.
Membrane Protein Preparation.
HeLa cell membrane proteins were prepared as described earlier51. Briefly, HeLa cell pellets were re-suspended in ice-cold homogenization buffer (20 mM Hepes, 1.5 mM MgCl2, 5 mM KCl, 150 mM NaCl, 15% glycerol, 0.25 M sucrose, 0.1 mM EDTA, 2 mM-mercaptoethanol, 1 mM PMSF). After disruption with a 3 ml syringe and 21G needle, the lysate was centrifuged at 400×g for 15 minutes. The supernatant was diluted with 2 times volume of PBS and centrifuged at 35,000 rpm for 1 hour in an ultracentrifuge. The membrane protein pellet was resuspended in solubilization buffer (50 mM Hepes, 5 mM MgCl2, 5 mM KCl, 150 mM NaCl, 15% glycerol, 0.25 M sucrose, 0.1 mM EDTA, 2 mM-mercaptoethanol, 1 mM PMSF, 0.5% Brij 96V(FLUKA™, St Louis, Mo.). The use of Brij96V as a detergent was intrumental as desmosomal proteins are highly insoluble.
Western Blot with Ad3 and PtDd.
Technical details for mass-spectroscopy analysis are described elsewhere51. To immunoprecipitate DSG2 from soluble crude membrane protein preparations, DSG2-specific mAb 6D8, and the Pierce Crosslink Immunoprecipitation Kit (Pierce Biotechnology, Rockford, Ill.) were used. Crude membrane proteins from HeLa cells were solubilized with 0.5% detergent Brij 96V, pre-incubated with control resin for 3 hours at 4° C. to reduce nonspecific binding, and then incubated with DSG2 antibody crosslinked proteinA/G agarose overnight at 4° C. Bound protein were eluted per manufacturer's instruction.
Surface Plasmon Resonance (SPR) Analyses.
Acquisitions were done on a BIACORE™ 3000 instrument. HBS-N (GE-HEALTHCARE™, Pittsburgh, Pa.) supplemented with 2 mM CaCl2 was used as running buffer in all experiments at a flow rate of 5 l min−1. Immobilisation on CM4 sensorchip (BIACORE™) was performed using DSG2 (LENICO TECHNOLOGY™, Inc.) at 0.1 mg ml−1 diluted in 10 mM sodium acetate buffer pH4.2 injected for 10 minutes on EDC-NHS activated flow-cell. A control flow-cell was activated by EDC-NHS and inactivated by ethanolamine. Different concentration of PtDd, BsDd, Ad3 fiber knob were injected for 5 minutes followed by 3 minutes dissociation time and the signal was automatically subtracted from the background of the ethanolamine deactivated EDC-NHS flow cell. For the adenovirus binding experiments, a similar protocol was used with the injection of wild-type Ad2, Ad3 and Ad5 at 5·109 vp per ml.
siRNA Studies.
A set of DSG2 specific siRNA was synthesized by DHARMACON™ (THERMO SCIENTIFIC™). The target sequences were CAAUAUACCUGUAGUAGAA (SEQ ID NO: 29), GAGAGGAUCUGUCCAAGAA(SEQ ID NO: 30), CCUUAGAGCUACGCAUUAA (SEQ ID NO: 31) and CCAGUGUUCUACCUAAAUA (SEQ ID NO: 32). Control siRNA was purchased from QIAGEN™, Valencia, Calif. siRNA transfection was performed using HyperFect transfection reagent (QIAGEN™)
DSG2 Expressing U937 Cells.
DSG2 cDNA (accession No. BC099657) from Capital Biosciences (Rockville, Md.) was cloned into the lentivirus vector pRRL-SIN52 under the control of the EF1 promoter. VSVG-pseudotyped lentivirus vectors were produced and titered as described earlier53.
Animal Studies:
All experiments involving animals were conducted in accordance with the institutional guidelines set forth by the University of Washington. Mice were housed in specific-pathogen-free facilities. Breast cancer xenografts were established by injecting cancer cells in matrigel (1:1 vol/vol) into the mammary fat pad of CB17 SCID-beige mice. Herceptin was injected intraperitoneally at a dose of 10 mg/kg. PtDd was given intravenously at a dose of 2 mg/kg. Tumor volumes were measured as described previously54.
Abstract
In Example 1, we identified desmoglein 2 (DSG2) as the main receptor for a group of species B adenoviruses (Ads), including Ad3, a serotype that is widely distributed in the human population. In this example, we have attempted to delineate structural details of Ad3 interaction with DSG2. For CAR- and CD46-interacting Ad serotypes, attachment to cells can be completely blocked by an excess of recombinant fiber knob protein, while soluble Ad3 fiber knob only inefficiently blocks Ad3 infection. We found that the DSG2 interacting domain(s) within Ad3 are formed by several fiber knob domains that have to be in the spatial constellation that is present in viral particles. Based on this finding, we generated a small recombinant, self-dimerizing protein containing the Ad3 fiber knob (Ad3-K/S/Kn). Ad3-K/S/Kn bound to DSG2 with high affinity and blocked Ad3 infection. We demonstrated by confocal immunofluorescence and transmission electron microscopy analyses that Ad3-K/S/Kn, through its binding to DSG2, triggered transient opening of intercellular junctions in epithelial cells. Pretreatment of epithelial cells with Ad3-K/S/Kn resulted in increased access to receptors that are localized in or masked by epithelial junctions, e.g. CAR or Her2/neu. Ad3-K/S/Kn treatment released CAR from tight junctions and thus increased transduction of epithelial cells by a serotype Ad5-based vector. Furthermore, pretreatment of Her2/neu-positive breast cancer cells with Ad3-K/S/Kn increased killing of cancer cells by the Her2/neu-targeting monoclonal antibody trastuzumab (Herceptin). This study widens our understanding of how Ads achieve high avidity to their receptors and infection of epithelial tissue. The small recombinant protein Ad3-K/S/Kn has practical implications for the therapy of epithelial cancer and gene/drug delivery to normal epithelial tissues.
Introduction
The protruding fiber is the moiety within the Ad capsid that mediates a high affinity binding to the primary attachment receptor. Each Ad capsid has 12 fibers linked to penton bases. Each fiber consists of a tail domain that is anchored within the penton base, a shaft domain consisting of repeats of up to 14 amino acids that form β-sheets (with the number of repeats ranging from 6 to 23 in different serotypes), and the C-terminal homo-trimeric knob domain. For CAR- and CD46-interacting Ads, the knob domain binds with high affinity to the receptor and soluble fiber knobs completely block infection. In this study, we have attempted to delineate structural details of Ad3 interaction with DSG2. We report that multimers of (trimeric) Ad3 fiber knobs are required for high affinity DSG2 binding. This is clearly different from the strategy of CAR- and CD46-interacting Ad serotypes to achieve infection. This specific mode of Ad3-DSG2 interaction is functionally crucial for Ad3, because it allows opening of epithelial junctions.
Material and Methods
Proteins and Antibodies.
Recombinant human DSG2 protein was from Leinco Technologies, Inc. (St. Louis, Mo.). The recombinant Ad3 fibers were produced in E. coli with N-terminal 6-His tags, using the pQE30 expression vector (QIAGEN™, Valencia, Calif.) and purified by Ni-NTA agarose chromatography as described elsewhere (34). Recombinant Ad3 penton-dodecahedra (PtDds) was produced in insect cells and purified as described previously (8).
The following antibodies were used for immunofluorescence studies: polyclonal goat anti-DSG2 (R&D Systems, Inc., Minneapolis, Minn.), mouse mAb anti-DSG2 (clone 6D8) (CELL SCIENCES™, Canton, Mass.), rabbit anti-Claudin 7 (ABCAM™, Cambridge, Mass.), FITC conjugated goat anti-adenovirus (MILLIPORE™ Billerica, Mass.), monoclonal anti-6×His (Serotec, MCA1396), rabbit anti-20-1 antibody (CELL SIGNALING TECHNOLOGY™ Inc., Beverly, Mass.). Polyclonal rabbit antibodies against purified recombinant Ad3 knob were produced by PICKCELL LABORITORIES™ B.V. (Amsterdam, The Netherlands). Monoclonal anti-DSG2 antibodies 20G1, 7H9, 13B11, 10D2 and 8E5 (12) were purified from hybridoma culture supernatants.
Recombinant Ad3 Fiber Knobs.
The recombinant Ad3 fiber knobs S/Kn, S2/Kn, S3/Kn, S4/Kn, S5/Kn, and S6/Kn were generated by PCR using Ad3 genomic DNA as a template. The PCR products were then cloned into the E. coli expression vector pQE30 as BclI/HindIII or BamHI/HindIII fragments. The following primers were used:
For generating the K- or E-coil containing Ad3 fiber knobs the following oligonucleotides were annealed and cloned into pQE30 as BamHI fragments.
To generate Ad3-E/S2/Kn and Ad3-K/S2/Kn, the following primers were used:
The PCR products were then cloned into the BamHI/HindIII site of pQE30-Kcoil and pQE30-E. coli. To generate Ad3-E/S/Kn and Ad3-K/S/Kn, the following primers were used:
The PCR products were than cloned into the BamHI/HindIII site of pQE30-Kcoil and pQE30-Ecoil. Cell lines. 293 (MICROBIX™, Toronto, Ontario, Canada), HeLa (American Type Culture Collection, ATTC) were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mmol/L L-glutamine (Glu), 100 units/mL penicillin, and 100 μg/mL streptomycin (P/S). BT474-M1 cells (16) were cultured in DMEM/F:12 with 10% FBS, 1% Pen/Strep and L-Glutamine. Colon cancer T84 cells (ATCC CCL-248)) were cultured in a 1:1 mixture of Ham's F12 medium and DMEM, 10% FBS, Glu and P/S. To achieve cell polarization, 1.4×105 T84 cells were cultured in 6.5 mm TRANSWELL™ inserts (0.4 μm pore size) (Costar TRANSWELL™ Clears) for more than 20 days until transepithelial resistance was stable.
Adenoviruses.
Propagation, methyl-3H thymidine labeling, purification and titering of Ads was performed as described elsewhere (31). Ad5/35-GFP and Ad5-GFP are Ad5 vectors containing Ad35 and Ad5 fibers and a CMV-GFP expression cassette (24). Ad3-GFP is a wild-type Ad3-based vector containing a CMV-GFP expression cassette inserted into the E3 region (33). Ad5/3L-GFP and Ad5/3S-GFP are E1/E3 deleted, Ad5-based vectors, containing the same CMV-GFP expression cassette inserted into the E3 region. The construction of chimeric Ad vectors followed the protocol described earlier (23). The primer sequences used to construct the chimeric fiber genes were as follows:
SpeI or XhoI restriction sites were introduced after the fiber stop codon for Ad5/3L-GFP or Ad5/3S-GFP, respectively. To generate full-length E1/E3-deleted vector genomes, the corresponding shuttle plasmid containing chimeric fiber genes and GFP expression cassette were inserted in pAdHM4 by homologous recombination in E. coli strain BJ1583. The resulting plasmids pAd5/3L-GFP and pAd5/3S-GFP were analyzed by restriction analysis and sequencing. To produce the corresponding viruses, pAd5/3L-GFP and pAd5/3S-GFP were digested with PacI to release the viral genomes and transfected into 293 cells as described before. Recombinant viruses were propagated in 293 cells and purified by standard methods. Ad particle (viral particle, VP) concentrations were determined spectrophotometrically by measuring the optical density at 260 nm (OD260) and plaque titering (plaque forming units, pfu) was performed using 293 cells as described elsewhere (24). The VP to pfu ratio was 20:1 for all virus preparations.
Attachment and Transduction Assays.
Adherent cells were detached from culture dishes by incubation with Versene and washed with PBS. A total of 1.8×105 cells/tube was resuspended in 100 μl of ice-cold adhesion buffer containing 3H-labeled Ad at a multiplicity of infection (M01) of 8,000 VP per cell. After 1 h of incubation at 4° C., cells were pelleted and washed twice with 0.5 ml of ice-cold wash-buffer (PBS, 1% FBS). After the last wash, the supernatant was removed, and the cell-associated radioactivity was determined with a scintillation counter. The number of viral particles (VP) bound per cell was calculated by using the virion specific radioactivity and the number of cells. For competition studies, 4.5 μg of competitor (PtDds, fiber knobs, antibodies) were allowed to attach for 60 min at 4° C. in adhesion buffer and non-bound competitor removed by washing cells twice with PBS before cells were resuspended in attachment buffer containing 3H-labeled Ad. For transduction studies, cells were exposed to Ad vectors at the indicated MOIs for one hour, washed, and GFP expression in 20,000 cells was measured by flow cytometry 18 hours later.
Ad5 Infection Studies.
In all transduction studies of HeLa, we have used conditions (MOI, virus concentration, exposure time) that have been optimized previously to be within the linear range of transduction (33).
Western Blot:
Mini-PROTEAN precast gels (BIO-RAD, Hercules, Calif.) with 4-15% gradient polyacrylamide were used. For the studies shown in
Native Polyacrylamide Gel Electrophoresis:
For the study shown in
Trastuzumab Cytotoxicity Assay.
5×104 BT474-M1 cells/well were plated in triplicate in 96 well plates and grown to confluence. Ad3 fiber knobs or monoclonal antibodies (5 μg/ml) were added to the inner chamber. One hour later, trastuzumab (GENENTECH™, San Francisco, Calif.) (15 m/ml) was added and cell viability was measured 2 hours later by WST-1 assay (ROCHE™, San Francisco, Calif.). Three independent studies were performed.
Immunofluorescence Analyses.
Cells were cultured in 8 chamber glass slides (BD Falcon), washed twice with ice-cold PBS and then fixed with methanol/aceton (1:1 vol/vol) for 15 min at 4° C. or with 4% paraformaldehyde for 30 min at 4° C. After fixation, cells were washed with PBS twice and blocked with 500 μl PBS/2% dry-milk powder for 20 min at room temperature. Antibody staining was performed in 100 μl PBS for 90 min at 37° C. or 4° C. overnight. If needed, secondary antibodies directed against the appropriate host, were applied after 3 washes with PBS for 45 min at room temperature. After 3 washes with PBS, glass slides were mounted using VECTASHIELD with DAPI (VECTOR LABS™). Photographs were taken with a Leica DFC300FX digital camera. Confocal images were taken on a ZEISS™ META confocal microscope using 40× or 100× oil lenses and ZEISS™ 510 software (ZEISS™ Microlmaging, Thornwood, N.Y.).
Electron Microscopy.
Polarized cells in TRANSWELL™ chambers were fixed with half-strength Karnovsky's fixative (2% paraformaldehyde, 2.5% glutaraldehyde, 0.2 M Cacodylate buffer) for one hour at room temperature. The fixative in the inner chamber contained 0.2% ruthenium red. The ruthenium red (Ruthenium(III)chloride oxide, ammoniated), was purchased from Alfa Aesar (Ward Hill, Mass.). Post-fixation was performed with 1% OsO4-phosphate buffer. The membranes were then cut out from the TRANSWELL™ chambers and embedded in Medcast (Ted Pella, Redding, Calif.). Ultrathin sections were stained with uranyl acetate and lead citrate. Processed grids were evaluated with a JEOL JEM1200EXII transmission electron microscope. Images were acquired with an Olympus SIS Morada Digital CCD camera using iTEM software for image processing.
siRNA Studies.
A set of DSG2 specific siRNA was synthesized by DHARMACON™ (THERMO SCIENTIFIC™). The target sequences were CAAUAUACCUGUAGUAGAA (SEQ ID NO: 29), GAGAGGAUCUGUCCAAGAA(SEQ ID NO: 30), CCUUAGAGCUACGCAUUAA (SEQ ID NO: 31) and CCAGUGUUCUACCUAAAUA (SEQ ID NO: 32). Control siRNA was purchased from QIAGEN™, Valencia, Calif. siRNA transfection was performed using HyperFect transfection reagent (QIAGEN™). A total of 1×105 HeLa were transfected with 1 ug DSG2 siRNA or control siRNA. Forty eight hours after siRNA transfection, cells were collected with versene, and the attachment of 3H-Ad3 or 3H-Ad35 virus was analyzed as described above. Forty eight hours after siRNA transfection, cells were infected with Ad vectors at an MOI of 50 pfu/cells, and GFP expression was analyzed 18 hours later.
Surface Plasmon Resonance (SPR) Analyses.
Acquisitions were done on a BIACORE™ X instrument. HBS-N (GE-HEALTHCARE™, Pittsburgh, Pa.) supplemented with 2 mM CaCl2 was used as running buffer in all experiments at a flow rate of 5 μl/min. Immobilization on CM4 sensorchip (BIACORE™) was performed using DSG2 (LENICO TECHNOLOGY™, Inc.) at 0.1 μg/ml diluted in 10 mM sodium acetate buffer pH4.2 injected for 10 minutes on EDC-NHS activated flow-cell. A control flow-cell was activated by ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) and inactivated by ethanolamine. Different concentration of Ad3 fiber knobs or PtDds were injected for 5 minutes association followed by 3 minutes dissociation time, and the signal was automatically subtracted from the background of the ethanolamine deactivated EDC-NHS flow cell.
For experiments using biotinylated ligands, two flow cells of a CM4 sensorchips were activated as described above and then coated by injection of streptavidin (0.1 μg/ml in acetate buffer pH4.1) for 5 minutes. Biotinylated ligands were then injected at 0.1 μg/ml in running buffer for 5 minutes on one of these two flow cells, the other being used for background subtraction during the run. Different concentrations of soluble DSG2 were then injected in running buffer on these flowcells and background was automatically subtracted.
Negative Stain Electron Microscopy.
Recombinant fiber knob proteins were visualized by negative-stain EM to assess their assembly status. The standard mica/carbone preparation was used with protein at 0.1 mg/ml. Samples were stained using 1% (w/v) sodium silicotungstate (pH 7.0) and visualized on Philips CM12 electron microscope at 100 kV.
Statistical Analysis:
All results are expressed as mean+/−SD. Wilcoxon signed-rank test was applied when applicable. A p-value <0.05 was considered significant.
Results
Chimeric Ad5 Vectors Containing Ad3 Fibers use DSG2 as a Receptor.
Our preliminary studies indicated that DSG2 interacting domain(s) within Ad3 are formed by the fiber or fiber/penton only in the spatial constellation that is present in viral particles, i.e. Ad3 virions or PtDds. To assess a potential role of Ad3 penton (which is present in PtDds) in binding to DSG2, we generated an Ad vector that contained Ad3 fibers (Ad5/3S-GFP), but had all other capsid proteins (including the penton) derived from Ad5. To evaluate whether the Ad3 fiber shaft had a cucial role in Ad3-DSG2 interaction, e.g., contained DSG2 additional binding sites, we also generated a chimeric Ad5/3 vector that had the Ad3 shaft substituted by the Ad5 shaft (Ad5/3L-GFP) (
Crosslinking of Ad3 Fiber Knobs is Required for Efficient Binding to DSG2.
We then focused our attention on the Ad3 fiber. We produced in E. coli a series of recombinant Ad3 fiber knob proteins, containing the fiber knob and increasing numbers of Ad3 shaft repeats (from one to six repeats) (
Ad3 Fiber Knob Dimers Block Ad3 Infection.
Crosslinking with antibodies enhanced the blocking effect of Ad3 fiber knobs containing fewer shaft motifs than the wild-type Ad3 fiber knob. For a potential therapeutic application of Ad3 fiber knobs as junction openers, we focused our further studies on decreasing the size of the molecule by evaluating fiber knob variants with the minimum number of shaft motifs, i.e. S2/Kn. Based on the finding that fiber knob cross-linking increased binding to DSG2, we generated dimers of S2/Kn by incorporating dimerization domains. To avoid the spontaneous fiber knob dimerization and potential formation of inclusion bodies during production in E. coli, we utilized a hetero-dimeric system consisting of E-coil and K-coil peptides, which interact with each other with high affinity (17). Two fiber knob variants containing five repeats of EVSALEK (SEQ ID NO:22) (K-coil) and KVSALKE (SEQ ID NO:23) (E-coil) respectively, a G/S rich-flexibility domain followed by two shaft motifs and the homotrimeric fiber knob domain were generated (
Binding of a Minimal Dimeric Ad3 Fiber Knob Protein to DSG2.
We then attempted to produce the smallest Ad3 fiber knob dimer, containing the K-coil or E-coil dimerization domain, only one shaft motif, and the homotrimeric Ad3 fiber knob (Ad3-K/S/Kn and Ad3-E/S/Kn) (
SPR Analysis of Dimeric Ad3 Fiber Knob Binding to DSG2.
To study the interaction of Ad3-K/S/Kn and Ad3-K/S/Kn+Ad3-E/S/Kn with DSG2 in more detail, we performed surface plasmon resonance (SPR) studies. We initially designed binding experiments, in which DSG2 molecules were allowed to bind to immobilized fiber knobs (
Interaction of Ad3 with several DSG2 molecules is supported by immunofluorescence analyses of epithelial cells (
Multimeric DSG2 Ligands (Ad3 Virions, PtDds, Ad3-K/S/Kn) Trigger Opening of Epithelial Junctions
Epithelial cells maintain several intercellular junctions (tight junctions, adherens junctions, gap junctions, and desmosomes), a feature which is often conserved in epithelial cancers in situ and in cancer cell lines (29).
Opening of epithelial junctions by Ad3-K/S/Kn was further confirmed by electron microscopy (EM) studies. EM images of untreated epithelial cells show intact tight and desmosomal junctions as judged by the exclusion of the apically applied dye ruthenium red from basolateral space (
In addition to leakage studies with ruthenium red, we used three functional assays to demonstrate opening of epithelial junctions by Ad3-K/S/Kn: i) Exposure of polarized epithelial cells to Ad3-K/S/Kn increased the transepithelial permeability within 30 minutes, as shown by 14C-PEG-4000 transflux studies (
Overall, our functional studies showed that Ad3-K/S/Kn can trigger opening of epithelial junctions, while ligands that are unable to multimerize had no effect on junctions.
Discussion
In this study, we describe two findings: i) multimerization of Ad3 fiber knob is required to achieve high-affinity and stable binding to DSG2 and ii) the multimeric mode of Ad3-fiber knob/DSG2 interaction triggers opening of epithelial junctions.
Most Ad infections, including infection by the CAR-interacting Ad5, the DSG2-interacting Ad3, and the CD46-interacting Ad35 (22) target the airway epithelium. Achieving a high avidity binding to receptors with a low dissociation rate appears to be crucial for Ad infection in order to maintain contact between virus and target cells, and more importantly, to trigger subsequent events that allow the virus to disrupt the epithelial barrier, enter the target cells, and spread within the target tissue. For CAR- and CD46-binding Ads, high avidity binding is achieved through interaction between the trimeric fiber knob and three receptor units. Furthermore, as shown for the interaction between Ad11 and CD46, initial Ad binding to the receptor can trigger conformational changes in the receptor to stabilize the binding (21); although it remains to be shown that the latter mechanism is used by Ads other than Ad11. For CAR— and CD46-interacting Ads, attachment involves the fiber knob and the receptor, and it can be completely blocked by an excess of soluble fiber knob. This is not the case for the DSG2-binding Ad3. We have shown that complete inhibition of Ad3 binding and infection requires the physical linkage and, most likely, a specific spatial constellation of at least two fiber knobs. This is achieved with Ad virions, Ad3 PtDds or dimeric Ad3-K/S/Kn. These ligands appear to achieve simultaneous binding to several DSG2 molecules, which, on the one hand, provides a high avidity and, on the other hand, is functionally relevant for opening of epithelial junctions. We are currently conducting crystal structure and mutagenesis studies to further support our findings on Ad3-DSG2 interaction.
In order to initiate infection, many pathogens have evolved mechanisms to disrupt junctional integrity. Vibrio cholera strains produce Zona occludens toxin (Zot), which possess the ability to reversibly alter intestinal epithelial junctions, allowing the passage of macromolecules through mucosal barriers (6). Clostridium perfringens enterotoxin removes claudins-3 and -4 from the tight junctions to facilitate bacterial invasion (25). Furthermore, oncoproteins encoded by human Ad, HPV, HTLV-1 can transiently open epithelial junctions by mislocalizing the junction protein ZO-1 (15). The latter mechanisms used by viruses appear to play a role in lateral viral spread. However, for an efficient initial infection, attachment of virus must be linked with triggering the opening of epithelial junctions.
In Example 1, using immunofluorescence, PI3K/MAPK phosphorylation, mRNA expression array, and metabolic pathway inhibition approaches, we reported that binding of DSG2 by Ad3 virions and Ad3 PtDds triggers an epithelial-to-mesenchymal transition in epithelial cells, resulting in transient opening of intercellular junctions. Intercellular junction opening mediated by interaction of Ad3 particles or recombinant PtDds with DSG2 was further supported by increased epithelial cell permeability and access to receptors that are trapped in intercellular junctions (e.g. Her2/neu). In the present study, we provide morphological (confocal immunofluorescence, EM) and functional (permeability, trastuzumab killing, Ad5 infection) data showing that Ad3-K/S/Kn also triggers junction opening. Importantly, other DSG2 ligands that are not able to multimerize and cluster DSG2 such as monomeric Ad3-E/S/Kn or monoclonal antibodies against different regions of DSG2 were unable to efficiently open junctions. It remains, however, the question how Ad3 can pass the tight junctions that are located at the apical side of DSG2/desmosomes. We speculate that efficient infection occurs through a positive feed-forward mechanism because the Ad-DSG2 mediated junction opening occurs within minutes thereby exposing DSG2 trapped in junctions to viral particles that are present at the site of infection. In the discussion of Ad3 infection mechanisms, it is also noteworthy that, during Ad3 replication, PtDds are formed at a massive excess of 5.5×106 PtDds per infectious virus (7). This suggests that PtDd formation is functionally important for Ad3, i.e. advantageous in spread or persistence of Ad3. A similar mechanism appears to take place in infection of epithelial cells by Ad5. During replication of Ad5, excess production of fiber results in the disruption of epithelial junctions either by interfering with CAR dimerization or by triggering intracellular signaling that leads to reorganization of intercellular junctions (4, 32). Recently, it has been suggested (based on in vitro studies) that CD46 is exposed apically on polarized epithelial respiratory cells and is therefore more likely to function as an Ad receptor for initial infection in vivo (9). We could not confirm this in human CD46 transgenic mice, where the CD46 expression pattern did not correlate with Ad transduction (19). Moreover, in polarized epithelial cancer cultures, we found CD46 trapped in intercellular junctions and not accessible to Ad35 applied to the cell surface (33). At this point, we believe that the question of how CD46-binding Ads disrupt the epithelial barrier remains to be answered.
The finding that Ad3-K/S/Kn triggers junction opening has practical implications because intercellular junctions represent physical obstacles for access and intratumoral dissemination of anti-cancer therapeutics (26-27). An epithelial junction opener would be relevant for cancer therapies with monoclonal antibodies directed against tumor-associated antigens that are trapped in epithelial junctions (e.g. Her2/neu or EGFR1) (33). A junction opener might also improve the efficacy of adoptive T-cell therapy (5) or treatment with liposomal chemotherapy drugs (10). Finally, Ad3-K/S/Kn might increase that efficacy of transduction of normal or malignant tissues by Ad5-based gene therapy vectors. The biotechnological applicability of Ad3-K/S/Kn is further underscored by the ease of its production and purification and by the fact that it spontaneously homodimerizes.
In conclusion, our study sheds light on the mechanisms of Ad3 infection of epithelial cells. The finding that Ad3-K/S/Kn, a small recombinant protein, triggers opening of epithelial junctions has implications for cancer therapy and drug delivery into epithelial tissues.
Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector. J Virol 74:2567-2583.
Abstract
The therapeutic efficacy of monoclonal antibodies (mAbs) for cancer treatment is limited by intercellular junctions that tightly link epithelial tumor cells to one another. We generated a small, recombinant adenovirus serotype 3 derived protein, junction opener 1 (JO-1), which binds to the epithelial junction protein desmoglein 2 (DSG2). We demonstrate in mouse models with tumors derived from Her2/neu- and EGFR-positive human cancer cell lines that within one hour after intravenous injection, JO-1 mediates the cleavage of DSG2 dimers in intercellular junctions as well as the activation of intracellular signaling pathways that lead to a decrease in the level of the tight junction protein E-cadherin. JO-1-triggered changes in epithelial junctions enabled better intratumoral penetration of the anti-Her2/neu mAb trastuzumab (Herceptin) as well as for improved access to its target receptor, Her2/neu, which is partly trapped in junctions. This directly translated in a better therapeutic efficacy of trastuzumab in xenograft models using Her2/neu-positive breast, gastric, and ovarian cancer cells. Furthermore, the combination of JO-1 with the EGFR-targeting mAb cetuximab (Erbitux) greatly improved the therapeutic outcome in a model of metastatic EGFR-positive lung cancer. A combination of JO-1/trastuzumab treatment with an additional approach that allows for the transient degradation of tumor stroma proteins resulted in complete tumor eradication. In addition to its clinical relevance for cancer treatment, this study also sheds light on the mechanism of adenovirus serotype 3 infection of epithelial cells.
Introduction
Monoclonal antibodies (mAbs) have emerged as a class of novel oncology therapeutics. To date, there are 21 marketed therapeutic mAbs for the treatment of cancer, with hundreds of more currently in clinical development. Among the marketed mAbs are trastuzumab (Herceptin) and cetuximab (Erbitux). Trastuzumab targets the human epidermal growth factor receptor 2 (Her2/ErbB-2). The receptor for cetuximab is the human epidermal growth factor receptor 1 (Her1/ErbB-1). Both receptors belong to the family of tyrosine kinase receptors and initiate signaling through several pathways which promote cell survival and proliferation (Harari et al., 2007). Trastuzumab is used as a first line therapy in Her2/neu positive breast cancer patients and has also been approved for metastatic Her2/neu positive gastric cancer. Current FDA-approved indications for cetuximab include colorectal, head and neck, lung, and pancreatic cancer (Wheeler et al., 2010). Most patients with early stage breast or colon cancer have a measurable tumor response to trastuzumab and cetuximab therapy. However, in patients with advanced or recurrent disease, the response rate to these mAbs is only 8% to 10% (Adams and Weiner, 2005).
The mechanisms of trastuzumab and cetuximab action include the activation of antibody-dependent or complement-dependent cytotoxicity, and interference with tyrosine kinase receptor signaling that is required for tumor cell survival (Wheeler et al., 2010). A unifying aspect among these mechanisms is that tumor cell growth inhibition is dependent on the binding of mAbs to their corresponding receptors. Therefore, molecules that prevent access and binding to the receptor, either by physically inhibiting intratumoral transport from blood vessels to malignant cells or masking of receptors, are predicted to block trastuzumab and cetuximab activity (Lesniak et al., 2009). Among these molecules are tumor stroma proteins such as collagen or laminin (Li et al., 2004). In a recent study, we demonstrated that transient degradation of these stroma proteins significantly improved trastuzumab therapy (Beyer et al., 2011).
In addition to obstacles formed by tumor stroma proteins, the epithelial phenotype of cancer cells also creates physical barriers to cancer therapy (Strauss and Lieber, 2009; Strauss et al., 2009). Several studies demonstrated that the expression or upregulation of epithelial proteins correlated with increased resistance to trastuzumab (Fessler et al., 2009) and cetuximab (Oliveras-Ferraros et al., 2011) therapy of breast and colorectal cancer, respectively. Epithelial cells maintain several intercellular junctions (tight junctions, adherens junctions, gap junctions, and desmosomes), a feature which is often conserved in epithelial cancers in situ and in cancer cell lines (Turley et al., 2008). Epithelial junctions are composed of adhesive dimers consisting of cadherin molecules derived from two neighboring cells (Koeser et al., 2003). Desmogleins 1, 2, and 3 (DSG1-3) and desmocollins 1, 2, and 3 (DSC1-3) are subclasses of cadherins. DSC2 and DSG2 are widely expressed and are found together in desmosomes of the basal layer of epithelial cells. The cytoplasmic tails of the desmosomal cadherins link the plasma membrane to the cytoskeleton through a complex of proteins, which include plakoglobin, desmoplakin, and plakophilins. Desmoglein 2 (DSG2) is overexpressed in a series of epithelial malignancies, including breast cancer (Wang et al., 2011b) (Suppl.
In Example 1, we demonstrated that a group of human adenoviruses (Ads) (Ad serotype 3, 7, 11, and 14) use DSG2 as a primary attachment receptor for the infection of cells. Importantly, in epithelial cells, Ad3 binding to DSG2 triggered activation of signaling pathways resulting in the transient opening of epithelial junctions. The opening of junctions was reflected by an increased transepithelial permeability and by the unmasking of proteins that are trapped in tight junctions, e.g., the coxsackie-adenovirus receptor or the zonula occludens-1 protein. The opening of the epithelial junctions was also achieved with recombinant subviral particles, such as the penton-dodecahedra (PtDd), consisting of 12 Ad3 fibers linked to their penton bases (Fuschiotti et al., 2006; Norrby et al., 1967). We subsequently generated a minimal Ad3-derived DSG2 ligand formed by two fiber knob domains. This protein, with a molecular weight of approximately 50 kDa, is produced in E. coli and can be easily purified. In a series of functional studies we demonstrated that this protein efficiently triggers the opening of the junction. In the following study, we therefore refer to this protein as junction opener-1 (JO-1).
In this study, we have partially delineated the in vivo mechanism of JO-1-mediated junction opening. We show that Her2/neu and EGFR are trapped in the intercellular junctions in xenograft tumors. JO-1 treatment greatly increased the permeation of mAbs in tumors and significantly increased the efficacy of trastuzumab and cetuximab therapy in a series of xenograft tumor models.
Results
JO-1 Triggers Opening of Epithelial Junctions.
In the examples above, we showed that Ad3 particles or recombinant Ad3 penton-dodecahedra (PtDd) (
The functional activity of JO-1 was tested on polarized colon cancer T84 cells. This demonstrated that incubation of cells with JO-1 triggered remodeling of epithelial junctions, as shown by confocal microscopy for Claudin 7 and DSG2 (
JO-1 Triggers Intracellular Signaling and Increases Penetration of mAb in Epithelial Tumors In Vivo.
A breast cancer xenograft model was used to study the effect of JO-1 on epithelial junctions in vivo. Human breast cancer HCC1954 cells were injected into the mammary fat pad of CB17-SCID/beige mice. The resulting tumors resembled the histology of breast cancer in humans (Li et al., 2004), i.e. tumors were vascularized and contained nests of epithelial cells glued together by epithelial junctions and surrounded by extracellular matrixes). When tumors reached a volume of ˜200 mm3, JO-1 was injected intravenously. JO-1 could be detected in the tumors by immunofluorescence microscopy as early as 1 hour post-injection. JO-1 accumulated in the tumors as is indicated by the increased immunofluorescence at 12 hours post injection (
In the examples above, it was found in in vitro studies that Ad3 binding to DSG2 of epithelial cells triggered intracellular signaling including pathways that are involved in epithelial-to-mesenchymal transition (EMT). EMT is a reprogramming process involved in embryonal development, but also in tumor metastasis. Among the feature that characterize EMT are decreased expression of epithelial markers, altered location of transcription factors, and activation of Erk1/2 (MAPK) (Turley et al., 2008). In our studies with xenograft tumors, we found less non-phosphorylated and phosphorylated forms of E-cadherin in tumors 12 hours after intravenous injection of JO-1 (
Overall, these studies indicate that JO-1 triggers activation of Erk1/2 pathways in vivo, in HCC1954 tumors. Based on this, we hypothesize that opening of epithelial junctions by binding of JO-1 to DSG2 involves at least two mechanisms: i) cleavage of the DSG2 ECD, and disruption of DSG2 dimers with subsequent internalization; and ii) induction of EMT-like events through the activation of Erk1/2. Next, we tested whether JO-1-triggered opening of epithelial junctions in tumors would increase the penetration of monoclonal antibodies into xenograft tumors. Trastuzumab, which is a humanized IgG1 mAb, was initially utilized for this study. As in previous studies, trastuzumab was injected intraperitoneally at a dose of 10 mg/kg. Trastuzumab was visualized in tumors with antibodies specific to human IgG. In tumor sections and Western blot analyses, trastuzumab was detectable 1 hour post-injection and at higher levels 12 hours after injection (
mAb Targets are Trapped in Epithelial Junctions.
In breast cancer xenograft sections and in cultured breast cancer cells, we found co-staining of Her2/neu and the adherens junction protein Claudin 7 (FIG. 19A,). Confocal microscopy of breast cancer BT474 cells confirmed the trapping of Her2/neu in lateral junctions. This is in agreement with an earlier study, demonstrating that Her2/neu is a basolateral protein that becomes accessible from the apical surface only when the tight junctions are disrupted (Vermeer et al., 2003). Incubation of the Her2/neu positive breast cancer cell lines BT474 (
Release of mAb receptors from trapping is supported by the enhanced killing of cancer cells by trastuzumab and cetuximab. In vitro killing of BT474 breast cancer and A549 lung cancer cells by trastuzumab and cetuximab, respectively, was inefficient (
JO-1 Improves Trastuzumab Therapy In Vivo.
JO-1's potential enhancement of trastuzumab therapy was first tested in an orthotopic breast cancer model based on Her2/neu positive BT474-M1 cells. JO-1 injection alone had no significant effect on tumor growth (
A second breast cancer model involved HCC1954 cells. Tumors derived from these cells are more resistant to trastuzmab (
To further consolidate the clinical relevance of JO-1 as a co-therapeutic for trastuzumab, we performed efficacy studies in a Her2/neu-positive gastric cancer (NCI-N87) model (
JO-1 Improves Cetuximab Therapy In Vivo.
A xenograft model with subcutaneous tumors derived from A549 lung cancer cells was initially utilized. Cetuximab treatment of mice with pre-established A549 tumors did not result in a significant delay of tumor growth when compared to treatment with PBS. JO-1 was injected intravenously or intraperitoneally followed by cetuximab 12 hours later. Both treatment approaches had a significant therapeutic effect and resulted in a decrease of tumor volumes (
Next, the co-therapy approach was tested in an orthotopic lung cancer model. To establish this model, A549 cells were injected intravenously. In this model, mice became morbid within 37 days of tumor cell transplantation with predominant tumor localization to the lung (
Combined Tumor Stroma Protein Degradation and Junction Opening.
We have recently shown in immunohistochemical studies of tumor sections from cancer patients and xenografts that the extracellular matrix proteins forming the tumor stroma tightly surround nests of malignant breast and colon cancer cells (Li et al., 2009). Transient degradation of tumor stroma proteins by intratumoral expression of the peptide hormone relaxin significantly enhanced trastuzumab therapy (Beyer et al., 2011). Here, we utilized the HCC1954 model to test whether additional transient tumor stroma protein degradation, would further increase the effect of JO-1 on trastuzumab therapy (
Effect in the Presence of Antibodies Against JO-1 on In Vitro Cytotoxicity Assays.
Ad3 is a widely distributed pathogen. As such, JO-1 is a viral protein, which can potentially be immunogenic, and can interfere with its therapeutic activity after repeated administration. Ten out of 30 serum samples from breast cancer patients tested positive for neutralizing Ad3 antibodies (data not shown). This is in agreement with reports that the serum prevalence for neutralizing anti-Ad3 antibodies in humans (by age 10) is ˜40% (Sakamoto et al., 1995). We show, however, that the anti-Ad3 antibody-positive human sera did not react with JO-1 (data not shown). This is not surprising as most neutralizing antibodies are directed against the Ad hexon (Sumida et al., 2005). To obtain anti-JO-1 positive serum, mice were vaccinated with JO-1 (data not shown). In in vitro cytotoxicity studies, no interference was found of the anti-JO-1 antibodies with the enhancing effect of JO-1 on trastuzumab killing of Her2/neu-positive BT474 cells (data not shown). Along this line, pooled human serum from healthy volunteers or breast cancer patients also did not interfere with the function of JO-1.
Discussion
JO-1 as New Co-Therapeutic.
Despite their success in many areas, the therapeutic efficacy of mAbs are limited, with only a minority of patients responding to these agents as monotherapies. The action of many anti-cancer mAbs, including trastuzumab and cetuximab, involve binding to the corresponding target receptors on tumor cells. Target receptor masking or preventing intratumoral dissemination of mAbs is a potential shielding and escape mechanism for cancer cells (Lesniak et al., 2009). In addition to the barriers formed by tumor stroma proteins, such as collagen or laminin, the epithelial phenotype of epithelial cancer also creates obstacles to mAb therapy. A recent study reported that the expression of epithelial features such as mucin 1 correlated with trastuzumab resistance (Fessler et al., 2009). Furthermore, the upregulation of E-cadherin contributed to resistance to cetuximab therapy of colorectal cancer (Oliveras-Ferraros et al., 2011).
While it has long been known that tumor invasion and metastasis is accompanied by EMT, recent studies have shown that this process can be reversed by a mesenchymal-to-epithelial transition (MET) (Thiery and Sleeman, 2006). Regaining epithelial features, including epithelial junctions, appears to represent a protection mechanism for the tumor that also shields it from anti-cancer treatment (Brennan et al., 2010; Strauss et al., 2009; Thiery and Sleeman, 2006). In this context, we found in studies on primary ovarian cancer cells that acquisition of chemoresistance and the formation of putative cancer stem cells involved processes reminiscent of MET (Strauss et al., 2011).
We hypothesized that an approach that allows for a transient dissolution of epithelial junctions could improve the therapeutic efficacy of mAbs such as trastuzumab and cetuximab. In the context of basic adenovirological studies, we designed the small recombinant protein JO-1. JO-1 binding to DSG2 resulted in the transient opening of tight junctions. This increased the penetration of trastuzumab in the tumor and allowed for better access to mAb target receptors, which, in turn, facilitated mAb therapy in a series of xenograft models involving human epithelial tumor cells. Potentially, the combination of JO-1 with trastuzumab and cetuximab might allow the reduction of the effective dose of these mAbs, thereby reducing critical side effects, i.e. trastuzumab-associated cardiotoxicity and acne-like rashes that often occur during cetuximab therapy.
JO-1 is an attractive therapeutic for development because it can be produced in high yields in E. coli. Notably, DSG2 ligands that are not able to cluster DSG2, such as monoclonal antibodies against DSG2, are inefficient in junction opening in the tumor models used in this study. To our knowledge, there is no other therapy in development that binds to DSG2, and is able to sensitize tumors to mAbs. A series of pathogens have evolved mechanisms to disrupt junction integrity. For example, Vibrio cholerae strains produce Zona occludens toxin (Zot), which possesses the ability to reversibly alter intestinal epithelial junctions, allowing the passage of macromolecules through mucosal barriers (Fasano et al., 1991). A Zot-derived hexapeptide (AT-1001) has been developed, however clinical testing in patients with coeliac disease was recently stopped due to safety concerns and the lack of efficacy.
Mechanisms of Action.
Our data suggest that JO-1 triggers junction opening in epithelial tumors through two, potentially connected mechanisms: the disruption of DSG2 dimers in intercellular junctions and/or intracellular signaling that leads to a decrease of E-cadherin and potentially other junction proteins. Because cadherins form dimers between neighboring cells, a number of pathogens have evolved mechanism to trigger cleavage within the extracellular domain of cadherins. Candida albicans, an organism able to invade the bloodstream via the gastrointestinal tract, binds to E-cadherin and triggers cleavage of both intra- and extracellular domains of E-cadherin, thereby destabilizing the homotypic interactions between adjacent epithelial cells (Frank and Hostetter, 2007). Furthermore, Bacteroides fragilis enterotoxin and Porphyromonas gingivalis gingipains have been associated with the degradation of E-cadherin (Katz et al., 2000; Wu et al., 1998). Both of these studies attributed the cleavage of E-cadherin to a specific bacterial protease. Our data suggest that Ad3 uses a similar mechanism to breach the epithelial barrier in the respiratory tract. We are currently studying the mechanisms of DSG2 cleavage. In ongoing studies, we are attempting to determine whether JO-1 binding triggers conformational changes within DSG2. Dissolution of DSG2 dimers might in turn destabilize more apically localized tight junctions as discussed recently (Koeser et al., 2003).
Based on in vitro studies above, Ad3 binding to DSG2 triggers the activation of Erk1/2 and other pathways involved in EMT. This study shows that Erk1/2 is activated in tumors within 1 hour after intravenous JO-1 injection. Our finding is in agreement with previous reports showing that cadherins engage in bidirectional signaling with the receptor tyrosine kinases to regulate intercellular junctions (Klessner et al., 2009). We are currently generating various DSG2 knockout models and truncated/mutated DSG2 variants to better delineated the role of DSG2 in maintaining epithelial junctions.
Side Effects on Normal Epithelial Tissues.
In humans and non-human primates, DSG2 is expressed in the gastro-intestinal and respiratory tracts (Li et al., 2011). As the mouse orthologue of DSG2 is not recognized by Ad3 or JO-1 (Wang et al., 2011b), safety studies in normal mice are relatively inadequate. We have therefore generated transgenic mice containing the human DSG2 locus. The expression pattern and level of human DSG2 in these animals were similar to those found in humans. Furthermore, we showed that JO-1 binding to human DSG2 in transgenic mouse epithelial cells triggered junction opening to a degree similar to data observed in human cells. In preliminary studies with DSG2-transgenic mice we did not find critical side effects of intravenous JO-1 injection (2 mg/kg) (Li et al., 2011). We speculate that DSG2 in normal epithelial cells is not readily accessible to intravenously applied JO-1. On the other hand, greater leakage of tumor-associated blood vessels and the lack of strict cell polarization might make epithelial tumors more responsive to JO-1. Lack of toxicity after intravenous injection of JO-1 ligands is also underscored by studies with adenoviruses containing Ad3 fibers. These viruses bind to DSG2 and act on junctions the same way as JO-1. Intravenous injection of Ad5/3 or Ad3 oncolytic vectors into humans was found to be safe (Hemminki et al., 2010; Koski et al., 2010). We are currently conducting detailed toxicity studies in non-human primates and DSG2-transgenic mice to validate the potential of JO-1 as a safe therapeutic.
JO-1 Immunogenicity.
As JO-1 is a viral protein, adaptive immune responses might develop in humans, particularly after repeated injection. This might, however, not be a clinical problem. The first-line treatment of Her2/neu-positive metastatic breast cancer is trastuzumab in combination with chemotherapeutic drugs (doxorubicin, cyclophosphamide, paclitaxel, or docetaxel). Also, cetuximab is used in combination with chemotherapeutics (vinorelbine plus cisplatin) in non-small cell lung cancer patients. It is likely that the immunosuppressive chemotherapy prevents immune responses against JO-1. Again, this expectation is supported by studies with oncolytic adenovirus vectors. In these studies, immunosuppression allowed for repeated vector application (Thomas et al., 2008). On the other hand, it would be beneficial if JO-1 could be used in immunocompetent cancer patients. For example, JO-1 can theoretically act as an immunostimulatory monotherapy by increasing the access and intratumoral penetration of pre-existing or transplanted anti-tumor T-cells. Tumor-specific T-cells are frequently found in untreated breast cancer patients, even at late stages of disease, and transfer of these cells into mice has been shown to reject xenotransplanted autologous tumors (Beckhove et al., 2004; Disis et al., 1994; Disis et al., 2000; Feuerer et al., 2001; Toso et al., 1996). Therefore, we started to investigate in more detail JO-1 immunogenicity. Despite the fact that approximately one third of humans have neutralizing antibodies against Ad3, our studies showed these antibodies did not interact with JO-1 (Suppl.
In agreement with other studies (Wang et al., 2011b), (Biedermann et al., 2005), (Harada et al., 1996), (Schmitt et al., 2007), (Trojan et al., 2005), (Abbod et al., 2009), we found a higher DSG2 expression in malignant tissues than in the surrounding normal epithelial tissue. Furthermore, a recent study on squamous cell cervical cancer demonstrated that in contrast to all other desmosomal proteins, DSG2 was up-regulated in invasive cancer (Kurzen et al., 2003). There are, however, also studies reporting a reduction in the amounts of DSG2 in invasive pancreatic or gastric cancer (Ramani et al., 2008; Yashiro et al., 2006). The latter, as well as the finding that JO-1 triggers EMT-like signaling, might raise the question of whether JO-1 would facilitate metastasis. Notably, in all models used in this study, we did not see stimulation of tumor growth or macroscopic/microscopic signs of metastasis in animals treated with JO-1 alone. Tumor invasion and metastasis requires more than transient activation of EMT pathways. Detachment from epithelial cancers and migration of tumor cells is only possible after long-term crosstalk between malignant cells and the tumor microenvironment, resulting in changes in the tumor stroma and phenotypic reprogramming of epithelial cells into mesenchymal cells (Guarino, 2007).
In summary, the epithelial junction opener JO-1 has the potential to improve mAb therapies of cancer both in term of efficacy and safety, i.e. by allowing lower therapeutic mAbs doses. This study also sheds light on the mechanisms of Ad3 infection of epithelial cells.
Material and Methods
Proteins and Antibodies.
The generation of JO-1 (also known as Ad3-K/S/Kn) has been described previously (Wang et al., 2011a). JO-1 was produced in E. coli using the pQE30 expression vector (QIAGEN™, Valencia, Calif.) and purified by Ni-NTA agarose chromatography as described elsewhere (Wang et al., 2007). Recombinant Ad3 penton-dodecahedral (PtDd) protein complexes were produced in insect cells and purified as described elsewhere (Fender et al., 1997).
The following antibodies were used for immunofluorescence studies or Western blot: polyclonal goat anti-DSG2 (R&D Systems, Inc., Minneapolis, Minn.), mouse mAb anti-DSG2 (clone 6D8) (Cell Sciences, Canton, Mass.), rabbit anti-Claudin 7 (ABCAM™, Cambridge, Mass.), anti-human IgG-FITC (Santa Cruz), rabbit anti-EGFR (ABCAM™), mouse anti-Her2/neu (ABCAM™), mouse anti-E-cadherin (Cell Signaling), mouse anti-human IgG Fc (R&D Systems), mouse mAb against phospho-p44/42 MAPK (Erk1/2)(Thr202/Tyr204) (Cell Signaling), mouse anti-Erk1/2 (Cell Signaling), mouse anti-phospho-E-cadherin (pS838/840) (Epitomics, Inc.), mouse anti-6-His (QIAGEN™)
Cell Lines.
BT474-M1 cells were cultured in DMEM/F12 with 10% FBS, 1% Pen/Strep and 2 mM L-Glutamine. HCC1954 breast cancer, A549 lung cancer and NCI-N87 cells were cultured in RMPI with 10% FBS and 1% Pen/Strep. SKOV3-ip1 cells were cultured in MEBM medium. Colon cancer T84 cells were cultured in a 1:1 mixture of Ham's F12 medium and DMEM, with the addition of 10% FBS, Glu and Pen/Strep. To achieve cell polarization, 1.4×105 T84 cells were cultured in collagen-coated 6.5 mm TRANSWELL™ inserts (0.4 μm pore size) (Costar TRANSWELL™ Clears) for a period of 14 to 20 days until transepithelial resistance was stable (Wang et al., 2011b).
Immunofluorescence Analyses.
Cells were cultured in chamber glass slides (BD Falcon), washed twice with ice-cold PBS and then fixed with methanol/acetone (1:1 vol/vol) for 15 min at 4° C., or with 4% paraformaldehyde for 30 min at 4° C. Cells were washed twice with PBS after fixation and subsequently blocked with PBS containing 2% dry-milk powder (BIORAD™) for 20 min at room temperature. Antibodies were diluted in PBS (Claudin7 1:100, DSG2 1:100, Her2/neu 1:50, EGFR 1:50, E-cadherin 1:100, Human IgG 1:400, phospho-p44/42 MAPK 1:400, Penta-His 1:500) and cells were stained for 90 min at room temperature or at 4° C. overnight. When required, suitably directed secondary antibodies directed were applied after 3 washes with PBS for a period of 30 min at room temperature. Glass slides were mounted using VECTASHIELD with DAPI (VECTOR LABS™). Photographs were obtained with a Leica DFC300FX digital camera. Confocal images were taken on a ZEISS™ META Confocal Microscope using 40× or 100× oil lenses and ZEISS™ 510 software (ZEISS™ Microlmaging, Thornwood, N.Y.).
Tissues.
Paraffin sections of human breast cancers were deparaffinized and rehydrated. Antigen retrieval was performed with Vector Antigen Unmasking solutions pH 6.0 (VECTOR LABORATORIES™, Inc., Burlingame, Calif.). Mouse anti-DGS2 antibody (3G132) (ABCAM™) was diluted 1:10. Immunohistochemistry was performed using the Polink-2 HRP Broad Kit (Golden Bridge International, Inc. Mukilteo, Wash.) and DAB as a substrate.
Western Blot.
Xenograft tumor tissue was dissected, manually homogenized (tissue disruptor) and incubated for 30 min in protein lysis buffer [20 mM Hepes (pH 7.5), 2 mM EGTA, 10% glycine, 1% TritonX100, 150 mM NaCl (all from SIGMA-ALDRICH™), and protease/phospatase inhibitors (Complete Protease Inhibitor Cocktail and PhosSTOP, Roche)] on ice. Confluent cultured cells were washed twice with ice-cold PBS and then lysed for 30 min in protein lysis buffer on ice. Samples were pelleted (10 min, 4° C., 15,000 RPM, and the protein containing supernatant stored at −80° C. A total of 80 μg of total protein was used for the Western blot procedure. Protein samples were boiled (5 min at 95° C.) and separated by polyacrylamide gel electrophoresis (PAGE) using 10% Bis-Tris gels and MOPS buffer (Novex, INVITROGEN™), followed by transfer onto nitrocellulose membranes according to the supplier's protocol (iBlot, INVITROGEN™). Membranes were blocked in PBS containing 0.1% Tween20 (PBS-T, Sigma) and 5% dry milk powder. Incubation times for primary and secondary antibodies were 16 h at 4° C. and 1 h at room temperature, respectively. Antibodies were diluted [Human IgG 1:1000, DSG2 1:500, Ad3-K serum 1:2000, p-E-cadherin 1:2000, E-cadherin 1:1000, Claudin7 1:500, Vimentin 1:2000, phospho-p44/42 MAPK (Erk1/2) 1:2000, Erk1/2 1:2000] in PBS-T and 5% dry-milk powder. Membranes were washed 5 times in PBS-T between antibody incubations, and films were developed using ECL plus (AMERSHAM™) or the Odyssey system (LI-COR BIOSCIENCES™)
DSG2 mRNA PCR:
Reverse transcription was performed on 1 μg of total RNA using the Quantitect Reverse Transcription Kit (QIAGEN™) to evaluate the expression levels of DSG2 in breast cancer. The expression of the housekeeping gene GAPDH was used as reference. The quantitative PCR (qPCR) was run in triplicates using the SensiMix SYBR Kit (Quantace) on a 7900HT Fast Real-Time PCR System (APPLIED BIOSYSTEMS™/LIFE TECHNOLOGIES™). The following primers were used:
The qPCR was performed under the following conditions: after an initial 10 min enzyme activation step at 95° C., 40 amplification cycles were run, each consisting of 95° C. for 15 s and 60° C. for 1 min. Lastly, a final elongation step was performed for two minutes at 60° C.
Permeability Assay.
A total of 5×105 T84 cells were seeded on 12 mm TRANSWELL™ inserts [PET membrane, with 0.4 m pore size (Corning, N.Y.)] and cultured for 14-20 days until transepithelial resistance was stable. Culture medium was changed every 2-3 days. The cells were exposed to DSG2 ligands (20 g/ml) in adhesion medium (DMEM, 1% FBS, 2 mM MgCl2, 20 mM HEPES) for 15 min at room temperature. Subsequently, 1 mCi of [14C] polyethylene glycol-4000 (PEG-4000) (PERKIN ELMER™, Covina Calif.) diluted with DMEM/F12 medium, was added to the inner chamber. Medium aliquots were harvested from the inner and outer chambers at 15 and 30 min and measured by a scintillation counter. Permeability was calculated as described elsewhere (Yang et al., 2004).
Trastuzumab and Cetuximab Cytotoxicity Assay.
BT474 or A549 cells were plated at a density of 5×104 cells/well in triplicate in 96-well plates and grown to confluence. JO-1 (500 ng/ml) was added to the medium. 12 h later, trastuzumab or cetuximab (15 g/ml) were added and cell viability was measured 2 h later by WST-1 assay (ROCHE™, San Francisco, Calif.). Three independent studies were performed.
Electron Microscopy.
Polarized cells in the TRANSWELL™ chambers were fixed with half-strength Karnovsky's fixative (2% paraformaldehyde, 2.5% glutaraldehyde, and 0.2 M Cacodylate buffer) for 1 h at room temperature. The fixative in the inner chamber contained 0.2% ruthenium red. The ruthenium red [Ruthenium(III)chloride oxide, ammoniated], was purchased from Alfa Aesar (Ward Hill, Mass.). Post-fixation was performed with 1% OsO4-phosphate buffer. The membranes were cut out from the TRANSWELL™ chambers and embedded in Medcast (Ted Pella, Redding, Calif.). Ultra-thin sections were stained with uranyl acetate and lead citrate. Processed grids were evaluated with a JEOL JEM1200EXII transmission electron microscope. Images were acquired with an Olympus SIS Morada Digital CCD camera using iTEM software for image processing.
Animal Studies:
All experiments involving animals were conducted in accordance with the institutional guidelines set forth by the University of Washington. Mice were housed in specific-pathogen-free facilities. Breast cancer xenografts were established by injecting 4×106 cancer cells into the mammary fat pad of CB17 SCID-beige mice. Trastuzumab was injected intraperitoneally (i.p.) at a dose of 10 mg/kg. PtDd or JO-1 was given intravenously (i.v.) at a dose of 2 mg/kg. Tumor volumes were measured as described previously (Tuve et al., 2007). Mice were sacrificed when the tumor volume reached 1,000 mm3 or ulcerated. Lung cancer xenografts were established by injecting 4×106 A549 subcutaneously (s.c.) into the right flank of CB17 SCID beige mice. Cetuximab was injected at 10 mg/kg i.p. 10 h after JO-1 i.v. injection at day 12 and repeated after 3 days. For the disseminated lung tumor model, eight week old male CB17 SCID-beige mice were intravenously injected with 2×106 A549 cells on day 1. The treatment with JO-1 (2 mg/kg i.v) and cetuximab (10 mg/kg i.p.) was started at day 10 and repeated every three days. Animals were monitored for weight loss and signs of dyspnea. Animals were sacrificed when the first mouse of the non-treatment group was moribund. India ink (15% in PBS) was injected intratracheally prior to the removal of the lungs. Metastases appeared unstained (white) against the black normal tissue. To produce anti-JO-1 antibodies, mice were injected subdermally with JO-1 mixed with Freund's adjuvant as described elsewhere (Li et al., 2009).
HSC-based Relaxin Expression:
The protocol has been described elsewhere (Beyer et al., 2011). Briefly, transplant recipients were 6- to 10-week old, female CB17 SCID-beige mice, sublethally irradiated with 350 cGy immediately before tail vein injection with 6×105 lentivirus vector-transduced bone marrow cells from 5-FU-treated mice. After engraftment of cells in the recipients' bone marrow was confirmed, a total of 4×106 HCC1954 were injected into the mammary fat pad. The lentivirus vector expressing relaxin under the control of doxycycline (Dox) has been described previously (Beyer et al., 2011).
Neutralizing Antibody Assay:
Serum samples from cancer patients were taken before patients underwent chemotherapy. Briefly, 293 cells were plated in 96-well plates at 4×104 cells per well and incubated at 37° C. The following day, serum samples were heat inactivated at 56° C. for 30 min and serially diluted from 1:2 to 1:1,204 in MEM containing 2% FCS. A total of 20 plaque forming units (PFU) per cell of wild-type Ad5 or Ad11 in 10 μl of MEM, was incubated with 100 μl of each serum dilution for 1 h at 37° C. Medium was removed from cells plated the previous day, and 55 μl of virus-containing serum was added to the cells along with 45 μl of 293 growth medium. A further 100 μl of 293 growth medium was added to the cells at 3 and 6 days postinfection. At 8 days postinfection, cells were analyzed for the presence of cytopathic effect (CPE), and serum samples were scored positive for the presence of neutralizing antibodies if no CPE was seen at a dilution of 1:2 or higher.
Statistical Analysis:
All results are expressed as mean+/−SD. Student's t-test or 2-Way ANOVA for multiple testing, were applied when applicable. A p-value <0.05 was considered significant.
This application claims the benefit of U.S. Provisional Application No. 61/353,652, filed Jun. 10, 2010, U.S. Provisional Application No. 61/430,091, filed Jan. 5, 2011, and U.S. Provisional Application No. 61/470,663, filed Apr. 1, 2011, all of which are incorporated herein by reference in their entirety.
This invention was made with U.S. Government support under R01CA080192 and RO1 HLA078836 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 7560527 | Legrand et al. | Jul 2009 | B1 |
| Number | Date | Country |
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| 9941359 | Aug 1999 | WO |
| 0042208 | Jul 2000 | WO |
| 2005040333 | May 2005 | WO |
| WO 2005040333 | May 2005 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20140107014 A1 | Apr 2014 | US |
| Number | Date | Country | |
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| 61470663 | Apr 2011 | US | |
| 61430091 | Jan 2011 | US | |
| 61353652 | Jun 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13158246 | Jun 2011 | US |
| Child | 14066353 | US |
