WO2011156637A1 - Pphox and rubicon peptides and methods of use - Google Patents

Abstract

This disclosure provides compositions and method of diminishing or inhibiting reactive oxygen species (ROS) production by administering a p22phox peptide, retro-inverso or an equivalent thereof that includes amino acids 3-10 which competitively binds Rubicon but does not activate ROS-producing NADPH oxidase complex.

Description

PPHO AND RUBICON PEPTIDES AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 1 19(e) of U.S.

Provisional Application Nos. 61/393,302, filed October 14, 2010, and 61/353,632, filed June 10, 2010, the contents of which are hereby incorporated by reference in their entirety into the present disclosure.

FIELD OF THE INVENTION

[0002] The present invention relates to the use of p22phox and Rubicon peptides to regulate phagocytosis and autophagy.

BACKGROUND OF THE INVENTION

[0003] Phagocytosis and autophagy cooperate together as part of the host's first- line of immune defense against microbial invasions. Specifically, the reactive oxygen species (ROS)-producing NADPH oxidase complex of phagocytes, containing the integral membrane protein p22p/?o as a common subunit, translocates to the phagosomal membrane upon microbial infection to produce ROS critical for the elimination of invading microbes. Autophagy facilitates phagocytosis by promoting phagosome maturation and rapid acidification, and by preventing pathogens from escaping into the cytosol, but details of how autophagy machinery directly couples with and facilitates phagocytosis remains elusive.

[0004] Overproduction of ROS leads to various diseases and conditions such as acute inflammatory disease, influenza-induced acute lung injury, asthma, sepsis, hypertension or atherosclerosis.

[0005] Autophagy is an active homeostatic degradation process of removal or turnover of cytoplasmic components from a cell. Misregulation of autophagy is also implicated in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and transmissible spongiform encephalopathies.

Autophagy is also implicated in the process of carcinogensis and pathogen infection.

For example, autophagy-like structures appear after viral infection, and in the case of herpes simplex virus, autophagy is induced following infection through the activation of a double-stranded RNA-activated protein kinase R. However, during viral replication, autophagy is not induced, because the protein kinase R that induces autophagy is inactivated. It is also suggested that microbial virulence may be determined in part by the ability of pathogens to successfully antagonize host autophagy. Consequently, induction of autophagy is a promising strategy for eliminating replicating intracellular viruses.

SUMMARY

[0006] Applicants have discovered that the amino acids 3-10 of p22phox are sufficient for binding to Rubicon. Peptides that include amino acids 3-10 but do not include the C-terminal half of the p22phox protein which is responsible for binding gp91 phox_^ therefore, can compete with endogenous p22phox in binding to Rubicon, thereby inhibiting p22p/?ox-Rubicon interaction and thus inhibiting Rubicon-mediated activation of the reactive oxygen species (ROS)-producing NADPH oxidase complex.

[0007] Applicants have also discovered the direct crosstalk between autophagy and phagocytosis machineries by demonstrating that the Run/cysteine-rich- domaincontaining Beclinl -interacting autophagy protein (Rubicon) is an essential positive regulator of the NADPH oxidase complex, inducing ROS production upon microbial infection or plasma membrane Toll-like receptor (TLR) activation. While Rubicon primarily associates with the Beclinl -UVRAG-containing autophagy complex under normal and stressed conditions, it periodically interacts with the p22p/?ox of NADPH oxidase complex, upon microbial infection or TLR2/4 activation, facilitating the stabilization and phagosomal translocation of the p22p/?ox-NADPH oxidase complex to induce a ROS burst, inflammatory cytokine production, and thereby, potent anti-microbial activities. Consequently, the expression or depletion of the Rubicon gene in macrophage cell lines, primary bone marrow-derived macrophages and mice profoundly affected ROS and inflammatory cytokine production, and subsequent anti-microbial activities.

[0008] It's also been discovered by the Applicants that Rubicon's actions in the Beclinl -UVRAG-containing autophagy complex and in the p22p/?ox-containing NADPH oxidase complex are functionally and genetically separable. These results indicate that Rubicon orchestrates two ancient innate immune machineries, autophagy and phagocytosis, toggling between the two depending on the environmental stimuli, ultimately generating an optimal intracellular immune milieu against microbial infection.

[0009] GST pulldown (GST-PD) studies showed that Beclinl and p22phox efficiently bound the central coiled-coil domain (CCD, aa505-557) and the serine-rich region (SR, aa558-625) of Rubicon, respectively. The ACCD mutant induced NADPH oxidase activity, ROS production, pro-inflammatory cytokine production, and bacterial killing activity as strongly as wildtype (WT). In striking contrast, the expression of the ASR or ACDD/SR mutants led to the suppression of NADPH oxidase activity and pro-inflammatory cytokine production with no effect on bacterial killing activity under the same conditions, suggesting that its p22p/?ox-binding activity, and not its Beclinl -binding activity, is essential for Rubicon-mediated induction of a robust anti-bacterial phagocytic response. Expression of the Rubicon WT or the ASR mutant, both capable of binding Beclinl , showed increased LC3-II and p62 protein levels upon rapamycin or starvation treatment, the indications of the suppression of autophagosome maturation step. In contrast, expression of the ACCD mutant, which lost its Beclinl binding ability, showed no effect on autophagosome maturation as evidenced by similar levels of LC3-II and p62 protein compared to those of vector control cells.

[0010] Accordingly, peptide fragments of Rubicon that either include the p22phox binding sequence (SR) or the Beclinl binding sequence (CCD) can affect the ROS production and autophagy pathways independently, respectively, without interfering with the other. More specifically, a peptide fragment of Rubicon that includes SR but not CCD (e.g. ACCD), can be used to activate ROS production. Likewise, a peptide fragment of Rubicon that includes CCD but not SR (e.g., ASR) can bind Beclinl and inhibits autophagy. As cancer cell may rely on autophagy for survival, the peptide fragment that includes CCD can be useful in suppressing cancer cell growth.

[0011] Thus, in one embodiment, the present disclosure provides an isolated or recombinant peptide comprising, or alternatively consisting essentially of, or yet alternatively consisting of, amino acids 3-10 of SEQ ID NO: 1 or an equivalent or retro-inverso thereof, wherein the peptide does not bind gp91 phox. In one aspect, the peptide does not include the C-terminal half of SEQ ID NO: 1 .

[0012] In one aspect, the peptide further comprises a transduction domain, e..g, a cell penetrating peptide. In one aspect, the cell penetrating peptide comprises a TAT peptide. In another aspect, the peptides with or without the transduction domain is less than or equal to 30 amino acids long. Alternatively, these peptides are less than or equal to 22 amino acids long, and in a particular aspect, the peptides are 8 amino acids long. In one aspect, the peptide comprises SEQ ID NO: 8.

[0013] In another embodiment, the present disclosure provides a polynucleotide that encodes the isolated or recombinant peptide of any of the above embodiments or a complement thereof, as well as equivalents of the polynuceotides or

complements..

[0014] Further provided is an antibody that specifically recognizes the peptides as described above, e.g., amino acids 3-10 of SEQ ID NO: 1 or an equivalent or retro- inverso thereof. In one embodiment, the antibody is a monoclonal antibody. In another aspect, the antibody is a humanized antibody. The antibody can be of any appropriate species, e.g., mammalian and more specifically murine or human.

[0015] The present disclosure, in one embodiment, provides a method for inhibiting production of reactive oxygen species in a cell or a tissue, comprising, or alternatively consisting essentially of, or yet alternatively consisting of, contacting the cell or the tissue with an agent that inhibits the interaction between p22phox and Rubicon, thereby inhibiting production of reactive oxygen species in the cell or the tissue. The contacting can be in vivo or in vitro. In one aspect, the cell is

macrophage cell. The cell can be of any appropriate species, e.g., mammalian and more specifically human.

[0016] Further provided, in one embodiment, is a method for treating a condition in a subject in need thereof wherein the condition is one or more of an acute

inflammatory disease, influenza-induced acute lung injury, asthma, sepsis, hypertension or atherosclerosis, the method comprising, or alternatively consisting essentially of, or yet alternatively consisting of, administering to the subject an effective amount of an agent that inhibits the interaction between p22phox and Rubicon, thereby treating the condition in the subject. The subject can be of any appropriate species, e.g., mammalian and more specifically human.

[0017] The present disclosure also provides the agents that inhibit the interaction between p22p/?ox and Rubicon, including but not limited to:

(a) an isolated or recombinant peptide of any of the above embodiments,

(b) a polynucleotide of any of the above embodiments,

(c) a cell of any of the above embodiments,

(d) an antibody directed to p22phox,

(e) an antibody of any of the above embodiments

(f) an antisense or siRNA directed to p22phox, or

(g) a small molecule inhibitor of Rubicon-p22p/?ox binding.

[0018] In another embodiment, the present disclosure provides use of an agent that inhibits the interaction between p22phox and Rubicon for the manufacture of a medicament for treating a condition in a subject in need thereof wherein the condition is one or more of an acute inflammatory disease, influenza-induced acute lung injury, asthma, sepsis, hypertension or atherosclerosis.

[0019] Also provided is a method of identifying an agent that inhibits the interaction between p22p/?ox and Rubicon, comprising, or alternatively consisting essentially of, or yet alternatively consisting of, contacting a candidate agent with a cell under a condition that stimulates production of reactive oxygen species, wherein a reduction of production of reactive oxygen species and an increased free p22p/?ox or Rubicon level compared to a suitable control indicates that the candidate agent inhibits the interaction between p22p/?ox and Rubicon.

[0020] In one embodiment, the present disclosure provides an isolated or recombinant peptide comprising, or alternatively consisting essentially of, or yet alternatively consisting of, a Rubicon variant wherein the variant comprises a p22p/?ox-binding sequence and does not have a Belcinl -binding sequence. In one aspect, the Rubicon variant is a Rubicon sequence having a deletion of the Belcinl - binding sequence. [0021] The present disclosure, in yet another embodiment, provides a method for inhibiting production of reactive oxygen species in a cell or a tissue, comprising, or alternatively consisting essentially of, or yet alternatively consisting of, contacting the cell or the tissue with a Rubicon variant or a polynucleotide encoding the Rubicon variant, thereby inhibiting production of reactive oxygen species.

[0022] In one embodiment, the present disclosure provides a method for treating infection in a cell or a tissue, comprising, or alternatively consisting essentially of, or yet alternatively consisting of, contacting the cell or the tissue with a Rubicon variant or a polynucleotide encoding the Rubicon variant, thereby treating infection in the cell or the tissue.

[0023] In one embodiment, the present disclosure provides an isolated or recombinant peptide comprising, or alternatively consisting essentially of, or yet alternatively consisting of, a Rubicon variant wherein the variant comprises a Belcinl -binding sequence and does not have a p22p/?ox-binding sequence.

[0024] The present disclosure, in yet another embodiment, provides a method of inhibiting autophagy in a cell or a tissue, comprising contacting the cell or the tissue with a Rubicon variant that includes a Belcinl -binding sequence and does not have a p22p/?ox-binding sequence or a polynucleotide encoding the variant or a cell transformed with each thereof, thereby inhibiting autophagy in the cell or the tissue.

[0025] Also provided is a method of suppressing growth of a cancer cell, comprising contacting the cancer cell with a Rubicon variant that includes a Belcinl - binding sequence and does not have a p22p/?ox-binding sequence, a polynucleotide encoding the variant or a cell transformed with each thereof, thereby suppressing growth of the cancer cell.

[0026] The present disclosure, in yet another embodiment, provides a method of identifying an agent that inhibits the interaction between Beclinl and Rubicon, comprising contacting a candidate agent with a cell under a condition that stimulates autophagy, wherein a reduction of autophagy compared to a suitable control indicates that the candidate agent inhibits the interaction between Beclinl and Rubicon. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 , panels a through h, show Rubicon interaction with the p22phox- gp91 phox NADPH oxidase complex, (a) Differential interactions of Rubicon with the Beclinl -containing autophagy complex and the p22p/?ox-gp91 phox complex.

Raw264.7 cells containing vector or Flag-Rubicon were labeled with S³⁵-Met/Cys for 6h, and stimulated with zymosan (100 pg/ml) or rapamycin (2 μΜ) for the indicated times, followed by IP with aFlag for autoradiography. Specific proteins are labeled with arrows, (b) Rubicon interaction with p22phox. At 48 h posttransfection with Flag- Rubicon (left) or Flag-p22p/?ox (right), Raw264.7 cells were stimulated with or without zymosan for 30 min and lysates were used for immunoprecipitation (IP), followed by immunoblotting (IB) as indicated, (c) Binding mapping, (top) Schematic diagram of Rubicon and p22phox. S-R, serinerich; CCD, coiled-coil domain; C-R, cystein-rich; and TM, transmembrane, (bottom) At 48 h posttransfection with mammalian GST or GST-Rubicon constructs together with V5-p22p/?ox (left) or V5- Beclinl (middle) or GST or GST-p22p/?ox constructs together with AU1 -Rubicon (right), HEK293T cells were used for GST pulldown, followed by IB with aV5, aGST or aAU1 . (d) Differential Rubicon interactions. Raw264.7 cells containing vector, Flag-Rubicon WT or its mutant were stimulated with zymosan for the indicated times, followed by IP with aFlag and IB with aBeclinl , aUVRAG, agp91 phox, ap22p/?ox or aFlag. The right panels show the densitometry results of all three independent co-IP assays, (e) Increased levels of p22phox and gp91 phox by Rubicon expression. THP- 1 cells containing vector, Rubicon WT or ASR were stimulated with or without zymosan and used for flow cytometry analysis to detect intracellular p22phox (left) or surface expressing gp91 phox (right), (f) Expression or depletion of Rubicon affects the colocalization of p22phox with L. monocyfogenes-containing phagosomes. At 48 h postinfection with lenti-GFP or lenti-GFP-Rubicon (left) at MOI=100 or with lenti- NS-shRNA or lenti-Rubicon-shRNA (right) at MOI=50, Raw264.7 cells were infected with heatkilled (HK)-TRITC-labeled L. monocytogenes (MOI=1 ) for 30 min, followed by confocal microscopy with Ap22phox. Images are representative of three

independent experiments. Bar, 2 μηη. (g and h) Rubicon enhances the co- localization of p22p/70x with L. monocytogenes or zymosan particles-containing phagosomes in a p22p/?oxbinding-dependent manner. Images are representative of three independent experiments, (g) Raw264.7 cells containing vector, Rubicon WT, ACCD or ASR were infected with HK-GFP-L monocytogenes for 30 min, followed confocal microscopy with ap22phox or aFlag. Bar, 2 μηη. (h) Raw264.7 cells containing vector, Rubicon WT, ACCD or ASR were stimulated with Texas-Red labeled opsonized-zymosan particles for 30 min, followed by confocal microscopy with ap22phox or aFlag. Bar, 2 μιτι. The colocalization frequencies of the

immunofluorescence results are quantitated (Fig S6, S9 and S13). (i) Rubicon enhances phagocytosis in a p22p/?ox-binding-dependent manner. Raw264.7 cells containing vector, Rubicon WT, ACCD or ASR were infected with HK-GFP-L.

monocytogenes (MOI=1 ) for the indicated times, followed by flow cytometry analysis to detect internalized HK-GFP-L monocytogenes.

[0028] FIG. 2, panels a through g, show that Rubicon activates NADPH oxidase activity in a p22p/?ox-binding-dependent manner. The following figures show that Rubicon activates ROS production. Raw264.7 cells or BMDMs were incubated with DHE (a), CM-H2DCFDA (b), or 4,5-DAF-2 DA (c) to detect O₂ ^", H₂O₂, and NO production, respectively, with or without 100 g/ml zymosan for 30 min. Live cells were washed with serum-free medium and imaged using a confocal microscope. Enhanced O₂ ^~, H₂O₂, and NO was abolished by pretreatment with 20 μΜ DPI, 20 mM NAC or 100 mM L-NMMA, respectively. Images are representative of three independent experiments. SC, solvent control. Bar, 10 μιτι. (c) Luminometry of NADPH oxidase activity of Raw264.7 cells containing vector or Rubicon after treatment with 100 g/ml zymosan or 100 ng/ml BLP, or infected with L.

monocytogenes (MOI=1 ). Quantitative data are the mean ± SD of values from three experiments, (d) Rubicon enhances NADPH oxidase activity (left) and NO synthase activity (middle and right). BMDMs infected with lenti-GFP or lenti-GFP-Rubicon were analyzed for NADPH oxidase activity upon zymosan or BLP treatment (left). BMDMs infected with lenti-GFP or lenti-GFP-Rubicon (MOI=100) or THP-1 cells expressing vector or Rubicon were analyzed for NO synthase activity upon treatment with zymosan or BLP (middle) or infection with L. monocytogenes or M. bovis BCG (MOI=1 , right). NADPH oxidase and NO synthese activities were abolished by pretreatment with 20 μΜ DPI, 0.5 mM AEBSF or 100 mM L-NMMA or L-NMAE, respectively. Quantitative data are the mean ± SD of values from three experiments. (e) Rubicon expression enhances downstream signaling. (Top panel) Raw264.7 cells containing vector or Rubicon were stimulated with 100 g/ml zymosan for the indicated times and then subjected to IB analysis or nuclear/cytoplasm fractionations to detect the phosphorylated and total forms of p38, p42/p44 MAPK, JNK, ΙκΒ-α, or NF-KB p65. (Bottom panel) The same cells were stimulated with L. monocytogenes (MOI=1 ) and/or TNFa (10 ng/ml) and then subjected to nuclear/cytoplasm

fractionation to detect NF-κΒ p65. The same blots were washed and blotted for actin, or Lamin A C. (f) Increase of cytokine production by Rubicon, (top) Raw264.7 cells containing vector or Rubicon were stimulated with 100 g/ml zymosan for the indicated times and the supernatants were analyzed for cytokine production using ELISA. (middle and bottom) BMDMs infected with lenti-GFP or lenti-GFP-Rubicon were stimulated with 100 μg ml zymosan or 100ng/ml BLP for 18h and the

supernatants were subjected to cytokine ELISA. Cytokine production was abolished by pre-treatment with 20 μΜ DPI or 0.5 mM AEBSF. Values are the mean ± SD of triplicate samples. ^*, p <0.05; ^**, p<0.01 ; ^***, p<0.001 compared with vector control cultures, (g) Rubicon enhances bacterial killing activity. Raw246.7, THP-1 or BMDMs containing vector or Rubicon were infected with L. monocytogenes (left) or M. bovis BCG (right) at a MOI=1 for the indicated times and then lysed to determine

intracellular bacterial loads. CFU, colony-forming units. The data are the mean ± SD of values from three experiments. ^**, p<0.01 ; ^***, p<0.001 compared with the vector control.

[0029] FIG. 3, panels a through g, show Rubicon's effect on ROS and

inflammatory cytokine production in NADPH oxidase-mediated anti-microbial pathway Depletion of Rubicon gene expression leads to the reduction of ROS production (a), NADPH oxidase activity (b, left), and NO synthase activity (b, right). At 48 h postinfection with lentivirus-shRNA-NS or lentivirus-shRNA-Rubicon

(MOI=50), Raw264.7 cells or BMDMs were incubated with DHE to detect O₂ ^~ production with or without 100 g/ml zymosan for 30 min with or without

pretreatment with 20 μΜ DPI (a) or were analyzed for NADPH oxidase and NO synthase activity upon treatment with zymosan, or infection with L. monocytogenes (MOI=1 ) or M. bovis BCG (MOI=1 ) (b) as described in FIG. 2. Bar, 10 μηη.

Quantitative data are the mean ± SD of values from three experiments. ^**, p<0.01 ; ^***, p<0.001 compared with the lentivirus-shRNA-NS culture, (c) Reduction of cytokine production by Rubicon gene depletion. At 48h postinfection with lentivirus- shRNA-NS or lentivirus-shRNA-Rubicon, Raw264.7 cells or BMDMs were stimulated with zymosan, BLP, L. monocytogenes or M. bovis BCG for 18h and the

supernatants were analyzed for cytokine production using ELISA. Values are the mean ± SD of triplicate samples. ^**, p<0.01 ; ^***, p<0.001 compared with the lentivirus-shRNA-NS culture, (d) Rubicon gene depletion reduces bacterial killing activity. At 48h postinfection with lentivirus-shRNA-NS or lentivirus-shRNA-Rubicon, Raw264.7 cells or BMDMs were infected with L. monocytogenes (top) or M. bovis BCG (bottom) at a MOI=1 for indicated times and then lysed to determine

intracellular bacterial loads. The data are the mean ± SD of values from three experiments. ^**, p<0.01 ; ^***, p<0.001 compared with the lentivirus-shRNA-NS culture, (e, f and g) Rubicon plays a primary role in the TLR-NADPH oxidase- mediated anti-microbial pathway. Raw264.7 cells containing vector or Rubicon were stimulated with zymosan (100 pg/ml), IFNy (10 ng/ml), and/or L. monocytogenes (MOI=1 ) and assayed for NADPH oxidase (e), NO synthase activity (f) and intracellular bacterial loads (g) as described in FIG. 2.

[0030] FIG. 4, panels a through g, show that Rubicon enhances mortality after L. monocytogenes infection in mice and activates ROS-mediated bacterial killing activity in a p22p/?ox-binding-dependent and Beclinl -binding independent manner, (a, b, and c) Alteration of Rubicon gene expression affects mice mortality after L. monocytogenes infection. At 48h post-injection with Ad-vector (1 x10¹³ pfu/kg), Ad- shRubicon (1 x10¹² pfu/kg), or Ad-Rubicon (1 x10¹³ pfu/kg) twice i.v via tail vein, mice were infected with L. monocytogenes (1x10⁷ CFU/kg) and mortality was measured for n=23 mice per group (a). Bacterial loads of infected mice (n=5 per group) in spleen and liver (b) or serum cytokine levels (c) were determined at 5 days p.i. with L. monocytogenes (1x10⁶ CFU/kg). (d) Rubicon enhances ROS production in a p22p/?ox-binding-dependent manner. Raw246.7 cells expressing vector, Rubicon WT, ACCD, ASR, or ACCD/SR mutant were incubated with DHE to detect O₂ ^~ production (left) or used for measuring NADPH oxidase activity and NO synthase activity (right) with or without zymosan for 30 min, as described in FIG. 2.

Quantitative data are the mean ± SD of values from three experiments. Bar, 10 μιτι. (e) Rubicon increases cytokine production. Raw264.7 cells containing vector, Rubicon WT or its mutant were stimulated with zymosan, L. monocytogenes, or M. bovis BCG for 18h and the supernatants were analyzed for cytokine production using ELISA. Quantitative data are the mean ± SD of values from three experiments, (f) Rubicon enhances bacterial killing activity. Raw246.7 containing vector, Rubicon WT or its mutant were infected with L. monocytogenes (top) or M. bovis BCG (bottom) at a MOI=1 for the indicated times and then lysed; intracellular bacteria were plated to determine CFU. The data are the mean ± SD of values from three experiments. ^***, p<0.001 compared with the vector control, (g) Rubicon plays distinctive roles in conventional and TLR-signaling-mediated autophagy. Raw246.7 cells expressing vector, Rubicon WT, ACCD, or ASR mutant were treated with rapamycin, starvation, zymosan, BLP or L. monocytogenes for indicated times and their cell lysates were used for IB with al_C3, ap62, or aactin.

[0031] FIG. 5, panels a through c, show that the N-terminal (amino acids 1 -10) of the retro-inverso sequence of p22phox is sufficient for binding to the serine rich (aa 558-625) region of Rubicon.

[0032] FIG. 6 is a list of p22phox peptide sequences of N10 (ten amino acids) and N8 (eight amino acids) and their mutants. It's shown that the N-terminal 8 amino acids of p22phox are sufficient to bind to Rubicon. The left 12 amino acids are HIV TAT for membrane penetration.

[0033] FIG. 7, panels a through I, demonstrate that N8 peptide (30 uM) efficiently blocks Rubicon and p22phox interaction (a and b) and thereby suppresses zymosan (TLR2 ligand)-induced or bacterial infection-induced inflammatory cytokine production (c and d) and ROS production (I). The N8 peptide mutants (W6A and W9A) no longer inhibit Rubicon and p22phox interaction (f and h) and no longer block the inflammatory cytokine production (I).

[0034] FIG. 8, panels a through c, show that treatment with the p22phox peptide reduced ROS production upon Zymosan or L. monocytogenes infection and a proposed regulatory network centered at Rubicon (bottom right corner). [0035] FIG. 9, panels a and b, show that treatments with the p22phox peptides fused to Tat reduced ROS production upon Zymosan stimulation.

[0036] FIG. 10, panels a through C, show that, in the absence of Tat, the p22phox peptide (aa 3-10) is inhibitory of Rubicon-p22p/?ox interaction.

[0037] FIGS. 11 A and B show that pre- or post-treatment with p22phox peptides significantly rescued mice from Gram-negative bacterial LPS-induced lethal shock.

[0038] FIG. 12, panels a through c, show that pre- or post-treatment with p22phox peptides significantly rescued mice from Gram-negative bacterial LPS- induced lethal shock.

[0039] FIG. 13, panels a through c, show the effects of p22phox peptides in cecal ligation and puncture (CLP)-induced sepsis: The N8 peptide efficiently blocks sepsis development by single (a, 6 hr after LPS injection), double (b, 6 and 18hr after LPS injection) and triple (c, 6, 12 and 18 hr after LPS injection) injection of N8 peptide. The middle panels are the suppressions of inflammatory cytokine and ROS (NOX activity) productions upon the N8 peptide treatment, and the right panels are the suppressions of COX-2 expression upon the N8 peptide treatment. The bottom panel shows the immunohistochemistry of COX-2 staining of liver and spleen of CLP- induced sepsis mice with or without TAT or TAT-N8 peptide injection.

[0040] FIGS. 14 A and B show that treatment with p22phox peptides suppressed the NOX activity and cytokine production of lung epithelial cells induced by LPS or influenza infection.

[0041] FIGS. 15 A and B show that post-treatment with p22phox peptides significantly rescued mice from Flu-induced acute lung injury.

[0042] FIG. 16, panels A through E show that treatment with p22phox peptides significantly rescued mice from Flu-indcued acute lung injury.

[0043] FIGS. 17 A and B show that treatment with p22phox peptides significantly rescued mice from asthma related airway hyperreactivity (AHR). [0044] FIG. 18 illustrates the regions on p22phox that is responsible for binding gp91 phox.

DETAILED DESCRIPTION

[0045] Before the compositions and methods are described, it is to be understood that the disclosure is not limited to the particular methodologies, protocols, cell lines, assays, and reagents described, as these may vary. It is also to be understood that the terminology used herein is intended to describe particular embodiments of the present disclosure, and is in no way intended to limit the scope of the present disclosure as set forth in the appended claims.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices, and materials are now

described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0047] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001 ) Molecular Cloning: A Laboratory

rd

Manual, 3 edition; the series Ausubel et al. eds. (2007) Current Protocols in

Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991 ) PCR 1 : A Practical Approach (IRL Press at Oxford

University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of

Animal Cells: A Manual of Basic Technique, 5^th edition; Gait ed. (1984)

Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization;

Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and

Expression in Mammalian Cells; and Mayer and Walker eds. (1987)

Immunochemical Methods in Cell and Molecular Biology (Academic Press, London).

[0048] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1 . It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0049] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.

Definitions

[0050] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0051] As used herein, the term "comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0052] The term "isolated" as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term "isolated peptide fragment" is meant to include peptide fragments which are not naturally occurring as fragments and would not be found in the natural state as well as substantially purified from an extract thereof. The term "isolated" is also used herein to refer to polypeptides, antibodies, proteins, host cells and polynucleotides that are isolated from other cellular proteins or tissues and is meant to encompass both purified and recombinant polypeptides, antibodies, proteins and polynucleotides. In other embodiments, the term "isolated" means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature and can include at least 80 %, or alternatively at least 85 %, or alternatively at least 90 %, or alternatively at least 95%, or alternatively at least 98 %, purified from a cell or cellular extract. For example, an isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. An isolated cell, for example, is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide,

polypeptide, protein, antibody or fragment(s) thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart.

[0053] The term "binding" or "binds" as used herein are meant to include

interactions between molecules that may be detected using, for example, a hybridization assay. The terms are also meant to include "binding" interactions between molecules. Interactions may be, for example, protein-protein, antibody- protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid in nature. This binding can result in the formation of a "complex" comprising the interacting molecules. A "complex" refers to the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces.

[0054] The term "polypeptide" is used interchangeably with the term "protein" and in its broadest sense refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. As used herein the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein. The term "peptide fragment" as used herein, also refers to a peptide chain.

[0055] The phrases "equivalent of a peptide or polypeptide," "biologically equivalent polypeptide" or "biologically equivalent peptide or peptide fragment" refer to a protein or a peptide fragment which is homologous to the exemplified protein or peptide fragment and which exhibit similar biological activity in vitro or in vivo, e.g., approximately 100%, or alternatively, over 90% or alternatively over 85% or alternatively over 70%, as compared to the standard or control biological activity. Additional embodiments within the scope of this disclosure are identified by having more than 60%, or alternatively, more than 65%, or alternatively, more than 70%, or alternatively, more than 75%, or alternatively, more than 80%, or alternatively, more than 85%, or alternatively, more than 90%, or alternatively, more than 95%, or alternatively more than 97%, or alternatively, more than 98% or 99% sequence identity or homology. Percentage homology can be determined by sequence comparison using programs such as BLAST run under appropriate conditions. In one aspect, the program is run under default parameters.

[0056] As understood by those of skill in the art, a "retro-inverso" refers to an isomer of a linear peptide in which the direction of the sequence is reversed ("retro") and the chirality of each amino acid residue is inverted ("inverso"). Compared to the parent peptide, a helical retro-inverso peptide can substantially retain the original spatial conformation of the side chains but has reversed peptide bonds, resulting in a retro-inverso isomer with a topology that closely resembles the parent peptide, since all peptide backbone hydrogen bond interactions are involved in maintaining the helical structure. See Jameson et al., (1994) Nature 368:744-746 (1994) and Brady et al. (1994) Nature 368:692-693. The net result of combining D-enantiomers and reverse synthesis is that the positions of carbonyl and amino groups in each amide bond are exchanged, while the position of the side-chain groups at each alpha carbon is preserved. Unless specifically stated otherwise, it is presumed that any given L-amino acid sequence of the disclosure may be made into an D retro-inverso peptide by synthesizing a reverse of the sequence for the corresponding native L- amino acid sequence.

[0057] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.

Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of

polynucleotides: a gene or gene fragment (for example, a probe, primer, or EST), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, RNAi, siRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified

nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by

non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0058] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinfornnatics applications such as functional genomics and homology searching.

[0059] "Homology" or "identity" or "similarity" are synonymously and refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0060] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology.

Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and

BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address:

http://www.ncbi.nlm.nih.gov/blast/Blast.cgi, last accessed on November 26, 2007. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity.

[0061] Alternatively, sequence identify of nucleic acids can also be demonstrated by hybridization of the nucleic acids under stringent conditions. A non-limiting example of stringent hybridization conditions is at a temperature of 42° C in a solution consisting of 50% formamide, 5xSSC, and 1 % SDS, and washing at 65° C in a solution consisting of 0.2 X SSC and 0.1 % SDS.

[0062] A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotide or polypeptide sequences described herein may be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

[0063] The term "express" refers to the production of a gene product such as RNA or a polypeptide or protein.

[0064] As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.

[0065] A "gene product" or alternatively a "gene expression product" refers to the RNA when a gene is transcribed or amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.

[0066] "Short interfering RNA" (siRNA) refers to sequence-specific or gene specific suppression of gene expression (protein synthesis) that is mediated by double- stranded RNA molecules, generally, from about 10 to about 30 nucleotides long that are capable of mediating RNA interference (RNAi). As used herein, the term siRNA includes short hairpin RNAs (shRNAs).

[0067] The term "encode" as it is applied to polynucleotides refers to a

polynucleotide which is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced there from.

[0068] Applicants have provided herein the polypeptide and/or polynucleotide sequences for use in gene and protein transfer and expression techniques described below. It should be understood, although not always explicitly stated that the sequences provided herein can be used to provide the expression product as well as substantially identical sequences that produce a protein that has the same biological properties. These "biologically equivalent" or "biologically active" polypeptides are encoded by equivalent polynucleotides as described herein. They may possess at least 60%, or alternatively, at least 65%, or alternatively, at least 70%, or

alternatively, at least 75%, or alternatively, at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or alternatively at least 98%, identical primary amino acid sequence to the reference polypeptide when compared using sequence identity methods run under default conditions. Specific polypeptide sequences are provided as examples of particular embodiments.

Modifications to the sequences to amino acids with alternate amino acids that have similar charge.

[0069] "Cell penetrating peptides", "cell permeable proteins", "CPPs", or "Protein transduction domains", as used herein, refer to short peptides that facilitate cellular uptake of various molecular cargos (from small chemical molecules to nanosize particles and large fragments of DNA). A "cargo", such as a protein, is associated with the peptides either through chemical linkage via covalent bonds or through non- covalent interactions. The function of the CPPs are to deliver the cargo into cells, a process that commonly occurs through endocytosis with the cargo delivered to the endosomes of living mammalian cells. CPPs typically have an amino acid

composition containing either a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. In 1988, Frankel and Pabo found that the human immunodeficiency virus transactivator of transcription (HIV-TAT) protein can be delivered to cells using a CPP (Frankel et al. 1988a and Frankel et al. 1988b).

[0070] A CPP employed in accordance with one aspect of the invention may include 3 to 35 amino acids, preferably 5 to 25 amino acids, more preferably 10 to 25 amino acids, or even more preferably 15 to 25 amino acids.

[0071] A CPP may also be chemically modified, such as prenylated near the C- terminus of the CPP. Prenylation is a post-translation modification resulting in the addition of a 15 (farneysyl) or 20 (geranylgeranyl) carbon isoprenoid chain on the peptide. A chemically modified CPP can be even shorter and still possess the cell penetrating property. Accordingly, a CPP, pursuant to another aspect of the invention, is a chemically modified CPP with 2 to 35 amino acids, preferably 5 to 25 amino acids, more preferably 10 to 25 amino acids, or even more preferably 15 to 25 amino acids.

[0072] A CPP suitable for carrying out one aspect of the invention may include at least one basic amino acid such as arginine, lysine and histidine. In another aspect, the CPP may include more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such basic amino acids, or alternatively about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% of the amino acids are basic amino acids. In one embodiment, the CPP contains at least two consecutive basic amino acids, or alternatively at least three, or at least five consecutive basic amino acids. In a particular aspect, the CPP includes at least two, three, four, or five consecutive arginine. In a further aspect, the CPP includes more arginine than lysine or histidine, or preferably includes more arginine than lysine and histidine combined.

[0073] CPPs may include acidic amino acids but the number of acidic amino acids should be smaller than the number of basic amino acids. In one embodiment, the CPP includes at most one acidic amino acid. In a preferred embodiment, the CPP does not include acidic amino acid. In a particular embodiment, a suitable CPP is the HIV-TAT peptide.

[0074] CPPs can be linked to a protein recombinantly, covalently or non- covalently. A recombinant protein having a CPP peptide can be prepared in bacteria, such as E. coli, a mammalian cell such as a human HEK293 cell, or any cell suitable for protein expression. Covalent and non-covalent methods have also been developed to form CPP/protein complexes. A CPP, Pep-1 , has been shown to form a protein complex and proven effective for delivery (Kameyama et al. (2006) Bioconjugate Chem. 17:597-602).

[0075] CPPs also include cationic conjugates which also may be used to facilitate delivery of the proteins into the progenitor or stem cell. Cationic conjugates may include a plurality of residues including amines, guanidines, amidines, N-containing heterocycles, or combinations thereof. In related embodiments, the cationic conjugate may comprise a plurality of reactive units selected from the group consisting of alpha-amino acids, beta-amino acids, gamma-amino acids, cationically functionalized monosaccharides, cationically functionalized ethylene glycols, ethylene imines, substituted ethylene imines, N-substituted spermine, N-substituted spermidine, and combinations thereof. The cationic conjugate also may be an oligomer including an oligopeptide, oligoamide, cationically functionalized oligoether, cationically functionalized oligosaccharide, oligoamine, oligoethyleneimine, and the like, as well as combinations thereof. The oligomers may be oligopeptides where amino acid residues of the oligopeptide are capable of forming positive charges. The oligopeptides may contain 5 to 25 amino acids; preferably 5 to 15 amino acids; more preferably 5 to 10 cationic amino acids or other cationic subunits.

[0076] A "gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, micelles, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

[0077] A polynucleotide of this disclosure can be delivered to a cell or tissue using a gene delivery vehicle. "Gene delivery," "gene transfer," "transducing," and the like as used herein, are terms referring to the introduction of an exogenous

polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well- known techniques such as vector-mediated gene transfer (by, e.g., viral

infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.

[0078] A "viral vector" is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.

[0079] As used herein, "retroviral mediated gene transfer" or "retroviral

transduction" carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.

[0080] Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0081] In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene.

Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071 . Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos. WO 95/00655 and WO 95/1 1984. Wild-type AAV has high infectivity and specificity integrating into the host cell's genome. See, Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0082] Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, Wl). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.

[0083] Gene delivery vehicles also include DNA/liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this disclosure. To enhance delivery to a cell, the nucleic acid or proteins of this disclosure can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens. In addition to the delivery of polynucleotides to a cell or cell population, direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins of this disclosure are other non-limiting techniques.

[0084] The terms "culture" or "culturing" refer to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell. [0085] A "composition" is intended to mean a combination of active polypeptide, polynucleotide or antibody and another compound or composition, inert (e.g. a detectable label) or active (e.g. a gene delivery vehicle) alone or in combination with a carrier which can in one embodiment be a simple carrier like saline or

pharmaceutically acceptable or a solid support as defined below.

[0086] A "pharmaceutical composition" is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0087] As used herein, the term "pharmaceutically acceptable carrier"

encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton ).

[0088] The phrase "solid support" refers to non-aqueous surfaces such as "culture plates" "gene chips" or "microarrays." Such gene chips or microarrays can be used for diagnostic and therapeutic purposes by a number of techniques known to one of skill in the art. In one technique, oligonucleotides are arrayed on a gene chip for determining the DNA sequence by the hybridization approach, such as that outlined in U.S. Patent Nos. 6,025,136 and 6,018,041 . The polynucleotides of this disclosure can be modified to probes, which in turn can be used for detection of a genetic sequence. Such techniques have been described, for example, in U.S. Patent Nos. 5,968,740 and 5,858,659. A probe also can be affixed to an electrode surface for the electrochemical detection of nucleic acid sequences such as described by Kayem et al. U.S. Patent No. 5,952,172 and by Kelley et al. (1999) Nucleic Acids Res. 27:4830-4837.

[0089] Various "gene chips" or "microarrays" and similar technologies are know in the art. Examples of such include, but are not limited to, LabCard (ACLARA Bio Sciences Inc.); GeneChip (Affymetric, Inc); LabChip (Caliper Technologies Corp); a low-density array with electrochemical sensing (Clinical Micro Sensors); LabCD System (Gamera Bioscience Corp.); Omni Grid (Gene Machines); Q Array (Genetix Ltd.); a high-throughput, automated mass spectrometry systems with liquid-phase expression technology (Gene Trace Systems, Inc.); a thermal jet spotting system (Hewlett Packard Company); Hyseq HyChip (Hyseq, Inc.); BeadArray (lllunnina, Inc.) GEM (Incyte Microarray Systems); a high-throughput microarrying system that can dispense from 12 to 64 spots onto multiple glass slides (Intelligent Bio-Instruments); Molecular Biology Workstation and NanoChip (Nanogen, Inc.); a microfluidic glass chip (Orchid biosciences, Inc.); BioChip Arrayer with four PiezoTip piezoelectric drop-on-demand tips (Packard Instruments, Inc.); FlexJet (Rosetta Inpharmatic, Inc.); MALDI-TOF mass spectrometer (Sequnome); ChipMaker 2 and ChipMaker 3 (TeleChem International, Inc.); and GenoSensor (Vysis, Inc.) as identified and described in Heller (2002) Annu. Rev. Biomed. Eng. 4:129-153. Examples of "gene chips" or a "microarrays" are also described in U.S. Patent Publ. Nos.: 2007- 01 1 1322, 2007-0099198, 2007-0084997, 2007-0059769 and 2007-0059765 and U.S. Patent Nos.: 7,138,506, 7,070,740, and 6,989,267.

[0090] In one aspect, "gene chips" or "microarrays" containing probes or primers homologous to a polynucleotide, polypeptide or antibody described herein are prepared. A suitable sample is obtained from the patient, extraction of genomic DNA, RNA, protein or any combination thereof is conducted and amplified if necessary. The sample is contacted to the gene chip or microarray panel under conditions suitable for hybridization of the gene(s) or gene product(s) of interest to the probe(s) or primer(s) contained on the gene chip or microarray. The probes or primers may be detectably labeled thereby identifying the gene(s) of interest.

Alternatively, a chemical or biological reaction may be used to identify the probes or primers which hybridized with the DNA or RNA of the gene(s) of interest. The genotypes or phenotype of the patient is then determined with the aid of the aforementioned apparatus and methods.

[0091] Other non-limiting examples of a solid phase support include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polynucleotide, polypeptide or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. or alternatively polystyrene beads. Those skilled in the art will know many other suitable carriers for binding protein, peptide, antibody or antigen, or will be able to ascertain the same by use of routine experimentation..

[0092] A "subject," "individual" or "patient" is used interchangeably herein, and refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, rabbits, simians, bovines, ovines, porcines, canines, felines, farm animals, sport animals, pets, equines, and primates, particularly humans.

[0093] "Cell," "host cell" or "recombinant host cell" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. The cells can be of any one or more of the type murine, rat, rabbit, simian, bovine, ovine, porcine, canine, feline, equine, and primate, particularly human. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0094] The terms "disease" and "disorder" are used inclusively and refer to any condition associated with regulation of autophagy. In the context of this disclosure the disease may be associated with cancer, a neurodegenerative disorder, or a pathogenic infection. As used herein, "cancer" may refer both to precancerous cells as well as cancerous cells of a tumor such as a solid tumor. Examples of

neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, and transmissible spongiform

encephalopathies. Examples of pathogenic infections include, but are not limited to, infection by bacteria such as group A Streptococcus, Mycobacterium tuberculosis, Shigella flexneri, Salmonella enterica, Listeria monocytogenes, Francisella

tularensis, and infection by viruses such as herpes simplex virus.

[0095] "Treating," "treatment," or "ameliorating" of a disease includes: (1 ) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.

[0096] The term "suffering" as it related to the term "treatment" refers to a patient or individual who has been diagnosed with or is predisposed to a disease. A patient may also be referred to being "at risk of suffering" from a disease. This patient has not yet developed characteristic disease pathology, however are know to be predisposed to the disease due to family history, being genetically predispose to developing the disease, or diagnosed with a disease or disorder that predisposes them to developing the disease to be treated.

Descriptive Embodiments

Isolated Peptide Fragments and Compositions

[0097] This disclosure provides isolated peptide fragments and variants of the p22phox and Rubicon proteins. In one aspect, this disclosure provides an isolated or recombinant peptide comprising, or alternatively consisting essentially of, or yet further consisting of, contiguous amino acids 3-10 of SEQ ID NO: 1 or an equivalent or retro-inverso thereof, wherein the peptide does not bind gp91 phox. Non-limiting examples of such include this peptide, wherein the peptide does not consist of the C-terminal half of SEQ ID NO: 1 . These peptides can further comprise, or alternatively consist essentially of, or yet further consist of a cell penetrating peptide, e.g., a TAT peptide. In one aspect, the the peptide is less than or equal to 30 amino acids long. In another aspect, the peptide is less than or equal to 22 amino acids long. In a yet further aspect, the peptide is 8 amino acids long. In one specific embodiment, the peptide comprises SEQ ID NO: 8.

[0098] Any of the aforementioned peptides can be further modified by conjugation to poly(ethyleneglycol) (PEG) to facilitate delivery and reduce an immune response.

[0099] Various proteins are disclosed herein with their GenBank Accession

Numbers for their human proteins and coding sequences. However, the proteins are not limited to human-derived proteins having the amino acid sequences represented by the disclosed GenBank Accession numbers, but may have an amino acid sequence derived from other animals, particularly, a warm-blooded animal (e.g., rat, guinea pig, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.).

[0100] As used herein, the term "p22phox", or "CYBA cytochrome b-245, alpha polypeptide" refers to a protein having an amino acid sequence substantially identical to any of the representative p22phox sequences of GenBank Accession Nos. NP_000092 (human), NP_031832 (mouse) or NP_077074 (rat). Suitable cDNA encoding p22phox are provided at GenBank Accession Nos. NM_000101 (human), NM_007806 (mouse) or NM_024160. [0101] The representative p22phox protein sequences are shown below.

Human p22phox: NP_000092 (SEQ ID NO: 1)

1 MGQIEWAMWA NEQALASGLI LITGGIVATA GRFTQWYFGA YSIVAGVFVC

51 LLEYPRGKRK KGSTMERWGQ KYMTAVVKLF GPFTRNYYVR AVLHLLLSVP

101 AGFLLATILG TACLAIASGI YLLAAVRGEQ WTPIEPKPRE RPQIGGTIKQ

151 PPSNPPPRPP AEARKKPSEE EAAVAAGGPP GGPQVNPIPV TDEVV

Mouse p22phox: NP_031832 (SEQ ID NO: 2)

1 MGQIEWAMWA NEQALASGLI LITGGIVATA GRFTQWYFGA YSIAAGVLIC

51 LLEYPRGKRK KGSTMERCGQ KYLTSVVKLF GPLTRNYYVR AALHFLLSVP

101 AGFLLATILG TVCLAIASVI YLLAAIRGEQ WTPIEPKPKE RPQVGGTIKQ

151 PPTNPPPRPP AEVRKKPSEG EEEAASAGGP QVNPMPVTDE VV

Rat p22phox: NP_077074 (SEQ IN NO: 3)

1 MGQIEWAMWA NEQALASGLI LITGGIVATA GRFTQWYFGA YSIVAGVLIC

51 LLEYPRGKRK KGSTMERCGQ KYLTAVVKLF GPLTRNYYVR AVLHLLLSVP

101 AGFLLATILG TVCLAIASVI YLLAAIRGEQ WTPIEPKPKE RPQVGGTIKQ

151 PPTNPPPRPP AEVRKKPSEA EEEAASAGGP QVNPIPVTDE VV

[0102] p22phox proteins are homologous to each other, as shown in the multi- alignment below. Accordingly, an equivalent of a p22phox sequence, in one embodiment, includes any p22phox sequence from any species, or a sequence that shares the reserved amino acids as shown the multi-alignment.

SEQ ID NO: 2 MGQIEWAMWANEQALASGLILITGGIVATAGRFTQWYFGAYSIAAGVLIC 50

SEQ ID NO: 3 MGQIEWAMWANEQALASGLILITGGIVATAGRFTQWYFGAYSIVAGVLIC 50

SEQ ID NO:l MGQIEWAMWANEQALASGLILITGGIVATAGRFTQWYFGAYSIVAGVFVC 50

Identity_" ******************************************* ***..* SEQ ID NO:: 2 LLEYPRGKRKKGSTMERCGQKYLTSVVKLFGPLTRNYYVRAALHFLLSVP 100

SEQ ID NO: : 3 LLEYPRGKRKKGSTMERCGQKYLTAVVKLFGPLTRNYYVRAVLHLLLSVP 100

SEQ ID NO: : 1 LLEYPRGKRKKGSTMERWGQKYMTAVVKLFGPFTRNYYVRAVLHLLLSVP 100

Identity :

SEQ ID NO: : 2 AGFLLATILGTVCLAIASVIYLLAAIRGEQWTPIEPKPKERPQVGGTIKQ 150

SEQ ID NO: : 3 AGFLLATILGTVCLAIASVIYLLAAIRGEQWTPIEPKPKERPQVGGTIKQ 150

SEQ ID NO: : 1 AGFLLATILGTACLAIASGIYLLAAVRGEQWTPIEPKPRERPQIGGTIKQ 150

Identity :

SEQ ID NO: : 2 PPTNPPPRPPAEVRKKPSEGEEEAASAG GPQVNPMPV DEVV 192

SEQ ID NO: : 3 PPTNPPPRPPAEVRKKPSEAEEEAASAG GPQVNPI PV DEVV 192

SEQ ID NO: : 1 PPSNPPPRPPAEARKKPSEEEAAVAAGGPPGGPQVNPIPVTDEVV 195

Identity :

[0103] The present disclosure shows that the amino acids 1 -10, in particular 3-10, and the corresponding retro-inverso sequences are adequate for p22p/?o to bind Rubicon. Accordingly, peptides that include these sequences are capable of binding Rubicon (Table 1 ).

Table 1 . Exemplary peptides that include amino acids 3-10 of p22phox.

[0104] It is further discovered that when either of amino acids 6 and 9 is replaced by a different amino acid, the binding activity of the peptides decreased. Therefore, an equivalent of such peptides are contemplated to include peptides that have "W" at position 6 and "W" at position 9 but may have variations or modifications at other positions, or the retro-inverso sequences thereof.

[0105] It is also discovered that the sequence on p22phox responsible for binding gp91 phox is at the C-terminal half, including about amino acid 94 to 195 (see FIG. 54). Accordingly, a p22phox variant or fragment of the equivalent thereof that does bind gp91 phox does not include all or part of the gp91 phox binding sequence.

[0106] As used herein, the term "Rubicon", or "run domain Beclin-1 interacting and cystein-rich containing protein" refers to a protein having an amino acid sequence substantially identical to any of the representative Rubicon sequences of GenBank Accession Nos. NP_001 1391 14 (isoform 1 ; SEQ ID NO: 10) or NP_055502 (isoform 2; SEQ ID NO: 1 1 ) (human) or NP_766203 (mouse; SEQ ID NO: 12). Suitable cDNA encoding Rubicon are provided at GenBank Accession Nos. NM_001 145642 or NM_014687 (human) or NM_172615 (mouse).

Sequences of these representative protein sequences and the multi-alignment are shown below.

SEQ ID NO 10

SEQ ID NO 11 MRPEGAGMELGGGE-ERLPEESRREHWQLLGNLKTTVEGLVSTNSPNVWS 49

SEQ ID NO 12 MRPEGAGMDLGGGDGERLLEKSRREHWQLLGNLKTTVEGLVSANCPNVWS 50

SEQ ID NO 10 MQSILYHGLIRDQACRRQTDYWQFVKDIRWLSPHSALHV 39

SEQ ID NO 11 KYGGLERLCRDMQSILYHGLIRDQACRRQTDYWQFVKDIRWLSPHSALHV 99

SEQ ID NO 12 KYGGLERLCRDMQNILYHGLIHDQVCCRQADYWQFVKDIRWLSPHSALHV 100

Identity

SEQ ID NO 10 EKFISVHENDQSSADGASERAVAELWLQHSLQYHCLSAQLRPLLGDRQYI 89

SEQ ID NO 11 EKFISVHENDQSSADGASERAVAELWLQHSLQYHCLSAQLRPLLGDRQYI 149

SEQ ID NO 12 EKFISLHESDQSDTDSVSERAVAELWLQHSLQCHCLSAQLRPLLGDRQYI 150

Identity

SEQ ID NO 10 RKFYTDAAFLLSDAHVTAMLQCLEAVEQNNPRLLAQIDASMFARKHESPL 139

SEQ ID NO 11 RKFYTDAAFLLSDAHVTAMLQCLEAVEQNNPRLLAQIDASMFARKHESPL 199

SEQ ID NO 12 RKFYTETAFLLSDAHVTAMLQCLEAVEQNNPRLLAQIDASMFARKQESPL 200

Identity

SEQ ID NO 10 LVTKSQSLTALPSSTYTPPNSYAQHSYFGSFSSLHQS-VPNNGSERRSTS 188

SEQ ID NO 11 LVTKSQSLTALPSSTYTPPNSYAQHSYFGSFSSLHQS-VPNNGSERRSTS 248

SEQ ID NO 12 LVTKSQSLTALPGSTYTPPASYAQHSYFGSSSSLQSMPQSSHSSERRSTS 250

Identity

SEQ ID NO 10 FPLSGPPRKPQESRGHVSPAEDQTIQAPPVSVSALARDSPLTPNEMSSST 238

SEQ ID NO 11 FPLSGPPRKPQESRGHVSPAEDQTIQAPPVSVSALARDSPLTPNEMSSST 298

SEQ ID NO 12 FSLSGPSWQPQEDRECLSPAETQTTPAPLPSDSTLAQDSPLTAQEMSDST 300

Identity

SEQ ID NO 10 LTSPIEASWVSSQNDSPGDASEGPEYLAIGNLDPRGRTASCQSHSSNAES 288

SEQ ID NO 11 LTSPIEASWVSSQNDSPGDASEGPEYLAIGNLDPRGRTASCQSHSSNAES 348

SEQ ID NO 12 LTSPLEASWVSSQNDSPSDVSEGPEYLAIGNPAPHGRTASCESHSSNGES 350

Identity

SEQ ID NO 10 SSSNLFSSSSSQKPDSAASSLGDQEGGGESQLSSVLRRSSFSEGQTLTVT 338

SEQ ID NO 11 SSSNLFSSSSSQKPDSAASSLGDQEGGGESQLSSVLRRSSFSEGQTLTVT 398

SEQ ID NO 12 SSSHLFSSSSSQKLESAASSLGDQEEGRQSQAGSVLRRSSFSEGQTAPVA 400

Identity

SEQ ID NO 10 SGAKKSHIRSHSDTSIASRGAPGGPRNI I IVEDPIAESCNDKAKLRGPL 388

SEQ ID NO 11 SGAKKSHIRSHSDTSIASRGAP ESCNDKAKLRGPL 433

SEQ ID NO 12 SGTKKSHIRSHSDTNIASRGAA 422

Identity

SEQ ID NO 10 PYSGQSSEVSTPSSLYMEYEGGRYLCSGEGMFRRPSEGQSLISYLSEQDF 438

SEQ ID NO 11 PYSGQSSEVSTPSSLYMEYEGGRYLCSGEGMFRRPSEGQSLISYLSEQDF 483 SEQ ID NO 12 EGGQYLCSGEGMFRRPSEGQSLISYLSEQDF

Identity

SEQ ID NO 10

SEQ ID NO 11

SEQ ID NO 12

Identity **************************************************

SEQ ID NO 10

SEQ ID NO 11

SEQ ID NO 12

Identity ******************** *. *. *. *****************. ***. *

SEQ ID NO 10

SEQ ID NO 11

SEQ ID NO 12

Identity ***.. *..********* . * ** * * .*****************

SEQ ID NO 10

SEQ ID NO 11

SEQ ID NO 12

Identity **************************************************

SEQ ID NO 10

SEQ ID NO 11

SEQ ID NO 12

Identity **************************************************

SEQ ID NO 10

SEQ ID NO 11

SEQ ID NO 12

Identity *********************** . *** . ******************* . *

SEQ ID NO 10

SEQ ID NO 11

SEQ ID NO 12

Identity ********************************* ****************

SEQ ID NO 10

SEQ ID NO 11

SEQ ID NO 12

Identity **** ************** *****.****************.*******

SEQ ID NO 10

SEQ ID NO 11

SEQ ID NO 12

Identity ************** . * . ********************* . ***** *****

SEQ ID NO 10

SEQ ID NO 11

SEQ ID NO 12

Identity ******* **. **************. *******. ***. *

[0107] It is discovered by GST pulldown (GST-PD) studies that Beclinl and p22phox efficiently bound the central coiled-coil domain (CCD, aa505-557) and the serine-rich region (SR, aa558-625) of Rubicon (human isoform 2, SEQ ID NO: 1 1 ), respectively. As shown in the multi-alignments, the corresponding CCD region on SEQ ID NO: 10 (human Rubicon isoform 1 ) is aa460-512 and on SEQ ID NO: 12 (mouse Rubicon) is aa 475-527. Likewise, the corresponding SR region on SEQ ID NO: 10 (human Rubicon isoform 1 ) is aa513-580 and on SEQ ID NO: 12 (mouse Rubicon) is aa528-595.

[0108] It is known to those skilled in the art that modifications can be made to any peptide by substituting one or more amino acids with one or more functionally equivalent amino acids that does not alter the biological function of the peptide. Thus, the peptides of this invention can have amino acid substitutions by an amino acid that possesses similar intrinsic properties including, but not limited to, hydrophobic, size, or charge. Methods used to determine the appropriate amino acid to be substituted and for which amino acid are know to one of skill in the art. Non-limiting examples include empirical substitution models as described by Layoff et al. (1978) In Atlas of Protein Sequence and Structure Vol. 5 suppl. 2 (ed. MR. Day off), pp. 345-352. National Biomedical Research Foundation, Washington DC; PAM matrices including Day off matrices (Layoff et al. (1978), supra, or JET matrices as described by Jones et al. (1992) Compute. Appl. Basic. 8:275-282 and Gannet et al. (1992) Science 256:1443-1 145; the empirical model described by Adak and

Hasegawa (1996) J. Mol. Evil. 42:459-468; the block substitution matrices

(BLOSSOM) as described by Henrico and Henrico (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Poisson models as described by Neil (1987) Molecular Evolutionary Genetics. Columbia University Press, New York.; and the Maximum Likelihood (ML) Method as described by Muller et al. (2002) Mol. Biol. Evil. 19:8-13.

[0109] Accordingly, in yet another aspect the isolated peptide fragment may comprise, or alternatively consisting essentially of, or yet further consisting of, or is a "biologically equivalent" or "biologically active" peptide fragment encoded by equivalent polynucleotides as described herein. They may possess at least 60%, or alternatively, at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively, at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or alternatively at least 98%, identical primary amino acid sequence to the reference polypeptide when compared using sequence identity methods run under default conditions. For example, one or more of the valise, isoleucine, leucine, methionine, phenylalanine, or tryptophan residues of the hydrophobic core of an alpha helix of a death effector domain may be modified or substituted with another hydrophobic residue such as valine, isoleucine, leucine, methionine, phenylalanine, or tryptophan.

[0110] Proteins and peptide fragments comprising, or alternatively consisting essentially of, or yet further consisting of, the amino acid sequences of the disclosure can be prepared by expressing polynucleotides encoding the polypeptide sequences of this disclosure in an appropriate host cell. This can be accomplished by methods of recombinant DNA technology known to those skilled in the art. Accordingly, this disclosure also provides methods for recombinantly producing the polypeptides of this disclosure in a eukaryotic or prokaryotic host cell, which in one aspect is further isolated from the host cell. The proteins and peptide fragments of this disclosure also can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin Elmer/Applied Biosystems, Inc., Model 430A or 431 A, Foster City, CA, USA. The synthesized protein or polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Accordingly, this disclosure also provides a process for chemically synthesizing the proteins of this disclosure by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence.

[0111] The protein and peptide fragments may be operatively linked to a transduction domain or cell penetrating peptide for facilitated cell entry. Protein transduction offers an alternative to gene therapy for the delivery of therapeutic proteins into target cells, and methods involving protein transduction are within the scope of the disclosure. Protein transduction is the internalization of proteins into a host cell from the external environment. The internalization process relies on a protein or peptide which is able to penetrate the cell membrane. To confer this ability on a normally non-transducing protein, the non-transducing protein can be fused to a transduction-mediating protein such as the antennapedia peptide, the HIV TAT protein transduction domain, or the herpes simplex virus VP22 protein. See Ford et al. (2001 ) Gene Ther. 8:1 -4. As such the polypeptides of the disclosure can, for example, include modifications that can increase such attributes as stability, half- life, ability to enter cells and aid in administration, e.g., in vivo administration of the polypeptides of the disclosure. For example, polypeptides of the disclosure can comprise, or alternatively consisting essentially of, or yet further consisting of, a protein transduction domain of the HIV TAT protein as described in Schwarze et al. (1999) Science 285:1569-1572, and exemplified below.

[0112] In a further aspect, any of the proteins or peptides of this disclosure can be combined with a detectable label such as a dye for ease of detection.

[0113] This disclosure also provides pharmaceutical composition for in vitro and in vivo use comprising, or alternatively consisting essentially of, or yet further consisting of a therapeutically effective amount of the p22phox or Rubicon peptide fragment that causes at least about 75%, or alternatively at least about 80%, or alternatively at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least about 99% effectiveness in the methods provided herein when applied in a molar concentration of less than about 10 micromolar, or alternatively less than about 9 micromolar, or alternatively less than about 8 micromolar, or alternatively less than about 7 micromolar, or alternatively less than about 6 micromolar, or alternatively less than about 5 micromolar, or alternatively less than about 4 micromolar, or alternatively less than about 3 micromolar, or alternatively less than about 2 micromolar, or alternatively less than about 1 micromolar, or alternatively less than about 0.5 micromolar, or alternatively less than about 0.25 micromolar concentration, as compared to a control that does not receive the composition. Comparative effectiveness can be determined by suitable in vitro or in vivo methods as known in the art and described herein.

[0114] In another embodiment, the polypeptides of the present disclosure can be PEGylated (i.e. comprising PEG (poly(ethyleneglycol)). The PEG can be covalently attached to the polypeptides, in some aspects. In one aspect, attached is a methoxpoly(ethyleneglycol) polymer having a molecular weight of approximately 20,000 Da. In one aspect, the PEG is attached to the glutamic acid residue of the peptide. Methods of preparing and attached PEG to polypeptides are well known in the art. See, for example, Veronese, F. M. ; Pasut, G. (2005), "PEGylation, successful approach to drug delivery", Drug Discovery Today 10 (21 ): 1451-1458. It is specifically contemplated that PEGylation decreases the immunogenicity of the peptides of the present disclosure and thus facilitate delivery and increase

therapeutic effects.

[0115] This disclosure also provides compositions for in vitro and in vivo use comprising, or alternatively consisting essentially of, or yet further consisting of one or more of the isolated peptide fragments or a variant or hybrid or a biological equivalent of each thereof, described herein and a pharmaceutically acceptable carrier. In one aspect, the compositions are pharmaceutical formulations for use in the therapeutic methods of this disclosure. In a further aspect, the disclosure provides a pharmaceutical composition comprising, or alternatively consisting essentially of, or yet further consisting of, the isolated peptide fragment or a variant or hybrid or a biological equivalent of each thereof, in a concentration such that a therapeutically effective amount of the or pharmacological dose of the composition causes at least a 75%, or alternatively at least a 80%, or alternatively at least a 85%, or alternatively at least a 90%, or alternatively at least a 95% or alternatively at least a 97% reduction in viral infectivity when applied in a molar concentration of less than

1 micromolar, to a culture of responsive virus (e.g., influenza virus) virion, as compared to a control that does not receive the composition. In alternative aspects, the pharmacological dose of the composition when applied to the is in the range of about 150 nM to about 2 micromolar, or about 200 nM to about 2 micromolar, or about 250 nM to about 2 micromolar, or about 300 nM to about 2 micromolar, or about 400 nM to about 2 micromolar, or about 450 nm to about 2 micromolar, or about 500 nM to about 2 micromolar, or about 550 nM to about 2 micromolar, or about 600 nM to about 2 micromolar, or about 700 nM to about 2 micromolar, or about 800 nM to about 2 micromolar, or about 900 nM to about 2 micromolar, or about 1 micromolar to about 2 micromolar, or about 1 .5 micromolar to about 2 micromolar, or about 50 nM to about 1 micromolar, or about 100 nM to about 1 micromolar, or about 150 nM to about 1 micromolar, or about 200 nM to about 1 micromolar, or about 250 nM to about 1 micromolar, or about 300 nM to about 1 micromolar, or about 400 nM to about 1 micromolar, or about 450 nm to about 1 micromolar, or about 500 nM to about 1 micromolar, or about 550 nM to about 1 micromolar, or about 600 nM to about 1 micromolar, or about 700 nM to about 1 micromolar, or about 800 nM to about 1 micromolar. Comparative effectiveness can be determined by suitable in vitro or in vivo methods as known in the art and described herein.

Isolated Polynucleotides and Compositions

[0116] This disclosure also provides isolated polynucleotides encoding the polypeptides and peptide fragments described above as well as the complementary sequences, sequences that hybridize under stringent conditions to the

polynucleotides or their complements, and biological equivalents of each thereof. In one aspect the isolated polynucleotides encode peptide fragments comprising the sequences (SEQ ID NOS: 1 through 12) and their biological equivalents, or variants. In another aspect, the polynucleotides or their biological equivalents, variants or hybrids are labeled with a detectable marker or label, such as a dye or radioisotope, for ease of detection. These polynucleotides can further comprise, or alternatively consist essentially of, or yet further consist of an expression or delivery vehicle, such as a liposome or viral vector.

[0117] This disclosure also provides the isolated complementary polynucleotides to the sequences identified above, their biological equivalents or their complements. Complementarity can be determined using traditional hybridization under conditions of moderate or high stringency. As used herein, the term polynucleotide intends DNA and RNA as well as modified nucleotides. For example, this disclosure also provides the anti-sense polynucleotide strand, e.g. antisense RNA or siRNA to these sequences or their complements. One can obtain an antisense RNA using the sequences that encode SEQ ID NOS: 1 through 8 or 41 through 47 using a methodology known to one of ordinary skill in the art wherein the degeneracy of the genetic code provides several polynucleotide sequences that encode the same polypeptide or the methodology described in Van der Krol et al. (1988)

BioTechniques 6:958. In another aspect, the polynucleotides or their biological equivalents are labeled with a detectable marker or label, such as a dye or radioisotope, for ease of detection.

[0118] Also provided are isolated polynucleotides encoding substantially homologous and biologically equivalent peptide fragments to the inventive peptide fragments. Substantially homologous and biologically equivalent intends those having varying degrees of homology, such as at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively at least 80%, or alternatively, at least 85%, or alternatively at least 90%, or alternatively, at least 95%, or alternatively at least 97% homologous as defined above and which encode peptides having the biological activity to bind Atg3 as described herein. It should be understood although not always explicitly stated that embodiments to substantially homologous peptide fragments and polynucleotides are intended for each aspect of this disclosure, e.g., peptides, polynucleotides and antibodies.

[0119] The polynucleotides of this disclosure can be replicated using conventional recombinant techniques. Alternatively, the polynucleotides can be replicated using PCR technology. PCR is the subject matter of U.S. Patent Nos. 4,683,195;

4,800,159; 4,754,065; and 4,683,202 and described in PCR: The Polymerase Chain Reaction (Mullis et al. eds, Birkhauser Press, Boston (1994)) and references cited therein. Yet further, one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate the DNA. Accordingly, this disclosure also provides a process for obtaining the peptide fragments of this disclosure by providing the linear sequence of the polynucleotide, appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides. In a separate embodiment, these polynucleotides are further isolated. Still further, one of skill in the art can operatively link the polynucleotides to regulatory sequences for their expression in a host cell. The polynucleotides and regulatory sequences are inserted into the host cell (prokaryotic or eukaryotic) for replication and amplification. The DNA so amplified can be isolated from the cell by methods well known to those of skill in the art. A process for obtaining

polynucleotides by this method is further provided herein as well as the

polynucleotides so obtained.

[0120] In one aspect, the RNA is short interfering RNA, also known as siRNA. Methods to prepare and screen interfering RNA and select for the ability to block polynucleotide expression are known in the art and non-limiting examples of which are shown below. These interfering RNA are provided by this disclosure alone or in combination with a suitable vector or within a host cell. Compositions containing the RNAi are further provided. RNAi is useful to knock-out or knock-down select functions in a cell or tissue as known in the art and described infra. siRNA sequences can be designed by obtaining the target mRNA sequence and determining an appropriate siRNA complementary sequence. siRNAs of the disclosure are designed to interact with a target sequence, meaning they

complement a target sequence sufficiently to hybridize to that sequence. An siRNA can be 100% identical to the target sequence. However, homology of the siRNA sequence to the target sequence can be less than 100% as long as the siRNA can hybridize to the target sequence. Thus, for example, the siRNA molecule can be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the target sequence or the complement of the target sequence. Therefore, siRNA molecules with insertions, deletions or single point mutations relative to a target may also be used. The generation of several different siRNA sequences per target mRNA is recommended to allow screening for the optimal target sequence. A homology search, such as a BLAST search, should be performed to ensure that the siRNA sequence does not contain homology to any known mammalian gene.

[0121] In general, its preferable that the target sequence be located at least 100- 200 nucleotides from the AUG initiation codon and at least 50-100 nucleotides away from the termination codon of the target mRNA (Duxbury (2004) J. Surgical Res. 1 17:339-344).

[0122] Researchers have determined that certain characteristics are common in siRNA molecules that effectively silence their target gene (Duxbury (2004) J.

Surgical Res. 1 17:339-344; Ui-Tei et al. (2004) Nucl. Acids Res. 32:936-48). As a general guide, siRNAs that include one or more of the following conditions are particularly useful in gene silencing in mammalian cells: GC ratio of between 45- 55%, no runs of more than 9 G/C residues, G/C at the 5' end of the sense strand; A U at the 5' end of the antisense strand; and at least 5 A U residues in the first 7 bases of the 5' terminal of the antisense strand.

[0123] siRNA are, in general, from about 10 to about 30 nucleotides in length. For example, the siRNA can be 10-30 nucleotides long, 12-28 nucleotides long, 15-25 nucleotides long, 19-23 nucleotides long, or 21 -23 nucleotides long. When an siRNA contains two strands of different lengths, the longer of the strands designates the length of the siRNA. In this situation, the unpaired nucleotides of the longer strand would form an overhang. [0124] The term siRNA includes short hairpin RNAs (shRNAs). shRNAs comprise a single strand of RNA that forms a stem-loop structure, where the stem consists of the complementary sense and antisense strands that comprise a double-stranded siRNA, and the loop is a linker of varying size. The stem structure of shRNAs generally is from about 10 to about 30 nucleotides long. For example, the stem can be 10-30 nucleotides long, 12-28 nucleotides long, 15-25 nucleotides long, 19-23 nucleotides long, or 21 -23 nucleotides long. Tools to assist siRNA design are readily available to the public. For example, a computer-based siRNA design tool is available on the internet at www.dharmacon.com, last accessed on November 26, 2007.

[0125] This disclosure also provides compositions for in vitro and in vivo use comprising, or alternatively consisting essentially of, or yet further consisting of one or more of the isolated polynucleotide as described herein and a pharmaceutically acceptable carrier. In one aspect, the compositions are pharmaceutical formulations for use in the therapeutic methods of this disclosure. In a further aspect, the disclosure provides a pharmaceutical composition comprising, or alternatively consisting essentially of, or yet further consisting of, the isolated polynucleotide in a concentration such that a therapeutically effective amount of the or pharmacological dose of the composition causes at least a 75%, or alternatively at least a 80%, or alternatively at least a 85%, or alternatively at least a 90%, or alternatively at least a

95% or alternatively at least a 97% reduction in viral infectivity when applied in a molar concentration of less than 1 micromolar, to a culture of responsive virus (e.g., influenza virus) virion, as compared to a control that does not receive the

composition. In alternative aspects, the pharmacological dose of the composition when applied to the is in the range of about 150 nM to about 2 micromolar, or about

200 nM to about 2 micromolar, or about 250 nM to about 2 micromolar, or about 300 nM to about 2 micromolar, or about 400 nM to about 2 micromolar, or about 450 nm to about 2 micromolar, or about 500 nM to about 2 micromolar, or about 550 nM to about 2 micromolar, or about 600 nM to about 2 micromolar, or about 700 nM to about 2 micromolar, or about 800 nM to about 2 micromolar, or about 900 nM to about 2 micromolar, or about 1 micromolar to about 2 micromolar, or about 1 .5 micromolar to about 2 micromolar, or about 50 nM to about 1 micromolar, or about

100 nM to about 1 micromolar, or about 150 nM to about 1 micromolar, or about 200 nM to about 1 micromolar, or about 250 nM to about 1 micromolar, or about 300 nM to about 1 micromolar, or about 400 nM to about 1 micromolar, or about 450 nm to about 1 micromolar, or about 500 nM to about 1 micromolar, or about 550 nM to about 1 micromolar, or about 600 nM to about 1 micromolar, or about 700 nM to about 1 micromolar, or about 800 nM to about 1 micromolar. Comparative

effectiveness can be determined by suitable in vitro or in vivo methods as known in the art and described herein.

Synthesis of dsRNA and siRNA

[0126] dsRNA and siRNA can be synthesized chemically or enzymatically in vitro as described in Micura (2002) Agnes Chem. Int. Ed. Emgl. 41 :2265-2269; Betz (2003) Promega Notes 85:15-18; and Paddison and Hannon (2002) Cancer Cell. 2:17-23. Chemical synthesis can be performed via manual or automated methods, both of which are well known in the art as described in Micura (2002), supra. siRNA can also be endogenously expressed inside the cells in the form of shRNAs as described in Yu et al. (2002) Proc. Natl. Acad. Sci. USA 99:6047-6052; and

McManus et al. (2002) RNA 8:842-850. Endogenous expression has been achieved using plasmid-based expression systems using small nuclear RNA promoters, such as RNA polymerase III U6 or H1 , or RNA polymerase II U 1 as described in

Brummelkamp et al. (2002) Science 296:550-553 (2002); and Novarino et al. (2004) J. Neurosci. 24:5322-5330.

[0127] In vitro enzymatic dsRNA and siRNA synthesis can be performed using an RNA polymerase mediated process to produce individual sense and antisense strands that are annealed in vitro prior to delivery into the cells of choice as describe in Fire et al. (1998) Nature 391 :806-81 1 ; Donze and Picard (2002) Nucl. Acids Res. 30(10):e46; Yu et al. (2002) Proc. Natl. Acad. Sci. USA 99, 6047-52; and Shim et al. (2002) J. Biol. Chem. 277:30413-30416. Several manufacturers (Promega, Ambion, New England Biolabs, and Stragene) produce transcription kits useful in performing the in vitro synthesis.

[0128] In vitro synthesis of siRNA can be achieved, for example, by using a pair of short, duplex oligonucleotides that contain T7 RNA polymerase promoters upstream of the sense and antisense RNA sequences as the DNA template. Each

oligonucleotide of the duplex is a separate template for the synthesis of one strand of the siRNA. The separate short RNA strands that are synthesized are then annealed to form siRNA as described in Protocols and Applications, Chapter 2: RNA

interference, Promega Corporation (2005) accessible at

www.promega.com/paguide/chap2.htm.

[0129] In vitro synthesis of dsRNA can be achieved, for example, by using a T7 RNA polymerase promoter at the 5'-ends of both DNA target sequence strands. This is accomplished by using separate DNA templates, each containing the target sequence in a different orientation relative to the T7 promoter, transcribed in two separate reactions. The resulting transcripts are mixed and annealed post- transcriptionally. DNA templates used in this reaction can be created by PCR or by using two linearized plasmid templates, each containing the T7 polymerase promoter at a different end of the target sequence. Protocols and Applications, Chapter 2: RNA interference, Promega Corporation, (2005).

[0130] RNA can be obtained by first inserting a DNA polynucleotide into a suitable prokaryotic or eukaryotic host cell. The DNA can be inserted by any appropriate method, e.g., by the use of an appropriate gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation. When the cell replicates and the DNA is transcribed into RNA; the RNA can then be isolated using methods well known to those of skill in the art, for example, as set forth in Sambrook and Russell (2001 ) supra. For instance, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook and Russell (2001 ) supra or extracted by nucleic-acid-binding resins following the accompanying instructions provided by manufactures.

[0131] In order to express the proteins described herein, delivery of nucleic acid sequences encoding the gene of interest can be delivered by several techniques. Examples of which include viral technologies (e.g. retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like) and non- viral technologies (e.g. DNA/liposome complexes, micelles and targeted viral protein- DNA complexes) as described herein. Once inside the cell of interest, expression of the transgene can be under the control of ubiquitous promoters (e.g. EF-1 ) or tissue specific promoters (e.g. Calcium Calmodulin kinase 2 (CaMKI) promoter, NSE promoter and human Thy-1 promoter). Alternatively expression levels may controlled by use of an inducible promoter system (e.g. Tet on/off promoter) as described in Wiznerowicz et al. (2005) Stem Cells 77:8957-8961 .

[0132] Non-limiting examples of promoters include, but are not limited to, the cytomegalovirus (CMV) promoter (Kaplitt et al. (1994) Nat. Genet. 8:148-154), CMV/human y- globin promoter (Mandel et al. (1998) J. Neurosci. 18:4271 -4284), NCX1 promoter, yMHC promoter, MLC2v promoter, GFAP promoter (Xu et al. (2001 ) Gene Ther. 8:1323-1332), the 1 .8-kb neuron-specific enolase (NSE) promoter (Klein et al. (1998) Exp. Neurol. 150:183-194), chicken beta actin (CBA) promoter

(Miyazaki (1989) Gene 79:269-277) and the β-glucuronidase (GUSB) promoter (Shipley et al. (1991 ) Genetics 10:1009-1018), the human serum albumin promoter, the alpha-1 -antitrypsin promoter. To improve expression, other regulatory elements may additionally be operably linked to the transgene, such as, e.g., the Woodchuck Hepatitis Virus Post-Regulatory Element (WPRE) (Donello et al. (1998) J. Virol. 72: 5085-5092) or the bovine growth hormone (BGH) polyadenylation site.

[0133] The disclosure further provides the isolated polynucleotides of this disclosure operatively linked to a promoter of RNA transcription, as well as other regulatory sequences for replication and/or transient or stable expression of the DNA or RNA. As used herein, the term "operatively linked" means positioned in such a manner that the promoter will direct transcription of RNA off the DNA molecule.

Examples of such promoters are SP6, T4 and T7. In certain embodiments, cell- specific promoters are used for cell-specific expression of the inserted

polynucleotide. Vectors which contain a promoter or a promoter/enhancer, with termination codons and selectable marker sequences, as well as a cloning site into which an inserted piece of DNA can be operatively linked to that promoter are well known in the art and commercially available. For general methodology and cloning strategies, see Gene Expression Technology (Goeddel ed., Academic Press, Inc. (1991 )) and references cited therein and Vectors: Essential Data Series (Gacesa and Ramji, eds., John Wiley & Sons, N.Y. (1994)), which contains maps, functional properties, commercial suppliers and a reference to GenEMBL accession numbers for various suitable vectors. Preferable, these vectors are capable of transcribing RNA in vitro or in vivo. [0134] Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, etc. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and efficient transformation of cells both in vitro and in vivo. When a nucleic acid is inserted into a suitable host cell, e.g., a prokaryotic or a eukaryotic cell and the host cell replicates, the protein can be recombinantly produced. Suitable host cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells as described above and constructed using well known methods. See Sambrook and Russell (2001 ), supra. In addition to the use of viral vector for insertion of exogenous nucleic acid into cells, the nucleic acid can be inserted into the host cell by methods well known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; DEAE-dextran; electroporation; or microinjection. See Sambrook and Russell (2001 ), supra for this methodology.

[0135] The present disclosure also provides delivery vehicles suitable for delivery of a polynucleotide of the disclosure into cells (whether in vivo, ex vivo, or in vitro). A polynucleotide of the disclosure can be contained within a gene delivery vehicle, a cloning vector or an expression vector. These vectors (especially expression vectors) can in turn be manipulated to assume any of a number of forms which may, for example, facilitate delivery to and/or entry into a cell.

[0136] In one aspect when isolated polynucleotides encoding two or more peptides, at least one of which is SEQ ID NOS. 1 through 12, a biological equivalent or retro-inverso forms, are intended to be translated and optionally expressed, the polynucleotides encoding the peptides may be organized within a recombinant mRNA or cDNA molecule that results in the transcript that expresses on a single mRNA molecule the at least two peptides. This is accomplished by use of a polynucleotide that has the biological activity of an internal ribosome entry site (IRES) located between the polynucleotide encoding the two peptides. IRES elements initiate translation of polynucleotides without the use of a "cap" structure traditionally thought to be necessary for translation of proteins in eukaryotic cells. Initially described in connection with the untranslated regions of individual

picornaviruses, e.g. polio virus and encephalomyocarditis virus, IRES elements were later shown to efficiently initiate translation of reading frames in eukaryotic cells and when positioned downstream from a eukaryotic promoter, it will not influence the "cap"-dependent translation of the first cistron. The IRES element typically is at least 450 nucleotides long when in occurs in viruses and possesses, at its 3' end, a conserved "UUUC" sequence followed by a polypyrimidine trace, a G-poor spacer and an AUG triple.

[0137] As used herein, the term "IRES" is intended to include any molecule such as a mRNA polynucleotide or its reverse transcript (cDNA) which is able to initiate translation of the gene downstream from the polynucleotide without the benefit of a cap site in a eukaryotic cell. "IRES" elements can be identical to sequences found in nature, such as the picornavirus IRES, or they can be non-naturally or non-native sequences that perform the same function when transfected into a suitable host cell. Bi- and poly-cistronic expression vectors containing naturally occurring IRES elements are known in the art and described for example, in Pestova et al. (1998) Genes Dev. 12:67-83 and International Application No. WO 01/04306, which in turn on page 17, lines 35 to 38 references several literature references which include, but are not limited to Ramesh et al. (1996) Nucl. Acids Res. 24:2697-2700; Pelletier et al. (1988) Nature 334:320-325; Jan et al. (1989) J. Virol. 63:1651 -1660; and Davies et al. (1992) J. Virol. 66:1924-1932. Paragraph [0009] of U.S. Patent Appl. Publ. No.: 2005/0014150 A1 discloses several issued U.S. patents wherein a virally- derived IRES element was used to express foreign gene(s) in linear multi-cistronic mRNAs in mammalian cells, plant cells and generally in eukaryotic cells. U.S.

Patent Appl. Publ. No. 2004/0082034 A1 discloses an IRES element active in insect cells. Methods to identify new elements also are described in U.S. Patent No.

6,833,254.

[0138] Also within the term "IRES" element are cellular sequences similar to that disclosed in U.S. Patent No. 6,653,132. The patent discloses a sequence element (designated SP163) composed of sequences derived from the 5'-UTR of VEGF (Vascular Endothelial Growth Factor gene), which, was presumably generated through a previously unknown mode of alternative splicing. The patentees report that an advantages of SP163 is that it is a natural cellular IRES element with a superior performance as a translation stimulator and as a mediator of cap- independent translation relative to known cellular IRES elements and that these functions are maintained under stress conditions.

[0139] Further within the term "IRES" element are artificial sequences that function as IRES elements that are described, for example, in U.S. Patent Appl. Publ. No.: 2005/0059004 A1 .

[0140] Operatively linked to the IRES element and separately, are sequences necessary for the translation and proper processing of the peptides. Examples of such include, but are not limited to a eukaryotic promoter, an enhancer, a termination sequence and a polyadenylation sequence. Construction and use of such

sequences are known in the art and are combined with IRES elements and protein sequences using recombinant methods. Operatively linked" shall mean the juxtaposition of two or more components in a manner that allows them to junction for their intended purpose. Promoters are sequences which drive transcription of the marker or target protein. It must be selected for use in the particular host cell, i.e., mammalian, insect or plant. Viral or mammalian promoters will function in

mammalian cells. The promoters can be constitutive or inducible, examples of which are known and described in the art.

[0141] In one aspect, the peptides are transcribed and translated from a separate recombinant polynucleotide and combined into a functional protein in the host cell. This recombinant polynucleotide does not require the IRES element or marker protein although in one aspect, it may be present.

[0142] These isolated host cells containing the polynucleotides of this disclosure are useful in the methods described herein as well as for the recombinant replication of the polynucleotides and for the recombinant production of peptides and for high throughput screening.

Host Cells [0143] Also provided are isolated host cells comprising one or more of the polypeptides, peptide fragments and/or polynucleotides of this disclosure. Suitable cells containing the inventive polypeptides and/or polynucleotides include prokaryotic and eukaryotic cells, which include, but are not limited to bacterial cells, yeast cells, insect cells, animal cells, mammalian cells, murine cells, rat cells, sheep cells, simian cells and human cells. Examples of bacterial cells include Escerichia coli,

Salmonella enterica and Streptococcus gordonii. The cells can be purchased from a commercial vendor such as the American Type Culture Collection (ATCC, Rockville Maryland, USA) or cultured from an isolate using methods known in the art.

Examples of suitable eukaryotic cells include, but are not limited to 293T HEK cells, as well as the hamster cell line BHK-21 ; the murine cell lines designated NIH3T3, NSO, C127, the simian cell lines COS, Vera; and the human cell lines HeLa, PER.C6 (commercially available from Crucell) U-937 and Hep G2. A non-limiting example of insect cells include Spodoptera frugiperda. Examples of yeast useful for expression include, but are not limited to Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Torulopsis, Yarrowia, or Pichia. See e.g., U.S. Patent Nos. 4,812,405; 4,818,700; 4,929,555; 5,736,383; 5,955,349; 5,888,768 and 6,258,559.

[0144] In addition to species specificity, the cells can be of any particular tissue type such as neuronal or alternatively a somatic or embryonic stem cell such as a stem cell that can or can not differentiate into a neuronal cell, e.g., embryonic stem cell, an induced pluripotent embryonic stem cell (iPSC), adipose stem cell, neuronal stem cell and hematopoietic stem cell. The stem cell can be of human or animal origin, such as mammalian.

Therapeutic Antibody Compositions

[0145] This disclosure also provides an antibody capable of specifically forming a complex with a protein or peptide or peptide fragment of this disclosure, which are useful in the therapeutic methods of this disclosure, e.g. the proteins and peptide fragments identified in Tables 1 through 4 and 6, supra. The term "antibody" includes polyclonal antibodies and monoclonal antibodies, antibody fragments, as well as derivatives thereof (described above). The antibodies include, but are not limited to mouse, rat, and rabbit or human antibodies, limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes, etc. The antibodies are also useful to identify and purify therapeutic and/or diagnostic polypeptides. Also provided are hybridoma cell lines producing

monoclonal antibodies of this disclosure.

[0146] Polyclonal antibodies of the disclosure can be generated using conventional techniques known in the art and are well-described in the literature. Several methodologies exist for production of polyclonal antibodies. For example, polyclonal antibodies are typically produced by immunization of a suitable mammal such as, but not limited to, chickens, goats, guinea pigs, hamsters, horses, mice, rats, and rabbits. An antigen is injected into the mammal, which induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This IgG is purified from the mammal's serum. Variations of this methodology include modification of adjuvants, routes and site of administration, injection volumes per site and the number of sites per animal for optimal production and humane treatment of the animal. For example, adjuvants typically are used to improve or enhance an immune response to antigens. Most adjuvants provide for an injection site antigen depot, which allows for a slow release of antigen into draining lymph nodes. Other adjuvants include surfactants which promote concentration of protein antigen molecules over a large surface area and immunostimulatory molecules. Non-limiting examples of adjuvants for

polyclonal antibody generation include Freund's adjuvants, Ribi adjuvant system, and Titermax. Polyclonal antibodies can be generated using methods described in U.S. Patent Nos. 7,279,559; 7,1 19,179; 7,060,800; 6,709,659; 6,656,746; 6,322,788; 5,686,073; and 5,670,153.

[0147] The monoclonal antibodies of the disclosure can be generated using conventional hybridoma techniques known in the art and well-described in the literature. For example, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1 , NS2, AE-1 , L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1 , Sp2 SA5, U397, MLA 144, ACT IV, MOLT4, DA-1 , JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O) or the like, or heteromyelomas, fusion products thereof, or any cell or fusion cell derived there from, or any other suitable cell line as known in the art (see, e.g., www.atcc.org, www.lifetech.com., last accessed on November 26, 2007, and the like), with antibody producing cells, such as, but not linnited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or any other cells expressing heavy or light chain constant or variable or framework or CDR

sequences, either as endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the like or any combination thereof. Antibody producing cells can also be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest. Any other suitable host cell can also be used for expressing heterologous or endogenous nucleic acid encoding an antibody, specified fragment, hybrid or biological equivalent of each thereof, of the present disclosure. The fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods.

[0148] In one embodiment, the antibodies described herein can be generated using a Multiple Antigenic Peptide (MAP) system. The MAP system utilizes a peptidyl core of three or seven radially branched lysine residues, on to which the antigen peptides of interest can be built using standard solid-phase chemistry. The lysine core yields the MAP bearing about 4 to 8 copies of the peptide epitope depending on the inner core that generally accounts for less than 10% of total molecular weight. The MAP system does not require a carrier protein for

conjugation. The high molar ratio and dense packing of multiple copies of the antigenic epitope in a MAP has been shown to produce strong immunogenic response. This method is described in U.S. Patent No. 5,229,490.

[0149] Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from various commercial vendors such as Cambridge Antibody Technologies

(Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg, Del.), Biovation (Aberdeen, Scotland, UK) Biolnvent (Lund, Sweden), using methods known in the art. See U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternative methods rely upon immunization of transgenic animals (e.g., SCID mice, Nguyen et al. (1977) Microbiol. Immunol. 41 :901 -907 (1997); Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-1 18; Eren et al. (1998) Immunol. 93:154-161 that are capable of producing a repertoire of human antibodies, as known in the art and/or as described herein. Such techniques, include, but are not limited to, ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA 95:14130-14135); single cell antibody producing technologies (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052, Wen et al. (1987) J. Immunol. 17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems,

(Cambridge, Mass).; Gray et al. (1995) J. Imm. Meth. 182:155-163; and Kenny et al. (1995) Bio. Technol. 13:787-790); B-cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134).

[0150] Antibody derivatives of the present disclosure can also be prepared by delivering a polynucleotide encoding an antibody of this disclosure to a suitable host such as to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce such antibodies in their milk. These methods are known in the art and are described for example in U.S. Patent Nos. 5,827,690;

5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

[0151] The term "antibody derivative" includes post-translational modification to linear polypeptide sequence of the antibody or fragment. For example, U.S. Patent No. 6,602,684 B1 describes a method for the generation of modified glycol-forms of antibodies, including whole antibody molecules, antibody fragments, or fusion proteins that include a region equivalent to the Fc region of an immunoglobulin, having enhanced Fc-mediated cellular toxicity, and glycoproteins so generated.

[0152] Antibody derivatives also can be prepared by delivering a polynucleotide of this disclosure to provide transgenic plants and cultured plant cells (e.g., but not limited to tobacco, maize, and duckweed) that produce such antibodies, specified portions or variants in the plant parts or in cells cultured there from. For example, Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240:95-1 18 and references cited therein, describe the production of transgenic tobacco leaves expressing large amounts of recombinant proteins, e.g., using an inducible promoter. Transgenic maize have been used to express mammalian proteins at commercial production levels, with biological activities equivalent to those produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147 and references cited therein. Antibody derivatives have also been produced in large amounts from transgenic plant seeds including antibody fragments, such as single chain antibodies (scFv's), including tobacco seeds and potato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol. 38:101 -109 and reference cited therein. Thus, antibodies of the present disclosure can also be produced using transgenic plants, according to known methods.

[0153] Antibody derivatives also can be produced, for example, by adding exogenous sequences to modify immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic. Generally part or all of the non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions are replaced with human or other amino acids.

[0154] In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Humanization or engineering of antibodies of the present disclosure can be performed using any known method such as, but not limited to, those described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101 ; 5,585,089; 5,225,539; and 4,816,567.

[0155] Techniques for making partially to fully human antibodies are known in the art and any such techniques can be used. According to one embodiment, fully human antibody sequences are made in a transgenic mouse which has been engineered to express human heavy and light chain antibody genes. Multiple strains of such transgenic mice have been made which can produce different classes of antibodies. B cells from transgenic mice which are producing a desirable antibody can be fused to make hybridoma cell lines for continuous production of the desired antibody. (See, e.g., Russel et al. (2000) Infection and Immunity April 2000:1820- 1826; Gallo et al. (2000) European J. of Immun. 30:534-540; Green (1999) J. of Immun. Methods 231 :1 1 -23; Yang et al. (1999) J. of Leukocyte Biology 66:401 -410; Yang (1999) Cancer Research 59(6): 1236-1243; Jakobovits (1998) Advanced Drug Delivery Reviews 31 :33-42; Green and Jakobovits (1998) J. Exp. Med. 188(3):483- 495; Jakobovits (1998) Exp. Opin. Invest. Drugs 7(4):607-614; Tsuda et al. (1997) Genomics 42:413-421 ; Sherman-Gold (1997) Genetic Engineering News 17(14); Mendez et al. (1997) Nature Genetics 15:146-156; Jakobovits (1996) Weir's

Handbook of Experimental Immunology, The Integrated Immune System Vol. IV, 194.1 -194.7; Jakobovits (1995) Current Opinion in Biotechnology 6:561 -566;

Mendez et al. (1995) Genomics 26:294-307; Jakobovits (1994) Current Biology 4(8):761 -763; Arbones et al. (1994) Immunity 1 (4):247-260; Jakobovits (1993) Nature 362(6417):255-258; Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551 -2555; and U.S. Patent No. 6,075,181 .)

[0156] The antibodies of this disclosure also can be modified to create chimeric antibodies. Chimeric antibodies are those in which the various domains of the antibodies' heavy and light chains are coded for by DNA from more than one species. See, e.g., U.S. Patent No. 4,816,567.

[0157] Alternatively, the antibodies of this disclosure can also be modified to create veneered antibodies. Veneered antibodies are those in which the exterior amino acid residues of the antibody of one species are judiciously replaced or "veneered" with those of a second species so that the antibodies of the first species will not be immunogenic in the second species thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein is primarily dependent on the nature of its surface, the immunogenicity of an antibody could be reduced by replacing the exposed residues which differ from those usually found in another mammalian species antibodies. This judicious replacement of exterior residues should have little, or no, effect on the interior domains, or on the interdomain contacts. Thus, ligand binding properties should be unaffected as a consequence of alterations which are limited to the variable region framework residues. The process is referred to as "veneering" since only the outer surface or skin of the antibody is altered, the supporting residues remain undisturbed. [0158] The procedure for "veneering" makes use of the available sequence data for human antibody variable domains compiled by Kabat et al. (1987) Sequences of Proteins of Immunological Interest, 4th ed., Bethesda, Md., National Institutes of Health, updates to this database, and other accessible U.S. and foreign databases (both nucleic acid and protein). Non-limiting examples of the methods used to generate veneered antibodies include EP 519596; U.S. Patent No. 6,797,492; and described in Padlan et al. (1991 ) Mol. Immunol. 28(4-5):489-498.

[0159] The term "antibody derivative" also includes "diabodies" which are small antibody fragments with two antigen-binding sites, wherein fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain. (See for example, EP 404,097; WO 93/1 1 161 ; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. (See also, U.S. Patent No. 6,632,926 to Chen et al. which discloses antibody variants that have one or more amino acids inserted into a hypervariable region of the parent antibody and a binding affinity for a target antigen which is at least about two fold stronger than the binding affinity of the parent antibody for the antigen.)

[0160] The term "antibody derivative" further includes "linear antibodies". The procedure for making linear antibodies is known in the art and described in Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (V_H -C_H 1 -VH -C_H1 ) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0161] The antibodies of this disclosure can be recovered and purified from recombinant cell cultures by known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite

chromatography and lectin chromatography. High performance liquid

chromatography ("HPLC") can also be used for purification. [0162] Antibodies of the present disclosure include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells, or alternatively from a prokaryotic cells as described above.

[0163] If a monoclonal antibody being tested binds with protein or polypeptide, then the antibody being tested and the antibodies provided by the hybridomas of this disclosure are equivalent. It also is possible to determine without undue

experimentation, whether an antibody has the same specificity as the monoclonal antibody of this disclosure by determining whether the antibody being tested prevents a monoclonal antibody of this disclosure from binding the protein or polypeptide with which the monoclonal antibody is normally reactive. If the antibody being tested competes with the monoclonal antibody of the disclosure as shown by a decrease in binding by the monoclonal antibody of this disclosure, then it is likely that the two antibodies bind to the same or a closely related epitope. Alternatively, one can pre-incubate the monoclonal antibody of this disclosure with a protein with which it is normally reactive, and determine if the monoclonal antibody being tested is inhibited in its ability to bind the antigen. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or a closely related, epitopic specificity as the monoclonal antibody of this disclosure.

[0164] The term "antibody" also is intended to include antibodies of all isotypes. Particular isotypes of a monoclonal antibody can be prepared either directly by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class switch variants using the procedure described in

Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984; J. Immunol. Methods 74:307.

[0165] The isolation of other hybridomas secreting monoclonal antibodies with the specificity of the monoclonal antibodies of the disclosure can also be accomplished by one of ordinary skill in the art by producing anti-idiotypic antibodies. Herlyn et al. (1986) Science 232:100. An anti-idiotypic antibody is an antibody which recognizes unique determinants present on the monoclonal antibody produced by the hybridoma of interest. [0166] Idiotypic identity between monoclonal antibodies of two hybridomas demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant. Thus, by using antibodies to the epitopic determinants on a monoclonal antibody it is possible to identify other hybridomas expressing monoclonal antibodies of the same epitopic specificity.

[0167] It is also possible to use the anti-idiotype technology to produce monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the mirror image of the epitope bound by the first monoclonal antibody. Thus, in this instance, the anti-idiotypic monoclonal antibody could be used for immunization for production of these antibodies.

[0168] The disclosure also provides antibodies that not only bind to a peptide fragment as identified herein but are further characterized by blocking Rubicon p22phox binding. The blocking antibodies are identified using methods well know in the art.

[0169] Antibodies can be conjugated, for example, to a pharmaceutical agent, such as chemotherapeutic drug or a toxin. They can be linked to a cytokine, to a ligand, to another antibody. Suitable agents for coupling to antibodies to achieve an anti-tumor effect include cytokines, such as interleukin 2 (IL-2) and Tumor Necrosis Factor (TNF); photosensitizers, for use in photodynamic therapy, including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine;

131 90 212

radionuclides, such as iodine-131 ( I), yttrium-90 ( Y), bismuth-212 ( Bi),

213 99m 186

bismuth-213 ( Bi), technetium-99m ( Tc), rhenium-186 ( Re), and rhenium-188

188

( Re); antibiotics, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, cytotoxin from Chinese cobra (naja naja atra), and gelonin (a plant toxin); ribosome inactivating proteins from plants, bacteria and fungi, such as restrictocin (a ribosome inactivating protein produced by Aspergillus restrictus), saporin (a ribosome inactivating protein from Saponaria officinalis), and RNase; tyrosine kinase inhibitors;

Iy207702 (a difluorinated purine nucleoside); liposomes containing anti cystic agents (e.g., antisense oligonucleotides, plasmids which encode for toxins, methotrexate, etc.); and other antibodies or antibody fragments, such as F(ab).

[0170] The antibodies of the disclosure also can be bound to many different carriers. Thus, this disclosure also provides compositions containing the antibodies and another substance, active or inert. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the disclosure. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation.

Compositions for Therapy

[0171] One or more of the above host cell, antibody, antibody fragment, antibody derivative, protein, hybrid peptide, variant peptide, peptide fragment or

polynucleotide encoding these compositions can be further combined with a carrier, a pharmaceutically acceptable carrier or medical device which is suitable for use of the compositions in diagnostic or therapeutic methods. Thus, the compositions comprise, or alternatively consist essentially of, or yet further consists of, one or more of the above compositions described above in combination with a carrier, a pharmaceutically acceptable carrier or medical device.

[0172] The carrier can be a liquid phase carrier or a solid phase carrier, e.g., bead, gel, microarray, or carrier molecule such as a liposome. The composition can optionally further comprise at least one further compound, protein or composition.

[0173] Additional examples of "carriers" includes therapeutically active agents such as another peptide or protein (e.g., an Fab' fragment). For example, an antibody of this disclosure, derivative or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody {e.g., to produce a bispecific or a multispecific antibody), a cytotoxin, a cellular ligand or an antigen. Accordingly, this disclosure encompasses a large variety of antibody conjugates, bi- and multispecific molecules, and fusion proteins, whether or not they target the same epitope as the antibodies of this disclosure. [0174] Yet additional examples of carriers are organic molecules (also termed modifying agents) or activating agents, that can be covalently attached, directly or indirectly, to an antibody of this disclosure. Attachment of the molecule can improve pharmacokinetic properties (e.g., increased in vivo serum half-life). Examples of organic molecules include, but are not limited to a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. As used herein, the term "fatty acid" encompasses mono-carboxylic acids and di-carboxylic acids. A "hydrophilic polymeric group," as the term is used herein, refers to an organic polymer that is more soluble in water than in octane.

[0175] Hydrophilic polymers suitable for modifying antibodies of the disclosure can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone. A suitable hydrophilic polymer that modifies the antibody of the disclosure has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. The hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods. For example, a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.

[0176] Fatty acids and fatty acid esters suitable for modifying antibodies of the disclosure can be saturated or can contain one or more units of unsaturation.

Examples of such include, but are not limited to n-dodecanoate, n-tetradecanoate, n- octadecanoate, n-eicosanoate, n-docosanoate, n-triacontanoate, n-tetracontanoate, cis-A9-octadecanoate, all cis-A5,8,1 1 ,14-eicosatetraenoate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like.

Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group. The lower alkyl group can comprise from one to about twelve, preferably one to about six, carbon atoms.

[0177] The present disclosure provides a composition comprising, or alternatively consisting essentially of, or yet further consisting of, at least one antibody of this disclosure, derivative or fragment thereof, suitable for administration in an effective amount to increase or induce cell cancer death, eliminate viral particles associated with a viral infection, and/or treat or ameliorate a neurodegenerative disease. The compositions include, for example, pharmaceutical and diagnostic compositions/kits, comprising a pharmaceutically acceptable carrier and at least one antibody of this disclosure, variant, derivative or fragment thereof. As noted above, the composition can further comprise additional antibodies or therapeutic agents which in

combination, provide multiple therapies tailored to provide the maximum therapeutic benefit.

[0178] Alternatively, a composition of this disclosure can be co-administered with other therapeutic agents, whether or not linked to them or administered in the same dosing. They can be co-administered simultaneously with such agents (e.g., in a single composition or separately) or can be administered before or after

administration of such agents. Such agents can include Aricept® (donepezil), Razadyne® (galantamine), Nanenda® (mementine), Exalon® (rivastigmine),

Cognex® (tacrine), or other agents known to those skilled in the art. The

compositions can be combined with alternative therapies such as administration of tranquilizers, mood stabilizing medications, behavior treatments (including

treatments for aggressive behavior, incontinence, sleep difficulties, and wandering behavior), and individual activities and therapies (e.g., Reminiscence therapy) known to those skilled in the art.

Compositions for Diagnosis and Therapy

[0179] One or more of the above compositions can be further combined with a carrier, a pharmaceutically acceptable carrier or medical device which is suitable for use of the compositions in diagnostic or therapeutic methods.

[0180] The carrier can be a liquid phase carrier or a solid phase carrier, e.g., bead, gel, gene chip, microarray, or carrier molecule such as a liposome. The composition can optionally further comprise at least one further compound, protein or composition.

[0181] Additional examples of "carriers" includes therapeutically active agents such as another peptide or protein (e.g., an Fab' fragment). For example, an antibody of this disclosure, derivative or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., to produce a bispecific or a multispecific antibody), a cytotoxin, a cellular ligand or an antigen. Accordingly, this disclosure encompasses a large variety of antibody conjugates, bi- and multispecific molecules, and fusion proteins, whether or not they target the same epitope as the antibodies of this disclosure.

[0182] Yet additional examples of carriers are organic molecules (also termed modifying agents) or activating agents, that can be covalently attached, directly or indirectly, to an antibody of this disclosure. Attachment of the molecule can improve pharmacokinetic properties (e.g., increased in vivo serum half-life). Examples of organic molecules include, but are not limited to a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. As used herein, the term "fatty acid" encompasses mono-carboxylic acids and di-carboxylic acids. A "hydrophilic polymeric group," as the term is used herein, refers to an organic polymer that is more soluble in water than in octane.

[0183] Hydrophilic polymers suitable for modifying antibodies of the disclosure can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone. A suitable hydrophilic polymer that modifies the antibody of the disclosure has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. The hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods. For example, a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.

[0184] Fatty acids and fatty acid esters suitable for modifying antibodies of the disclosure can be saturated or can contain one or more units of unsaturation.

Examples of such include, but are not limited to n-dodecanoate, n-tetradecanoate, n- octadecanoate, n-eicosanoate, n-docosanoate, n-triacontanoate, n-tetracontanoate, cis-A9-octadecanoate, all cis-A5,8,1 1 ,14-eicosatetraenoate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like.

Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group. The lower alkyl group can comprise from one to about twelve, preferably one to about six, carbon atoms.

[0185] Also provided is a composition containing at least one antibody of this disclosure, derivative or fragment thereof, suitable for administration in an effective amount to modulate a neurodegenerative disorder correlative to the expression of the receptor or receptor complex. The compositions include, for example, pharmaceutical and diagnostic compositions/kits, comprising a pharmaceutically acceptable carrier and at least one antibody of this disclosure, variant, derivative or fragment thereof. As noted above, the composition can further comprise additional antibodies or therapeutic agents which in combination, provide multiple therapies tailored to provide the maximum therapeutic benefit.

[0186] Alternatively, a composition of this disclosure can be co-administered with other therapeutic agents, whether or not linked to them or administered in the same dosing. They can be co-administered simultaneously with such agents (e.g., in a single composition or separately) or can be administered before or after

administration of such agents. Such agents can include anticancer therapies such as irinotecan, 5-Fluorouracil, Erbitux, Cetuximab, FOLFOX, radiation therapy, or therapies for neurodegenerative disorders such as Aricept® (donepezil), Razadyne® (galantamine), Nanenda® (mementine), Exalon® (rivastigmine), Cognex® (tacrine), or other agents known to those skilled in the art. The compositions can be combined with alternative therapies such as administration of tranquilizers, mood stabilizing medications, behavior treatments (including treatments for aggressive behavior, incontinence, sleep difficulties, and wandering behavior), and individual activities and therapies (e.g., Reminiscence therapy) known to those skilled in the art. Also provided is a kit comprising any of the compositions of the disclosure and

instructions for use it for any method of the disclosure.

Methods of Use ofp22phox or Rubicon Fragments or Variants and Their

Compositions

[0187] In one embodiment, as described above, the present discloses p22phox fragments, variants, their retro-inverso sequences and equivalents. Such peptides include at least amino acids 3-10 of a p22phox protein or an equivalent thereor. Further such peptides lack the C-terminal gp91 phox binding sequence so that it compete with natural p22phox in binding to Rubicon, thereby inhibiting Rubicon's ability to activate the reactive oxygen species (ROS)-producing NADPH oxidase complex by interacting with p22phox. Accordingly, such peptides are useful in inhibiting ROS activation or treating diseases associated with ROS activation. Non- limiting examples of such diseases include acute inflammatory disease, influenza- induced acute lung injury, asthma, sepsis, hypertension or atherosclerosis.

[0188] In addition to these peptides, the present disclosure further contemplates an agent that inhibits the interaction between Rubicon and p22phox would be similarly used. Such agents may include, without limitation, a polynucleotide encoding these peptides, an antibody directed to p22phox, an antisense or siRNA directed to p22phox, or a small molecule inhibitor of Rubicon-p22p/?ox binding.

[0189] Overproduction of ROS leads to various diseases and conditions such as acute inflammatory disease, influenza-induced acute lung injury, asthma, sepsis, hypertension or atherosclerosis. The p22phox fragments and peptides, therefore, are useful in treating such diseases, which are further demonstrated by the examples described below.

[0190] Further, applicants have discovered the direct crosstalk between autophagy and phagocytosis machineries by demonstrating that the Run/cysteine-rich- domaincontaining Beclinl -interacting autophagy protein (Rubicon) is an essential positive regulator of the NADPH oxidase complex, inducing ROS production upon microbial infection or plasma membrane Toll-like receptor (TLR) activation. While Rubicon primarily associates with the Beclinl -UVRAG-containing autophagy complex under normal and stressed conditions, it periodically interacts with the p22p/?ox of NADPH oxidase complex, upon microbial infection or TLR2/4 activation, facilitating the stabilization and phagosomal translocation of the p22p/?ox-NADPH oxidase complex to induce a ROS burst, inflammatory cytokine production, and thereby, potent anti-microbial activities. Therefore, Rubicon's actions in the Beclinl - UVRAG-containing autophagy complex and in the p22p/?ox-containing NADPH oxidase complex are functionally and genetically separable.

[0191] Accordingly, peptide fragments of Rubicon that either include the p22phox binding sequence or the Beclinl binding sequences, can affect the ROS production and autophagy pathways, respectively, without interfering with the other. Such peptides, therefore, can be used to binding Beclinl or p22phox independently and carry our biological activities accordingly.

[0192] A protein or polypeptide of this method can be administered into cells (whether in vivo, ex vivo, or in vitro) using any delivery vehicle suitable for delivery of a polypeptide. The polypeptide can be directly administered, or the polypeptide can be covalently or non-covalently complexed to a macromolecular carrier, including, but not limited to, natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids.

Polypeptides of this disclosure also can be combined with various liquid phase carriers, such as sterile or aqueous solutions, pharmaceutically acceptable carriers, suspensions and emulsions.

[0193] In some embodiments, the polypeptide described herein that may be administered therapeutically by this method may comprise a substantially

homologous and biologically equivalent polypeptide having at least 80% homology, or alternatively at least 85% homology, or alternatively at least 90% homology, or alternatively, at least 95% homology or alternatively, at least 98% homology to SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 1 1 , SEQ ID NO. 12, or SEQ ID NO. 13, or variant or their biological equivalents, each as determined using methods known to those skilled in the art and identified herein, when run under default parameters. Preferred amino acid substitutions for the biologically equivalent peptides are described supra. Also within the scope of this disclosure are the retro-inverso forms of these peptides. In alternate embodiments, the polynucleotide encoding the polypeptide is administered or delivered to the cell or a subject in need thereof.

[0194] In therapeutic applications, a pharmaceutical composition containing one or more polypeptide or polypeptide described herein is administered to a patient suspected of, or already suffering from such a disease associated with the regulation of autophagy, wherein said composition is administered in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histological and/or behavioral), including its complication and intermediate

pathological phenotypes in development of the disease.

[0195] An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the polypeptide of this disclosure to decrease autophagy either in vitro or in vivo by at least 10%, 25%, 40%, 60%, 80%, 90% or 95% as compared to control.

Determination of these parameters is well within the skill of the art. These

considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.

[0196] The "therapeutically effective amount" will vary depending on the

polypeptide, the disease and its severity and the age, weight, etc., of the patient to be treated all of which is within the skill of the attending clinician. It is contemplated that a therapeutically effective amount of a polypeptide described herein will decrease levels of autophagy in the patient as compared to the levels of autophagy in the absence of treatment. As such, cancer growth is suppressed or decreased. A therapeutically effective amount is distinguishable from an amount having a biological effect (a "biologically effective amount"). A polypeptide of the present disclosure may have one or more biological effects in vitro or even in vivo. A biological effect, however, may not result in any clinically measurable therapeutically effect as described above as determined by methods within the skill of the attending clinician.

[0197] Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be found below.

[0198] The pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders. In addition to an agent of the present disclosure, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the disclosure.

[0199] More particularly, an agent of the present disclosure also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated. [0200] Ideally, the agent should be administered to achieve peak concentrations of the active agent at sites of disease. This may be achieved, for example, by the intravenous injection of the agent, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing the active ingredient. Desirable blood levels of the agent may be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue. The use of operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component agent than may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects.

[0201] While it is possible for the agent to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic agents. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0202] Formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0203] Formulations of the present disclosure suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. [0204] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin,

hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium

carboxymethyl cellulose) surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

[0205] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0206] Pharmaceutical compositions for topical administration according to the present disclosure may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol or oil. Alternatively, a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active ingredients and optionally one or more excipients or diluents.

[0207] If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound that enhances absorption or penetration of the agent through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues. [0208] The oily phase of the emulsions of this disclosure may be constituted from known ingredients in an known manner. While this phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at lease one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

[0209] Emulgents and emulsion stabilizers suitable for use in the formulation of the present disclosure include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

[0210] The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus, the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

[0211] Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the agent.

[0212] Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the agent, such carriers as are known in the art to be appropriate. [0213] Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered as a dry powder or in an inhaler device by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer, include aqueous or oily solutions of the agent.

[0214] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit- dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0215] It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this disclosure may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents. It also is intended that the agents, compositions and methods of this disclosure be combined with other suitable compositions and therapies.

Kits and Screens

[0216] Also disclosed herein are kits having components and instructions for inhibiting or activating ROS production or inhibition of autophagy in a cell or in a subject in need thereof. The kits contain instructions for use and one or more of any compositions described above. [0217] Yet further disclosed is a screen of identifying an agent that inhibits the interaction between p22p/?ox and Rubicon, comprising contacting a candidate agent with a cell under a condition that stimulates production of reactive oxygen species, wherein a reduction of production of reactive oxygen species and an increased free p22p/?ox or Rubicon level compared to a suitable control indicates that the candidate agent inhibits the interaction between p22phox and Rubicon.

[0218] The present disclosure, in yet another embodiment, provides a method of identifying an agent that inhibits the interaction between Beclinl and Rubicon, comprising contacting a candidate agent with a cell under a condition that stimulates autophagy, wherein a reduction of autophagy compared to a suitable control indicates that the candidate agent inhibits the interaction between Beclinl and Rubicon.

[0219] The following examples are intended to illustrate, and not limit, the disclosures disclosed herein.

Experimental Examples

Example 1

[0220] This example shows the direct crosstalk between autophagy and

phagocytosis machineries by demonstrating that the Run/cysteine-rich- domaincontaining Beclinl -interacting autophagy protein (Rubicon)^{9, 10} is an essential positive regulator of the NADPH oxidase complex, inducing ROS production upon microbial infection or plasma membrane Toll-like receptor (TLR) activation. While Rubicon primarily associates with the Beclinl -UVRAG-containing autophagy complex under normal and stressed conditions, it periodically interacts with the p22p/?ox of NADPH oxidase complex, upon microbial infection or TLR2/4 activation, facilitating the stabilization and phagosomal translocation of the p22p/?ox-NADPH oxidase complex to induce a ROS burst, inflammatory cytokine production, and thereby, potent anti-microbial activities.

[0221] Consequently, the expression or depletion of the Rubicon gene in macrophage cell lines, primary bone marrow-derived macrophages and mice profoundly affected ROS and inflammatory cytokine production, and subsequent anti-microbial activities. [0222] Remarkably, Rubicon's actions in the Beclinl -UVRAG-containing autophagy complex and in the p22p/?ox-containing NADPH oxidase complex are functionally and genetically separable. These results indicate that Rubicon

orchestrates two ancient innate immune machineries, autophagy and phagocytosis, toggling between the two depending on the environmental stimuli, ultimately generating an optimal intracellular immune milieu against microbial infection.

Methods

Measurement of intracellular ROS and determination of NADPH oxidase activity

[0223] The oxidative fluorescent dyes DHE (1 μΜ), CM-H2DCFDA (2 μΜ), and DAF-2 DA (10 μΜ) were used to detect O₂ ^~, H₂O₂, and NO production, respectively, using confocal microscope and the lucigenin (b/s-N-methylacridinium nitrate, 5 x 10^"6 M) chemiluminescence and Griess Reagent spectrophotometer assays to measure NADPH oxidase and NO synthase activities, respectively (Yang et al. (2007) J Neuroinflammation 4:27). The values are expressed as relative light units per 1 x 10⁵ cells.

Cell culture

[0224] Primary bone marrow-derived macrophages (BMDMs) from 6- to 8-week- old female C57BL/6 mice were prepared. All animals were maintained in a pathogen- free environment. Briefly, Bone marrow cells from the femur and tibia were cultured for 4 days in 10% L929 culture media (as a source of M-CSF)-containing Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL) containing 4 mM glutamine and 10% FBS. Mouse macrophage cell line RAW264.7 (ATCC TIB-71 ; American Type Culture Collection), Human lung epithelial cell line A549 (ATCC CCL-185) and HEK293T (ATCC-1 1268) were maintained in DMEM (Gibco-BRL) containing 10% FBS (Gibco- BRL), sodium pyruvate, nonessential amino acids, penicillin G (100 lU/ml), and streptomycin (100 pg/ml). Human monocytic THP-1 (ATCC TIB-202) cells were grown in RPMI 1640/glutamax supplemented with 10% FBS and treated with 20 nM PMA (Sigma-Aldrich) for 24 h to induce their differentiation into macrophage-like cells, followed by washing three times with PBS. Human umbilical vein endothelial cells were purchased from Lonza, and grown and maintained in endothelial growth medium. Transient transfections were performed with Lipofectamine 2000 (Invitrogen), or calcium phosphate (Clontech), according to the manufacturer's instructions. RAW264.7 and THP-1 stable cell lines were generated using a standard selection protocol with 2 g/ml of puromycin.

Reagents

[0225] LPS, β-1 ,3-glucan, PMA, PGN, CHX, BSA, IgG, Cytochalasin D, 3- Methyladenine, Bafilomycin A1 , and rapamycin were from Sigma; Zymosan, BLP (Pam2CSK4), and MDP were purchased from Invivogen; NAC, DPI, AEBSF, L- NMMA, L-NAME, DHE, CM-H2DCFDA, and DAF-2 DA were from Calbiochem;

recombinant mTNF-a, mlL-6, mlL-Ι β, and mlFN-γ were from eBioscience; Texas Red-conjugated zymosan A (S. cerevisiae) BioParticles and LysoTracker Red DND- 99 were from Invitrogen which were used in the in vitro assays.

Bacterial strains

[0226] L. monocytogenes and the strain expressing GFP were provided by D. Portnoy (University of California-Berkely) and M. bovis BCG strain were provided by Yi Luo (University of Iowa). L. monocytogenes and M. bovis BCG were grown at 37°C in brain-heart-infusion (BHI) broth medium (BD) and Middlebrook 7H9 medium supplemented with Tween 80, glycerol and OADC (Difco Laboratories), respectively. For all assays, mid-log-phase bacteria (absorbance, 0.9) were used. Batch cultures were aliquoted and stored at -80°C. Representative vials of L. monocytogenes and M. bovis BCG were thawed and enumerated for viable colony-forming unit (cfu) on BHI agar (BD) and Middlebrook 7H10 agar (Difco), respectively. The effective concentration of LPS was <50 pg/ml in those experiments with a bacterium-to-cell ratio of 10:1 . Heat-killed L. monocytogenes were obtained by heating for 30 min at 60°C.

Phagocytosis assays

[0227] In brief, L. monocytogenes was fluorescently labeled with

tetramethylrhodamine isothiocyanate (TRITC; Sigma) in 0.1 M carbonate (pH 9.5) and stored at 4°C. TRITC-labeled L monocytogenes or GFP- L. monocytogenes (10 cfu) were then used to infect cells in a 12-well plate for indicated times at 37°C in a 5% CO₂ incubator. Partially attached, non-ingested bacteria were removed by 1 h of treatment with 10 g/ml gentamycin using trypan blue as a quenching agent to exclude fluorescence from the cell surface. Samples were then fixed with 4% pformaldehyde and analysed using a laser-scanning confocal microscopy (Nickon Eclipse C1 ) or BD FACSCanto II (Becton Dickinson; minimum 10,000 cells per sample) to identify the proportion of cells associated with fluorescent bacteria, used as a marker for phagocytosis.

[0228] Zymosan or TRITC-labeled opsonized-zymosan particles (Molecular Probes) were fed to cells for indicated times, with brief centrifugation upon zymosan addition to ensure particle contact with the cells. Cells were lifted in PBS containing 1 mM EDTA, 1 mM sodium azide, and 2.4U/ml proteinase K (to remove bound but uninternalized zymosan particles) prior to analysis by confocal microscope or flow cytometry.

Autoradiography

[0229] Raw264.7 were labeled for 6h with S³⁵ (10 pCi/ml; MP Biomedicals, Inc.) in Met/Cys-free DMEM containing dialysed 10% FBS (Sigma) and 1 % L-glutamine (Gibco-BRL), then were stimulated for indicated times at 37 °C with zymosan or rapamycin and rinsed. 1 g of Flag antibody was added to 1 ml of cell lysates and incubated at 4°C for 18h for immunoprecipitation. After addition of protein A G agarose beads, incubation was continued for 2h. Immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by standing on RT for 30 min. After fixing and amplifying, gels were dried and radiolabeled gels were visualized by autoradiography for 18 h.

Plasmid construction

[0230] All constructs for transient and stable expression in mammalian cells were derived from the pEBG-GST mammalian fusion vector and the pEF-IRES-Puro expression vector. DNA fragments corresponding to the coding sequences of the human Rubicon, p22phox, gp91 phox, and p47 phox genes were amplified by polymerase chain reaction (PCR) and subcloned into pEFIRES-puro between the Aflll and Xbal sites and selected for stable transfectants. AU1 -tagged Rubicon and V5-tagged Beclinl were cloned into the Aflll and Xbal sites in pEF-IRES-Puro. GST- tagged Rubicon, p22phox, and mutant genes were cloned into a pEBG derivative encoding an N-terminal GST epitope tag between the BamHI and Notl sites. Flag- tagged truncated mutant constructs of Rubicon were created by subcloning the PCR products of cDNA fragments containing each domain of the target genes into pEF- IRES-Puro. All constructs were sequenced using an ABI PRISM 377 automatic DNA sequencer to verify 100% correspondence with the original sequence.

Isolation of cytosolic and nuclear protein fractions

[0231] Cells were mechanically scraped in PBS and washed, then resuspended in lysis buffer (15 mM KCI, 10 mM HEPES, 2 mM MgCI₂, 0.1 mM EDTA, 1 mM PMSF, 1 mM DTT, 10 pg/ml aprotinin, 2 g/ml leupeptin, 0.1 % NP-40, pH 7.6). Cell suspensions were then incubated for 15 min on ice with occasional vortexing, and centrifuged for 30 s to pellet nuclei. Supernatants with cytosolic proteins were collected and stored at -20°C until used for Western blotting. Nuclei were rinsed with wash buffer (2 mM KCI, 25 mM HEPES, 0.1 mM EDTA, 1 mM PMSF, 1 mM DTT, 10 g/ml aprotinin, 2 g/ml leupeptin, pH 7.6) and incubated at 4°C for 20 min. Nuclear extracts were then prepared by centrifugation at 20,000g for 15 min in lysis buffer (25 mM HEPES, 0.1 mM EDTA, 20% glycerol, pH 7.6) and stored at -80°C until used for Western blotting. Protein concentration in the cell cytosolic or nuclear lysates was determined using a bicinchoninic acid assay. Fraction purity was tested by Western blotting using actin as a cytoplasmic marker and Lamin A/C as a nuclear marker.

Yeast two-hybrid screen

[0232] Yeast transformation with library cDNA was performed as recommended by the manufacturers. Briefly, Yeast strain Y187 bearing Gal4-Rubicon full length or C- terminal region fusion gene plasmid was grown overnight in synthetic dropout (SD)/-

Trp medium to a density of approximately 107 cells/ml, then diluted in 1 liter of warmed YPD to an optical density (OD600) of 0.2 - 0.3 and grown to exponential stage. The cells were harvested and washed with 100 ml of water twice and TE

(Clontech) once. The pellet was resuspended in 8 ml of 10 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.1 M Li-acetate (LiOAc). The suspension was mixed with 1 mg of transforming DNA and 20 mg of single-stranded salmon sperm DNA, after which 60 ml of a solution of 40% polyethyleneglycol-4000 in Tris-EDTA-LiOAc was added and mixed thoroughly, followed by incubation at 30°C with agitation for 30 min. After a heat pulse at 42°C for 15 min, the cells were pelleted, washed with 50 ml of Tris- EDTA, and plated on selective medium. Library screening and recovery of plasmids were performed according to manufacturer's instructions (Clontech).

Confocal fluorescence microscopy

[0233] For immunostaining, RAW 264.7 and BMDMs cells were seeded on 12-well culture dishes that contained 18 mm diameter round glass coverslips (10⁵ cells per well). Cells were fixed with 4% paraformaldehyde in PBS at 4°C for 10 min and permeabilized with 0.25% Triton X-100 in PBS for 15 min before being treated with 10% BSA for 1 hr at 25°C. Cultures were then stained with primary antibodies, including goat anti-p22p/?ox (C-17; Santa Cruz Biotech), mouse antigp91 phox (54.1 ; Santa Cruz Biotechnology), rabbit anti-Flag (Sigma), and rabbit anti-Beclin1 (Cell Signaling) overnight at 4°C. After washing to remove excess primary antibodies, the cultures were incubated for 1 h at RT with the following fluorescently labeled secondary antibodies: anti-rabbit IgG-FITC or TRITC, anti-mouse IgG-FITC or TRITC, and anti-goat lgGCy5 (Molecular Probes). Cells were imaged with a laser- scanning confocal microscopy (Nickon Eclipse C1 ).

Preparation of Latex Beads

[0234] Polystyrene beads (1 .7 μιτι diameter, 2.5% suspension; Polysciences, Inc.) were coated with BSA (100 pg/ml), IgG (100 pg/ml), or Zymsan (1 mg/ml) for 1 h at 37 °C or incubated for the same time period in PBS. Beads were washed extensively in PBS before use.

Phagosome Purification

[0235] Phagosomes in Raw264.7 cells were formed by the internalization of latex beads in culture medium at 37°C for the indicated times. The cells were then washed in PBS (3 x 10 min) on ice, disrupted in homogenization buffer [3 mM imidazole, pH 7.4, containing 8.55% (w/w) sucrose, 2 mM phenylmethylsulfonyl fluoride, 1 g/ml chymostatin, 1 g/nnl E-64, 1 g/nnl leupeptin, 1 g/nnl pepstatin] by nitrogen cavitation for 20 min at 300 psi in a bomb (Parr Instrument) at 4°C. After

centrifugation at 1 ,500 rpm for 7 min to remove nuclei and unbroken cells, the supernatants containing the latex bead-containing phagosomal compartments (LBC) were subjected to stepwise sucrose gradient centrifugation. The LBC supernatant was adjusted to 40% sucrose by adding 62% sucrose solution, and then loaded on a 1 -ml cushion of 62% sucrose. Then, 2 ml of 35% sucrose, 2 ml of 25% sucrose, and 2 ml of 10% sucrose solution (pH 7.4) were layered. Centrifugation was carried out at 24,000 rpm for 1 h in a SW41 Beckman swinging rotor. The LBC fractions were collected from the 10/25% sucrose interface. The collected LBC fractions were washed with PBS. The purity of the final LBC fractions was evaluated by electron microscopy.

Flow cytometry

[0236] Cells were analysed by flow cytometry for the expression of p22phox and gp91 phox using a FACSCanto II flow cytometer as indicated by the manufacturer (Becton Dickinson). After two washes with PBS, cells were fixed in 2%

paraformaldehyde for 10 min at 37°C or permeabilized by adding ice-cold 100% methanol for 30 min on ice. Cells were stained with primary mAb against p22phox- alexa fluor 488 (CS9, AbD Serotech) or gp91 phox alexa fluor 488 (NL7, AbD

Serotech) for 30 min at 4°C (1 :200). After two washes with PBS, cells were fixed in 4% paraformaldehyde and immediately analysed. In each case, 10,000 cells were acquired. The data were analysed using BD FACSCanto II (Becton Dickinson).

Immunoblot analysis and immunoprecipitation

[0237] For immunoblotting, polypeptides were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane (Bio-Rad).

Immunodetection was achieved with V5 (Invitrogen), AU1 (Covance), LC3

(LC3. N0.6, Cosmo Bio Co), 4G10 (Upstate), VPS34 (Abgent), and Rubicon (Bethyl

Laboratories). Abs to Flag and UVRAG from Sigma, GST (Z-5), actin (C4), p22phox

(FL-195), gp91pftox (54.1 ), p47phox (H-195), p62/SQSTM1 (D-3), IkBa (C-21 ),

Lamin A/C (346), MyD88 (E-1 1 ), TLR2 (TL2.1 ), NOX4 (H-300), and NF-κΒ p65 (C-

20) were purchased from Santa Cruz Biotechnology, Inc. Specific Abs to phospho- (Thr202/Tyr204)-ERK1/2, phospho-(Thr180/Tyr182)-p38, phospho-(Thr183/Tyr185)- SAPK/JNK, phospho-lkBa, and Beclinl were purchased from Cell Signaling

Technology. These proteins were visualized by a chemiluminescence reagent (Pierce) and detected by a Fuji Phosphor Imager.

[0238] For GST Pulldown, cells were harvested and then lysed in NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). Post-centrifuged supernatants were precleared with protein A G beads at 4°C for 2 h. Pre-cleared lysates were mixed with 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction was incubated for 4 h at 4°C. Precipitates were washed extensively with lysis buffer. Proteins bound to glutathione beads were eluted with SDS loading buffer by boiling for 5 min.

[0239] For immunoprecipitation, cells were harvested and then lysed in NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). After pre- clearing with protein A G agarose beads for 1 h at 4°C, whole-cell lysates were used for immunoprecipitation with the indicated antibodies. Generally, 1 -4 g of commercial antibody was added to 1 ml of cell lysates and incubated at 4°C for 8 to 12 h. After the addition of protein A/G agarose beads for 1 h, immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 5 min.

Autophaav analysis

[0240] For starvation, cells were washed three times with PBS and incubated in Hank's solution (Invitrogen) for 4 h at 37°C. Cells were treated with Hank's solution or complete medium containing 2 μΜ rapamycin (Sigma) for 4 h. For the LC3 mobility shift assay or p62 immunoblot, cells were treated for 30 min on ice, lysed with 1 % Triton X-100, and then subjected to immunoblotting analysis with an antibody against LC3 or p62.

Enzyme-linked immunosorbent assay

[0241] Murine BMDMs, RAW264.7 cells, and THP-1 cells were treated as indicated and processed for analysis by sandwich ELISA. Cell culture supernatants and mice sera were analysed for cytokine content using BD OptEIA ELISA set (BD Pharmingen) for the detection of TNF-a, IL-6, ΙΙ_-1 β, IL-23, and IL-12p40. All assays were performed as recommended by the manufacturers.

RNA extraction and semi-quantitative PCR

[0242] RNA was isolated using an RNeasy mini kit (Qiagen) and treated with RNase-free DNase as per the manufacturer's instructions. Complementary DNA was reverse transcribed from 2 g of total RNA using Superscript II reverse transcriptase (Invitrogen) and oligo-dT-3' primer in a total volume of 20μΙ. Primer sequences were as follows: mp22pftox (forward: 5'-GGGCAGATCGAGTGGGCCATG-3\ reverse: 5'- TCACACGACCTCGTCGGTCAC-3'), mp40pft ox (forward: 5'-CAAAGTCTACAT GGGCGCAAA-3', reverse: 5' TGTCTTCATAGAAGTAGCATCGTAGCC-3'), mp47pftox (forward: 5'-ACATCACAGGCCCCATCATCCTTC-3', reverse: 5'- ATGGATTGTCCTTTGTGCC-3'), mp67pftox (forward: 5'-

CTATCTGGGCAAGGCTACGGTT-3', reverse: 5'-CACAAAGCCAAACAATACGCG- 3'), mp91pftox (forward: 5'-AGTCGGGATTTCTGACCGGTAT-3\ reverse: 5'- TCCAGTCTCCAACAATACGGATATG-3'), mUVRAG (forward: 5'- CTCCGACATCTCCGGAAC-3', reverse: 5'-GGCATGGATCTGCTGTCC-3'), mBeclinl (forward: 5'-CTCAATGTCACTG AG AATG AA-3' , reverse: 5'- TTCGTCATCCAACTCCAGCTG-3'), mlL-1 β (forward: 5'-TTGTG

GCTGTGGAGAAGCTGT-3', reverse: 5'-AACGTCACACACCAGCAGGTT-3'), mTNF-a (for ward: 5'-TCTCATCAGTTCTATGGCCC-3\ reverse: 5'- G G G AGTAG ACAAG GTACA-3') , mlL-6 (forward: 5'-TCCATCCAGTTGCCTTCTTGG- 3', reverse: 5'-CCACGATTTCCAGAA CTG-3'), mll_-12p40 (forward: 5'- CAGAAGCTAACCATCTCCTGGTTTG-3', reverse: 5'- TCG ATAATTTG GTGCTTCACAC-3' ) , mlL-12p35 (forward: 5'- CCACCCTTGCCCTCCTAAAC-3',reverse: 5'-GGCAGCTCCCTCTTGTTGTG-3'), mlL-23p19 (forward: 5'-TGGCATCGAAA TTGAGA-3', reverse: 5'- TCAGTTCGTATTGGTAGTCCTGTTA-3'), β-actin (forward: 5'-CCT

TTGCCAACACAGT-3' , reverse: 5'-AGCCACCGATCCACACAG-3'). The PCR products were resolved on 1 % agarose gels and were stained with ethidium bromide. Quantification of L. monocytogenes or M. bovis BCG growth

[0243] To ensure the reliable quantification of intracellular bacteria, colony-forming unit (CFU) was utilized. Cells were infected for 1 h with L. monocytogenes or 2h with M. bovis BCG at a different MOI. Then, cells were washed three times with PBS, and fresh medium containing gentamycin (10 pg/ml) and amikacin (200 g/ml) was added, respectively. After various periods of incubation, cells were lysed with 0.3% saponin (Sigma-Aldrich) to release the intracellular bacteria and the lysates of infected cells were then resuspended vigorously, transferred to screwcapped tubes, and sonicated in a preheated 37°C water bath sonicator (Elma) for 5 min. Aliquots of the sonicates were then diluted 10-fold in BHI broth (for Listeria) or 7H9 medium (for mycobacteria). Four dilutions of each sample were plated separately on BHI agar (for Listeria) or 7H10 agar (for mycobacteria) plates and incubated at 37°C with 5% CO₂ for 1 day (for Listeria) or 21 days (for mycobacteria).

Production of lentiviral shRubicon or Rubicon

[0244] For silencing of human Rubicon, Oligonucleotide sequences for shRNA interference with Rubicon expression are bp 513-536 of 5' - GAUCGAUGCGUCCAUGUUU- 3', followed by a 9-nucleotide non-complementary spacer (TTCAAGAGA) and the reverse complement of the initial 19-nucleotide sequence. These dsDNA oligonucleotides were cloned into the pGIPZ lentiviral vector (Open Biosystems). Lentiviruses were produced by transient transfection using packaging plasmids (psPAX2, pMD2.VSV-G, purchased from Addgene) after Lipofectamine 2000 mediated transient transfection into 293T cells. Control vector was constructed by inserting a sequence that expresses an shRNA with limited homology to the Rubicon sequences. DNA fragments corresponding to the coding sequences of the Rubicon genes were amplified by PCR and subcloned into pCDH- CMV vector (System Biosciences).

Transduction of lentiviral shRubicon or Rubicon

[0245] Lentiviruses were produced by transient transfection using packaging plasmids (pGag, pVSV-G, pRev, purchased from Addgene) after Lipofectamine 2000 mediated transient transfection into 293T cells. Viral-containing media were collected 72 hr posttransfection and harvested for the viral particles by passing the supernatants through a 0.45 μηη filter. The supernatants were used to infect 2x105 cells in six-well plates in presence of 8 g/ml Polybrene (Sigma). For lentivirus infection, cells (5x105 cells/ml) in DMEM + 10% FBS were seeded in 24-well plates. After 24h, cells were infected with lentiviral vectors at various MOIs in the presence of 8 g/ml Polybrene. On the following day, the medium was replaced with fresh medium, and cells were incubated for additional 4 days and then analyzed for Rubicon knockdown. A parallel experiment using a GFP encoding lentivirus indicated that a minimum of 80% of Raw264.7 and BMDMs cells were transduced by lentiviruses. Titration of the lentiviral vectors was determined using 293T cells.

[0246] Briefly, approximately 2x10⁵ cells were plated in each well of a six-well plate. On the following day, cells were infected with viral supernatants in the presence of 8 g/ml Polybrene. After 24h, medium was removed and replaced with fresh medium containing 5 g/ml puromycin that was replaced every 3 days. On day 14, cells were stained with crystal violet for 15 min and colonies were counted using a cutoff of 50 viable cells.

Construction of adenoviral shRubicon or Rubicon

[0247] An adenoviruses expressing short hairpin RNA (shRNA) to the Rubicon gene was constructed using the AdEasy system (Stategene). The shRNA

oligonucleotides sequences were as follows: 5'-gatcc cc gat aga cag tat ate aga a ttc aag aga t tct gat ata ctg tct ate ttttt a -3', 5'- g gg eta tct gtc ata tag tct t aag ttc tct a aga eta tat gac aga tag aaaaa ttcga-3'. These dsDNA oligonucleotides were cloned into the pSuper vector (OligoEngine) between the Bglll and Hind II I restriction sites containing the human H1 promoter. The double-strand shRNA oligonucleotides containing the termination signal were inserted at the 3' end of the human H1 promoter and subcloned into the pShuttle vector (Stategene) Notl and Hindlll restriction sites. Control vector was constructed by inserting a sequence that expresses an shRNA with limited homology to the Rubicon sequences.

[0248] DNA fragments corresponding to the coding sequences of the Rubicon genes were amplified by PCR and subcloned into the pShuttle-CMV vector

(Stategene) between the Notl and EcoRV restriction sites Adenovirus production

[0249] Recombinant adenoviruses were constructed using AdEasy system (Stategene): digested adenovirus vectors with the Pac I were transfected into the AD-293 producer cells in a 6-wellplate and cultured with fresh media until cytopathic effect (CPE) was observed. When 80% CPE were observed, recombinant adenoviruses were harvested by repeatedly freezing at -80 °C and thawing at 37 °C four times. Cell lysates were then centrifuged at 2,000g for 30 min at 25 °C and the supernatants containing recombinant adenovirus particles were stored at -80 °C.

[0250] All adenoviruses were propagated in AD-293 cells, purified and

concentrated by BD Adeno-XTM purification kit (BD Biosciences Clontech). The typical titers were in the range of 1012-1013 plaque-forming units (pfu)/ml_ as determined via plaque assay using 1 .25 % SeaPlaque GTG agarose (BioWhittaker Molecular Applications) overlay. A sterile carrier solution [phosphate-buffered saline (PBS)] was used for control injections and dilution of the viruses.

Mice and adenovirus injection

[0251] Six-week-old female C57BL/6 was purchased from Charles River

Laboratories. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. Recombinant adenoviruses were thawed immediately before injection at 25 °C and diluted with saline solution to a final volume of 100 μί per mouse. Mice were held down with in a restrainer and their tails were mildly heated with a heating lamp to achieve vasodilatation.

Adenoviruses were injected through the tail vein slowly with a 30 gauge needle.

[0252] After injection, mild pressure was applied at the spot of injection until no bleeding was achieved to prevent the backflow of virus solution. Mice were monitored daily for 10 d. For measurement of the bacterial burden in liver and spleen, mice were killed at 5 day after inoculation, organs were homogenized in PBS, and serial dilutions of the homogenates were plated on BHI agar plates, with colonies counted 24 h later. Statistical analysis

[0253] All data were analyzed using Student's t test with a Bonferroni adjustment or ANOVA for multiple comparisons and are presented as the mean ± SD.

Differences were considered significant at p < 0.05. Where indicated, Prism software (Graph Pad) was used for two-way analysis of variance and Kaplan-Meier survival analyses.

Results

[0254] The newest endosome/lysosome autophagy protein, called Rubicon (Run/cysteine-richdomain-containing Beclinl -interacting autophagy protein), was identified from a subpopulation of Beclinl complexes that suppress autophagosome maturation and endocytosis. Since engaging the autophagy pathway via TLR signaling enhances phagosome maturation and destruction of the engulfed microbes⁵, radio-actively labeled Rubicon complexes from mouse macrophage Raw264.7 cells containing vector or Flag-Rubicon were purified upon stimulation with zymosan, a TLR2 ligand (100 pg/ml), or rapamycin (2 μΜ). As previously reported, Rubicon (130k) was found to associate with several proteins, presumably Vps15 (150k), Vps34 (1 10k), UVRAG (85k), and Beclinl (60k) based on their molecular weights and immunoblotting analyses (FIG. 1a). Besides the Beclinl - containing autophagy complex, Rubicon also interacted with two additional proteins with the molecular weights of 91 k and 22k, and these interactions periodically increased at 5 and 30 min after zymosan stimulation (FIG. 1a). In contrast, Rubicon interaction with the Beclinl -containing autophagy complex was not detectably affected under zymosan-treatment conditions (FIG. 1a). Conversely, rapamycin stimulation slightly increased Rubicon interaction with the Beclinl -containing autophagy complex without affecting its interactions with the 22k and 91 k proteins (FIG. 1a).

[0255] In an effort to identify the 22k and 91 k proteins associated with Rubicon, yeast two-hybrid screens were performed utilizing the full length or C-terminal region

(aa471 -972) of Rubicon as baits. These screens identified the p22phox integral membrane subunit of the NADPH oxidase complex as a major binding partner of

Rubicon, indicating that p22phox and gp91 phox are likely the 22k and 91 k proteins associated with Rubicon. The phagocytic NADPH oxidase complex generates superoxides leading to microbial killing and inflammation, and consists of an integral membrane heterodimer [gp91 phox (NOX2) and p22phox], three cytosolic proteins [pAOphox, pA7phox, and p67phox], and Rac1 or Rac2, depending on the species and phagocytic cell.

[0256] Co-immunoprecipitation (co-IP) showed that Rubicon strongly interacted with exogenous and endogenous p22p/?ox and this interaction was made more evident upon stimulation with zymosan (FIG. 1 b). In order to map the binding regions, GST-Rubicon mammalian fusions (GST-Rub-N-i_-47o, GST-Rub-CCD₅o5-557, GST-Rub-SR₅₆7-625, GST-Rub-CS₅₀5-625, GSTRub-CR₈₈i-972 GST-Rub-C_{471 -97}2, and GST-Rub-F _-972) and GST-p22p/?ox mammalian fusions (GST-P221-10, GST-p22 _-6o, GST-p2210-60, GST-p2260-i96, GST-p22 "1-128, and GST-p22i_i96) were constructed. GST pulldown (GST-PD) studies showed that Beclinl and p22phox efficiently bound the central coiled-coil domain (CCD, aa505-557) and the serine-rich region (SR, aa558-625) of Rubicon, respectively, indicating that the interactions of Rubicon with Beclinl and p22p/?ox are genetically separable (FIG. 1c). Remarkably, the N- terminal ten-amino acid peptide (aa1 -10) of p22phox was sufficient for Rubicon binding (FIG. 1 c).

[0257] Intracellular endosomal or lysosomal proteins including EEA1 , NPC1

LAMPs, and cathepsins display a biphasic phagosomal recruitment profile. To further delineate the temporal interaction profile of Rubicon, Flag-Rubicon complexes were immuno-purified upon zymosan or autophagy stimulation and immunoblotted with various antibodies. This showed that Rubicon interactions with gp91 p/?ox and p22phox periodically increased at 5 and 30 min after zymosan stimulation, whereas its interactions with Beclinl and UVRAG were initially reduced (1 -3 min) but immediately recovered (FIG. 1 d). These interactions were specific as the Rubicon

ASR mutant lost the p22p/?ox-gp91 phox interaction but retained the Beclinl -UVRAG interaction, whereas the ACCD mutant lost the Beclinl -UVRAG interaction but retained the periodic interaction with p22p/?ox-gp91 phox (FIG. 1 d). In contrast, rapamycin stimulation slightly increased Rubicon interaction with the Beclinl -

UVRAG complex without affecting its interaction with the p22p/?ox-gp91 phox complex. When the p22phox complex or the gp91 phox complex was immuno- purified, Rubicon interactions with p22phox or gp91 phox also peaked at 5 and 30 min after zymosan stimulation, whereas Rubicon interaction with p47p/?ox was not detected. Furthermore, not only did Rubicon respond primarily to TLR-mediated stimulation and not intracellular soluble PMA-induced stimulation, but Rubicon gene expression was also induced only upon various TLR-mediated stimulations, not PMA-induced stimulation, with similar kinetics to NADPH oxidase complex genes (p22phox, gp91 phox, pAOphox, pAlphox, and p67phox) and autophagy complex genes (Beclinl and UVRAG). These results indicate that while Rubicon is primarily present in the Beclinl -UVRAG complex under normal and autophagy conditions, it is periodically recruited to the p22p/?ox-gp91 phox complex upon TLR activation.

[0258] In virtually all chronic granulomatous-disease (CGD) patients with cytochrome b558 mutations, neutrophils lack both gp91 p/?ox and p22phox, regardless of which subunit is affected by the mutation, suggesting that the formation of the p22p/?ox-gp91 phox heterodimer is important for the stable expression of each subunit15. It was found that the expression or depletion of the Rubicon gene markedly increased or decreased, respectively, the levels of endogenous and exogenous p22phox and gp91 phox expression, but not p47p/?ox, in a binding- specific manner since the ASR mutant did not do so (FIG. 1 e). Cycloheximide (CHX) treatment further confirmed that Rubicon expression significantly stabilizes p22phox level. The nmf333 mouse strain carrying the p22phox Y121 H mutation, which dramatically interferes with either the synthesis or stability of the p22phox protein, exhibits a compound phenotype consisting of both a CGD-like immune defect and a balance disorder. Remarkably, Rubicon also bound and stabilized the p22phox Y121 H mutant. These indicate that Rubicon effectively stabilizes p22p/?ox and gp91 phox expression in a binding-dependent manner.

[0259] The effect of Rubicon on p22phox localization upon heat-killed (HK)- TRITC-labeled Listeria (L.) monocytogenes infection was examined next. Rubicon expression apparently increased p22phox colocalization with HK-TRITC-L.

monocytogenes as well as the number of phagosomes containing HK-TRITC-L. monocytogenes (FIG. 1f). Conversely, the depletion of Rubicon expression not only suppressed p22phox colocalization with HK-TRITC-L. monocytogenes, but also reduced phagosome numbers (FIG. 1f). Rubicon's effect was specific to zymosan, a TLR2 ligand, since these increases were not observed with BSA- or IgGlatex bead treatment, and treatment with Cytochalasin D (Cyto D), a phagocytosis inhibitor, completely blocked Rubicon-mediated phagocytosis.

[0260] To further delineate the effect of Rubicon on p22phox localization,

Raw264.7 cells were generated expressing WT and mutant forms of Rubicon.

Expression of Rubicon WT and ACCD markedly increased the number of HK-GFP- L. monocytogenes-pos^'\t\ve phagosomes and both completely colocalized with p22phox (FIG. 1g). In contrast, not only was the Rubicon ASR mutant display diffuse cytosolic staining, but it also apparently prevented p22p/?ox from translocating into HK-GFP-L. monocytogenes-pos^'\t\ve phagosomes (FIG. 1g). Furthermore, upon time course exposure to either Texas-Red-labeled opsonized-zymosan particles, Rubicon WT and ACCD extensively colocalized with p22phox and gp91 phox in the zymosan- particlecontaining phagosomes and also significantly increased the phagosome numbers and maturation (FIG. 1 h). Surprisingly, the zymosan-induced translocation of p22phox and gp91 phox into phagosomes was not observed upon expression of the Rubicon ASR mutant (FIG. 1 h). Finally, the level of internalized GFP-L.

monocytogenes increased by nearly 5 fold upon Rubicon WT and ACCD mutant expression, but decreased by 5 fold upon ASR mutant expression (FIG. 1 i).

[0261] To further delineate Rubicon's actions in the phagosomal recruitment of the 22p/70xgp91 p/70x-containing NADPH complex, Raw246.7 cells were infected with Rubiconoverexpressing or Rubicon-specific shRNA-mediated knockdown

adenoviruses or p22p/?oxspecific or gp91p/?ox-specific shRNA-mediated knockdown lentiviruses and then stimulated with zymosan-coated or IgG-coated beads for various times. The latex bead-containing phagosome fractions were subsequently purified by sucrose-step-gradient-ultracentrifugations. Under normal conditions, Rubicon-p22p/?ox-gp91 phox was recruited to phagosomes at similar kinetics to Beclin1 -UVRAG-VPS34. Under Rubicon-overexpression conditions, Rubicon- p22p/?oxgp91 phox was recruited to phagosomes earlier (5 min of stimulation) than Beclin1 -UVRAGVPS34 (15 min). Lastly, under Rubicon-depletion conditions, the phagosomal recruitment of Rubicon-p22p/?ox-gp91 phox was delayed and

dramatically reduced, whereas the phagosomal recruitment of Beclinl -UVRAG- VPS34 was not affected. Additionally, the expression levels of Rubicon, p22phox and gp91p/?ox were directly correlated with their phagosomal recruitment levels, whereas p47p/?ox was independently recruited to hagosomes. Finally, Rubicon's effects were TLR2-signaling specific since they were not detected upon IgG stimulation. These results collectively indicate that Rubicon facilitates the

phagosomal recruitment of the p22p/?ox-gp91 phox complex in a binding-dependent manner and that the Rubicon-p22p/?ox-gp91 phox complex and the Beclinl -UVRAG- VPS34 complex are independently recruited to phagosomes.

[0262] The oxidative fluorescent dyes dihydroethidium (DHE), 5,6-chloromethyl- 2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA), and 4,5- Diaminofluorescein diacetate (DAF-2 DA) were used next to detect O2^", H2O2, and NO production, respectively, and the lucigenin chemiluminescence and Griess Reagent spectrophotometer assays were used to measure NADPH oxidase and NO synthase activities, respectively. The chemiluminescent signal intensities attributed to O2^", H2O2, and NO production profoundly increased in Rubiconexpressing

Raw264.7 and primary mouse bone marrow-derived macrophage cells (BMDMs) compared to vector control cells upon zymosan stimulation (FIG. 2ab). Contrastingly, the antioxidant N-acetyl-L-cysteine (NAC), the NADPH oxidase inhibitor

diphenyliodonium chloride (DPI), and the NO synthase inhibitor L-NG-monomethyl arginine (L-NMMA) or L-NGnitro arginine methyl ester (L-NAME) significantly attenuated Rubicon-induced O₂ ^~, H₂O₂, and NO production, respectively (FIG. 2ab). Additionally, Rubicon expression dramatically enhanced NADPH oxidase activity as well as NO synthase activity in Raw264.7, THP-1 , and mouse BMDMs upon stimulation with zymosan or bacterial lipoprotein (BLP), or infection with L.

monocytogenes or Mycobacterium bovis BCG (FIG. 2cd). The stimulatory effect of Rubicon on NADPH consumption and phagocytosis was nearly abolished by pre- treatment with the NADPH oxidase inhibitors DPI and 4-(2-aminoethyl)benzene- sulphonyl fluoride (AEBSF) (FIG. 2d). By contrast, no significant difference in the PMA, LPS-, hot-alkalidepleted zymosan- or NOD2-recognizing muramyl dipeptide (MDP)-mediated release of ROS was detected between vector- and Rubicon- expressing Raw264.7 cells, indicating that due to its expression and interaction with TLR218, gp91 phox (NOX2) is primarily responsible for activating Rubicon-dependent ROS production in macrophages. Furthermore, Rubicon expression increased TLR2 interaction with p22p/?ox-gp91 phox and its recruitment into zymosan-coated bead- containing phagosomes where Rubicon was also localized.

[0263] This action was specific since there was little or no effect on the TLR2- MyD88 interaction as well as MyD88 Y257 phosphorylation^. When A549 epithelial cells endogenously expressing NOX4 but not gp91 phox (NOX2) were subjected to BLP or LPS stimulation, Rubicon interacted with the p22p/?ox-NOX4-TLR4 complex as previously shown and efficiently enhanced LPS-induced NOX4 activity and co- localization, but showed no effect on BLP-induced signaling activities, suggesting that Rubicon targets p22phox, a common subunit of NOX family, and thereby affects various TLR-NOX pathways. Finally, Rubicon expression detectably enhanced the phosphorylation of p38 and ΙκΒ, the degradation of ΙκΒ, and the nuclear localization of NF-KB p65 subunit in Raw246.7 cells upon zymosan treatment; however, there was no significant effect on the phosphorylation of p42/44 MAPK and JNK (FIG. 2e).

[0264] NADPH oxidase signaling is known to regulate pro-inflammatory cytokine expression. To examine the effect of Rubicon expression on the production of proinflammatory cytokines in response to various stimuli, murine Raw264.7 or human THP-1 cells stably expressing Rubicon were stimulated with zymosan (100 pg/ml), BLP (1 OOng/ml), L. monocytogenes (MOI=1 ) or M. bovis BCG (MOI=1 ) and the supernatants were collected at the indicated intervals for cytokine analysis. Rubicon expressing Raw264.7 and THP-1 cells produced markedly higher amounts of TNF-a, IL-6, IL-Ι β, IL-12p40, and IL-23 than vector-containing control cells (FIG. 2f).

Furthermore, lentivirus-mediated expression of Rubicon in primary mouse BMDMs or Raw264.7 led to significant increases in TNF-a, IL-6, IL-Ι β, IL-12p40, and IL-23 production, whereas pre-treatment with the NADPH oxidase inhibitors DPI and AEBSF nearly abolished this stimulatory effect of Rubicon on cytokine production (FIG. 2f). No significant difference in the LPS-mediated activation of cytokine production was detected under Rubicon-depletion or -overexpression conditions. Finally, the viability and growth rate of intracellular L. monocytogenes and M. bovis BCG were drastically reduced in Rubicon-expressing Raw246.7, THP-1 , and mouse BMDM cells compared to vector-containing control cells (FIG. 2g). Note that either gp91 p/?ox or p22phox expression also reduced the viability and growth rate of intracellular L. monocytogenes and M. bovis BCG as strongly as Rubicon expression and that the suppressive effects of Rubicon or p22phox on the viability of the intracellular L. monocytogenes were nearly abolished by pre-treatment with DPI, AEBSF, and/or L-NMMA, L-NAME. Consistently, the shRNA-mediated reduction of endogenous Rubicon expression led to the significant attenuation of NADPH oxidase activity, ROS production, and pro-inflammatory cytokine production (FIG. 3abc), and thereby, a marked reduction of bacterial killing activity against intracellular L.

monocytogenes and M. bovis BCG in Raw246.7 and BMDM cells (FIG. 3d).

Conversely, the addition of inflammatory cytokines (TNF-a, IL-6 or IL-1 β) detectably increased bacterial killing activity against intracellular L. monocytogenes in vector cells, and this effect was further enhanced in Rubicon expressing cells.

[0265] Besides NADPH phagocyte oxidase pathways, IFN-y-inducible nitric oxide synthase (iNOS) pathways, that generate nitric oxide (NO) radicals, represent another important antimicrobial system in phagocytic cells. To compare Rubicon's effect on TLR2-NADPH oxidaseinduced ROS production vs IFN-y-iNOS-induced NO production for microbial killing activity, Raw264.7 cells were stimulated with IFN-γ, zymosan, L. monocytogenes infection or in combination. While Rubicon-expressing cells showed slightly higher basal levels of ROS and NO production than vector- containing cells, there were only minimal differences in ROS and NO production upon IFN- Y stimulation (FIG. 3ef). By contrast, L. monocytogenes infection or zymosan stimulation produced significantly higher levels of ROS and NO production in Rubicon-expressing cells than in vector-containing cells (FIG. 3ef). The

combination of IFN-γ stimulation and L. monocytogenes infection or IFN-γ

stimulation and Zymosan produced even higher levels of ROS and NO production in Raw246.7-Rubicon cells than in Raw246.7-vector cells, which is likely an indication of the convergence of the two signaling pathways (FIG. 3ef). In addition, the levels of anti-microbial activity were directly correlated with ROS and NO production (FIG. 3g). These results indicate that Rubicon plays a primary role in NADPH oxidase- mediated anti-microbial pathways rather than iNOS-mediated anti-microbial pathways, further supporting the physiological relevance of Rubicon and p22phox interaction. [0266] In order to assess whether the depletion or expression of Rubicon affects in vivo host responses to infection with L. monocytogenes, recombinant adenoviruses, Ad-vector, AdshRubicon, and Ad-Rubicon, were injected intravenously via the tail vein at a dose of -1 x10¹³ pfu/mouse twice and at 1 day postinfection, mice were challenged intraperitoneally with a lethal bacterial dose (1 x10⁷ CFU per mouse, n = 23 mice per each group). Mice infected with Advector showed a median survival of 6d, mice infected with Ad-shRubicon died detectably sooner (median survival, 4d), and mice infected with Ad-Rubicon showed a significantly delayed mortality rate (median survival, 8d) and increased survival rate (40% survival) (FIG. 4a). To determine whether these effects were due to impaired or enhanced bacterial clearance in the Rubicon-depleted or Rubicon-expressing mice, respectively, the bacterial loads, serum cytokine levels, and Rubicon expressions were measured in the liver and spleen at 5d after infection with a lower bacterial dose (1 x10⁶ CFU per mouse). Under these conditions, Rubicondepleted mice or Rubicon-expressing mice had respectively -1000 fold higher or -10-100 fold lower bacterial loads in both organs compared to mice infected with Ad-vector (FIG. 4b). Correlated with the bacterial loads, the serum levels of TNF-a and IL-6 were lower in Rubicondepleted mice and higher in Rubicon-expressing mice than in mice infected with Ad-vector (FIG. 4c). Furthermore, Rubicon-depleted mice showed more severe L.

monocytogenes nduced splenomegaly than mice infected with Ad-vector or Ad- Rubicon. Thus, host defenses against pathogenic L. monocytogenes are

substantially affected by the level of Rubicon expression.

[0267] To further delineate Rubicon's actions in the Beclinl -UVRAG-containing autophagy complex and in the p22p/?ox-gp91p/?ox-containing NADPH complex, Raw264.7 cells expressing Rubicon mutants were evaluated for their phagocytosis and autophagy activities in comparison to those of cells expressing WT. The ACCD mutant induced NADPH oxidase activity, ROS production, pro-inflammatory cytokine production, and bacterial killing activity as strongly as WT (FIG. 4def). In contrast, the expression of the ASR or ACDD/SR mutants led to the suppression of NADPH oxidase activity and pro-inflammatory cytokine production with no effect on bacterial killing activity under the same conditions, suggesting that its p22p/?ox-binding activity, and not its Beclinl -binding activity, is important for Rubicon-mediated induction of a robust anti-bacterial phagocytic response (FIG. 4def). As previously described, expression of the Rubicon WT or the ASR mutant, both capable of binding Beclinl , showed increased LC3-II and p62 protein levels upon rapamycin or starvation treatment, the indications of the suppression of autophagosome

maturation step (FIG. 4g). In contrast, expression of the ACCD mutant, which lost its Beclinl binding ability, showed no effect on autophagosome maturation as evidenced by similar levels of LC3-II and p62 protein compared to those of vector control cells (FIG. 4g). Contrastingly, Rubicon WT and its mutants showed no suppressive effect on autophagosome maturation upon zymosan treatment, BLP treatment or L. monocytogenes infection, likely due to the dissociation of Rubicon from the Beclinl -UVRAG-containing autophagy complex upon such treatments (FIG. 4g). Consistently, Rubicon expression specifically affected the levels of rapamycin- induced, but not zymosaninduced, p62 degradation and treatment with 3- methyladenine or Bafilomycin further enhanced Rubicon suppression of the autophagosome maturation step. These results indicate that Rubicon plays distinctive roles in conventional and TLR-signaling-mediated autophagy, and that Rubicon's actions in the Beclinl -UVRAG-containing autophagy complex and in the p22p/70x-gp91p/70x-containing NADPH complex are functionally and genetically separable.

[0268] This example desmonstrates that due to its ability to bind the Beclinl - UVRAG-containing autophagic complex and the p22p/70x-gp91p/70x-containing NADPH complex in a genetically separable manner, Rubicon rapidly travels between two ancient innate immune machineries, autophagy and phagocytosis, to efficiently orchestrate intracellular anti-microbial responses. By default, Rubicon functions as a gatekeeper of autophagy by interacting with the Beclinl -UVRAG complex to suppress the maturation step. Upon microbial infection, however, Rubicon shuttles from the Beclinl -UVRAG complex to the p22p/?ox-gp91 phox complex, promoting autophagosome maturation while also activating NADPH oxidase activity to induce oxidative microbicidal activity in a timely and efficient fashion. After the completion of this cycle, Rubicon may return to the Beclinl -UVRAG autophagic complex in preparation for the next round of anti-microbial activity. Example 2

[0269] This example shows that N-terminal of p22phox inhibits Rubicon-p22p/?ox interaction. Amino acids 3-10 of p22phox, in particular, is inhibitory of Rubicon- p22phox interaction even without Tat. Treatment with such peptides significantly rescued mice from Gram-negative bacterial LPS-induced lethal shock, Influenza- induced Acute Lung Injury, and Asthma model.

[0270] These results demonstrate that p22phox peptide, as a functional ligand of Rubicon, regulatesTLR-mediated ROS, inflammatory responses, and suggest a novel therapy for acute inflammatory dieases. Therefore, these findings provide new opportunities for the design and discovery of therapeutic drugs to treat infectious diseases and inflammation.

[0271] This example shows that treatment with the p22phox peptide reduced ROS production.

[0272] FIG. 5A-C show that the N-terminal (amino acids 1 -10) of the retro-inverso sequence of p22phox is sufficient for binding to the serine rich (aa 558-625) region of Rubicon. FIG. 6 is a list of p22phox peptide sequences of N10 (ten amino acids) and N8 (eight amino acids) and their mutants. It's shown that the N-terminal 8 amino acids of p22phox are sufficient to bind to Rubicon.

[0273] As shown in FIG. 7, N8 peptide (30 uM) efficiently blocks Rubicon and p22phox interaction (a and b) and thereby suppresses zymosan (TLR2 ligand)- induced or bacterial infection-induced inflammatory cytokine production (c and d) and ROS production (I). The N8 peptide mutants (W6A and W9A) no longer inhibit Rubicon and p22phox interaction (f and h) and no longer block the inflammatory cytokine production (I). Likewise, FIG. 8A-C show that treatment with the p22phox peptide reduced ROS production upon Zymosan or L. monocytogenes infection. Accordingly, Applicants propose a regulatory network centered at Rubicon (FIG. 8 bottom right corner). FIG. 18 illustrates the regions on p22p/?ox that is responsible for binding gp91 phox. [0274] Further, FIG. 9 shows that treatments with the p22phox peptides fused to Tat reduced ROS production upon Zymosan stimulation. In FIG. 10, it is shows that, in the absence of Tat, the p22phox peptide (aa 3-10) is inhibitory of Rubicon- p22phox interaction.

[0275] In a LPS induced sepsis model, FIG. 11 shows that pre- or post-treatment with p22phox peptides significantly rescued mice from Gram-negative bacterial LPS- induced lethal shock. FIG. 12 shows that pre- or post-treatment with p22phox peptides significantly rescued mice from Gram-negative bacterial LPS-induced lethal shock.

[0276] Moreover, FIG. 13 show the effects of p22phox peptides in cecal ligation and puncture (CLP)-induced sepsis: The N8 peptide efficiently blocks sepsis development by single (a, 6 hr after LPS injection), double (b, 6 and 18hr after LPS injection) and triple (c, 6, 12 and 18 hr after LPS injection) injection of N8 peptide. The middle panels are the suppressions of inflammatory cytokine and ROS (NOX activity) productions upon the N8 peptide treatment, and the right panels are the suppressions of COX-2 expression upon the N8 peptide treatment. The bottom panel shows the immunohistochemistry of COX-2 staining of liver and spleen of CLP- induced sepsis mice with or without TAT or TAT-N8 peptide injection.

[0277] Cecal ligation and puncture (CLP)-induced sepsis model was carried out as follows: following laparotomy the latter one third of the cecum was mobilized and ligated below the ileocecal valve and punctured through both surfaces twice with a 22-gauge needle, and a small amount of the bowel contents was extruded through the puncture holes. The midline incision was then closed in layers with 6-0 Ethilon (Ethicon, Somerville, N.J., USA), and the animals were resuscitated with saline (40 mL/kg body weight) subcutaneously, Sham-operated controls (sham) underwent the same surgical procedures, i.e., laporatomy and resuscitation, but the cecum was neither ligated nor punctured. Previously it has been demonstrated that blood cultures taken from mice are positive for both Gram-negative {e.g., Bacterioides fragilis, Escherichia colo, Klebsiella, Proteus mirabilis) and Gram-positive {e.g., Streptococcus bovis) bacteria as early as 1 hour following cecal ligation and puncture (CLP), illustrating the polymicrobial nature of this septic model. [0278] The effects of p22phox peptides were also tested in a LPS-induced acute lung injury model. As shown in FIG. 14, treatment with p22phox peptides

suppressed the NOX activity and cytokine production of lung epithelial cells induced by LPS or influenza infection. Similarly, in a flu-induced acute lung injury model, the post-treatment with p22phox peptides significantly rescued mice from Flu-induced acute lung injury (FIG. 15-16).

[0279] Further, FIG. 17 shows that treatment with p22phox peptides significantly rescued mice from asthma related airway hyperreactivity (AHR).

[0280] When the peptide includes amino acids 3-10 of p22phox only, Tat is not needed for the peptide to be efficient in disrupting p22phox Rubicon interaction. The p22phox peptides are effective in reducing Zymosan-induced ROS production and rescued mice from Gram-negative bacterial LPS-induced lethal shock, suppressed the NOX activity and cytokine production of lung epithelial cells induced by LPS or influenza infection, and rescued mice from asthma related airway hyperreactivity (AHR).

[0281] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the disclosure, which is delineated by the appended claims.

Claims

WHAT IS CLAIMED IS:

1 . An isolated or recombinant peptide comprising amino acids 3-10 of SEQ ID NO: 1 or an equivalent or retro-inverso thereof, wherein the peptide does not bind gp91 phox.

2. The isolated or recombinant peptide of claim 1 , wherein the peptide does not consist of the C-terminal half of SEQ ID NO: 1 .

3. The isolated or recombinant peptide of claims 1 -2, wherein the peptide

further comprises a cell penetrating peptide.

4. The isolated or recombinant peptide of claim 3, wherein the cell penetrating peptide comprises a TAT peptide.

5. The isolated or recombinant peptide of any one of claims 1 -4, wherein the peptide is less than or equal to 30 amino acids long.

6. The isolated or recombinant peptide of claim 5, wherein the peptide is less than or equal to 22 amino acids long.

7. The isolated or recombinant peptide of claim 5, wherein the peptide is 8

amino acids long.

8. The isolated or recombinant peptide of any one of claims 1 -7, wherein the peptide comprises SEQ ID NO: 8.

9. The isolated or recombinant peptide of any one of claims 1 -8, further

comprising a poly(ethyleneglycol).

10. A polynucleotide, wherein the polynucleotide is one or more of: one that

encodes the isolated or recombinant peptide of any one of claims 1 -9; a polynucleotide having at least about 80% sequence identity to a

polynucleotide that encodes the isolated or recombinant peptide of any one of claims 1 -9; a polynudeotie hybridizing under stringent conditions (stringent hybridization conditions is at a temperature of 42° C in a solution consisting of 50% formamide, 5xSSC, and 1 % SDS, and washing at 65° C in a solution consisting of 0.2 X SSC and 0.1 % SDS) to a polynucleotide that encodes the isolated or recombinant peptide of any one of claims 1 -9, or the complement thereof.

1 1 . The polynucleotide of claim 10, further comprising a vector.

12. A cell transformed with the peptide of any one of claims 1 -8 or the

polynucleotide of claim 10 or 1 1 .

13. An antibody that specifically recognizes at least one of: the peptide of any one of claims 1 -8, amino acids 3-10 of SEQ ID NO: 1 or an equivalent or retro-inverso thereof.

14. The antibody of claim 13, wherein the antibody is a monoclonal antibody.

15. The antibody of claim 13 or 14, wherein the antibody is a humanized

antibody.

16. A method for inhibiting production of reactive oxygen species in a cell or a tissue, comprising contacting the cell or the tissue with an agent that inhibits the interaction between p22p/?o and Rubicon, thereby inhibiting production of reactive oxygen species in the cell or the tissue.

17. The method of claim 16, wherein the contacting is in vivo or in vitro.

18. The method of claim 16 or 17, wherein the cell is macrophage cell.

19. A method for treating a condition in a subject in need thereof wherein the condition is one or more of an acute inflammatory disease, influenza-induced acute lung injury, asthma, sepsis, hypertension or atherosclerosis, the method comprising administering to the subject an effective amount of an agent that inhibits the interaction between p22p/?o and Rubicon, thereby treating the condition in the subject.

20. The method of any one of claims 16-19, wherein the agent is one or more of:

(a) an isolated or recombinant peptide of any one of claims 1 -9,

(b) a polynucleotide of claim 10 or 1 1 , (c) a cell of claim 12,

(d) an antibody directed to p22phox,

(e) an antibody of any one of claims 13-15,

(f) an antisense or siRNA directed to p22phox, or

(g) a small molecule inhibitor of Rubicon-p22p/?ox binding.

21 . The method of any one of claims 16-20, wherein the cell or subject is a

mammalian cell or subject.

22. The method of claim 21 , wherein the cell or subject is a human cell or

subject.

23. The method of any one of claims 16-22, further comprising administering to the subject an therapy suitable for treating the condition.

24. Use of an agent that inhibits the interaction between p22phox and Rubicon for the manufacture of a medicament for treating a condition in a subject in need thereof wherein the condition is one or more of an acute inflammatory disease, influenza-induced acute lung injury, asthma, sepsis, hypertension or atherosclerosis.

25. A kit for use in inhibiting production of reactive oxygen species in a cell or treating a condition in a subject in need thereof wherein the condition is one or more of an acute inflammatory disease, influenza-induced acute lung injury, asthma, sepsis, hypertension or atherosclerosis, comprising an agent that inhibits the interaction between p22p/?ox and Rubicon and instructions to use.

26. A method of identifying an agent that inhibits the interaction between

p22p/?ox and Rubicon, comprising contacting a candidate agent with a cell under a condition that stimulates production of reactive oxygen species, wherein a reduction of production of reactive oxygen species and an increased free p22phox or Rubicon level compared to a suitable control indicates that the candidate agent inhibits the interaction between p22phox and Rubicon. An isolated or recombinant peptide comprising a Rubicon variant wherein the variant comprises a p22p/?ox-binding sequence and does not have a Belcinl - binding sequence.

The isolated or recombinant peptide of claim 27, wherein the Rubicon variant is a Rubicon sequence having a deletion of the Belcinl -binding sequence.

The isolated or recombinant peptide of claim 27 or 28, wherein the Rubicon sequence is a mammalian Rubicon sequence.

The isolated or recombinant peptide of claim 29, wherein the Rubicon sequence is a human Rubicon sequence.

The isolated or recombinant peptide of claim 30, wherein the p22phox- binding sequence comprises amino acids 558-625 of the human Rubicon sequence.

The isolated or recombinant peptide of claim 30, wherein the Belcinl -binding sequence comprises amino acids 505-557 of the Rubicon sequence.

The isolated or recombinant peptide of any one of claims 27-32, wherein the Rubicon variant consists of the p22p/?ox-binding sequence of the Rubicon sequence.

The isolated or recombinant peptide of any one of claims 27-33, further comprising a cell penetrating peptide.

The isolated or recombinant peptide of claim 34, wherein the cell penetrating peptide comprises a TAT peptide.

A polynucleotide that encodes the isolated or recombinant peptide of any one of claims 27-35.

The polynucleotide of claim 36, further comprising a vector.

A cell transformed with the peptide of any one of claims 27-35 or the polynucleotide of claim 36 or 37. A method for inhibiting production of reactive oxygen species in a cell or a tissue, comprising contacting the cell or the tissue with a peptide of any one of claims 27-35, a polynucleotide of claim 36 or 37 or a cell of claim 38, thereby inhibiting production of reactive oxygen species.

A method for treating infection in a cell or a tissue, comprising contacting the cell or the tissue with a peptide of any one of claims 27-35, a polynucleotide of claim 36 or 37 or a cell of claim 38, thereby treating infection in the cell or the tissue.

An isolated or recombinant peptide comprising a Rubicon variant wherein the variant comprises a Belcinl -binding sequence and does not have a p22phox- binding sequence.

The isolated or recombinant peptide of claim 41 , wherein the Rubicon variant is a Rubicon sequence having a deletion of the p22p/?ox-binding sequence.

The isolated or recombinant peptide of claim 42, wherein the Rubicon sequence is a mammalian Rubicon sequence.

The isolated or recombinant peptide of claim 43, wherein the Rubicon sequence is a human Rubicon sequence.

The isolated or recombinant peptide of claim 44, wherein the p22phox- binding sequence comprises amino acids 558-625 of the Rubicon sequence.

The isolated or recombinant peptide of claim 45, wherein the Belcinl -binding sequence comprises amino acids 505-557 of the Rubicon sequence.

The isolated or recombinant peptide of any one of claims 41 -45, wherein the Rubicon variant consists of the Belcinl -binding sequence of the Rubicon sequence.

The isolated or recombinant peptide of any one of claims 41 -47, further comprising a cell penetrating peptide. The isolated or recombinant peptide of claim 48, wherein the cell penetrating peptide comprises a TAT peptide.

A polynucleotide that encodes the isolated or recombinant peptide of any one of claims 41 -49.

The polynucleotide of claim 50, further comprising a vector.

A cell transformed with the peptide of any one of claims 40-48 or the polynucleotide of claim 50 or 51 .

A method of inhibiting autophagy in a cell or a tissue, comprising contacting the cell or the tissue with a peptide of any one of claims 41 -49, a

polynucleotide of claim 50 or 51 or a cell of claim 52, thereby inhibiting autophagy in the cell or the tissue.

A method of suppressing growth of a cancer cell, comprising contacting the cancer cell with a peptide of any one of claims 41 -49, a polynucleotide of claim 50 or 51 or a cell of claim 52, thereby suppressing growth of the cancer cell.

A method of identifying an agent that inhibits the interaction between Beclinl and Rubicon, comprising contacting a candidate agent with a cell under a condition that stimulates autophagy, wherein a reduction of autophagy compared to a suitable control indicates that the candidate agent inhibits the interaction between Beclinl and Rubicon.