Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/44—Oxidoreductases (1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/17—Monocytes; Macrophages
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/19—Dendritic cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/24—Antigen-presenting cells [APC]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
  • C12Y111/01—Peroxidases (1.11.1)
  • C12Y111/01015—Peroxiredoxin (1.11.1.15)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55516—Proteins; Peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention is related generally to the field of immunotherapy, and is more specifically related to enhancing an immune response against an antigen by using Prxl as an adjuvant.
• Prxl is a member of the typical 2-cysteine peroxiredoxin family, whose major intracellular functions are as a regulator of hydrogen peroxide signaling through its peroxidase activity and as a protein chaperone. Prxl expression is elevated in various cancers, including esophageal, pancreatic, lung, follicular thyroid, and oral cancer. Elevated Prxl levels have been linked with poor clinical outcomes and diminished overall patient survival. Recent studies have demonstrated that Prxl can be secreted by non-small cell lung cancer cells, possibly via a non-classical secretory pathway. However, until now, the function of secreted Prxl is unknown and has not been previously exploited for therapeutic purposes.
• the present invention provides compositions and methods for stimulating immune responses.
• the compositions comprise an antigen and isolated Prxl protein.
• the antigen and the Prxl protein can be provided in a complex, or they may be covalently linked to one another.
• the Prxl protein in the complex can be present in a multimer.
• the multimer is a decamer.
• the compositions may further comprise antigen presenting cells that have been exposed to the antigen and/or the Prxl protein.
• the antigen can be any antigen against which a stimulated immune response is desired, including but not necessarily limited to antigens expressed by cancer cells or infectious agents.
• the antigen is expressed by tumor cells.
• the method for stimulating an immune response to an antigen in an individual comprising administering to the individual a composition comprising the antigen and an isolated Prxl protein.
• the stimulated immune response can be greater than the immune response stimulated by the antigen in the absence of administering isolated Prxl protein.
• the stimulated immune response can comprise a cell mediated immune response, a humoral immune response and combinations thereof.
• the individual in whom an immune response to an antigen is stimulated is an individual who is a risk for, is suspected of having or has been diagnosed with cancer.
• TG-elicited macrophages were analyzed by flow cytometry for expression of CDl lb, Grl, and F4/80. A representative histogram of 3 independent isolations is shown and depicts Grl and F4/80 expression by CDl lb + cells. Numbers in the insets indicate the percentages of CDl lb + cells in each quadrant.
• B TG-elicited macrophages were incubated with stimulants for 24 h; supernatants were harvested and analyzed for TNF-a (open bars) and IL-6 levels (gray bars). Results are shown as pg/ml and are representative of three independent experiments; error bars represent standard deviation.
• TG-elicited macrophages were incubated for 24h with media only (black bars), 100 nM LPS or 2000 nM Prxl (open bars), 100 nM LPS or 2000 nM Prxl pre-incubated for 20 minutes with 10 ug/mL polymyxin B (hatched bars), or 100 nM LPS or denatured 2000 nM Prxl (gray bars).
• media only black bars
• 100 nM LPS or 2000 nM Prxl open bars
• 100 nM LPS or denatured 2000 nM Prxl gray bars.
• Asterisks indicate P ⁇ 0.01 as compared to cells treated with Prxl or LPS alone.
• TG-elicited macrophages were incubated with media alone, Prxl (50 nM) or LPS (100 nM) for 24 h in the presence (gray bars) or absence (open bars) of 10% FBS. Supernatants were harvested and analyzed for IL-6 levels. Results are shown as pg/ml; error bars represent standard deviation.
• Immature bone marrow derived dendritic cells were incubated with media alone, 20-200 nM Prxl or 100 nM LPS for 24h.
• A Following incubation cells were analyzed by flow cytometry for expression of CDl lc and CD86. Results are shown as percent total cells; error bars represent standard deviation.
• B Supernatants were harvested and analyzed for TNF-a. Results are shown as pg/ml and are representative of three independent experiments; error bars represent standard deviation.
• TG-elicited macrophages were incubated with media harvested from prostate tumor cell lines that were transfected with cDNA encoding for either control shRNA (Scramble) or shRNA specific for Prxl (shPrxl) or in media harvested from cells expressing Prxl specific shRNA to which 50 nM exogenous Prxl had been added (shPrxl + Prxl). Following 24h incubation, supernatants were harvested and analyzed for TNF-a. Results are shown as pg/ml and are representative of three independent experiments; error bars represent standard deviation.
• iBMDCs were isolated from C57BL/6 (TLR4 +/+ ; open bars) and C57BL/10ScNJ (TLR4 _/ ; closed bars) mice and stimulated with 200 nM Prxl, 100 nM LPS, or 100 mM Pam 3 Cys. Supernatants were collected and analyzed by IL-6 ELISA kits.
• TG-elicited macrophages were isolated from C57BL/6 (TLR4 +/+ ; open bars) and C57BL/10ScNJ (TLR4 "/_ ; closed bars) mice and stimulated with 200 nM Prxl, 100 nM LPS, or 100 mM Pam 3 Cys.
• FIG. 4 Interaction of Prxl with TLR4 is dependent upon CD14 and MD2
• A TG- elicited macrophages were isolated from C57BL/6 mice and stimulated with 50 nM Prxl in the presence or absence of control or blocking antibodies to Prxl, CD 14 or MD2 for 24h. Supernatants were collected and analyzed by IL-6 ELISA kits. Results are presented as pg/ml; error bars represent SEM; asterisks indicate P values less that 0.01.
• TG-elicited macrophages were harvested and cell lysates were precipitated with antibodies to TLR4, TLR2, and mouse/goat IgG as described in Materials and Methods; resulting precipitates were separated by SDS-PAGE and probed by Western blot analysis for the presence of Prxl . Blots were also probed with antibodies to TLR4 or TLR2 as a loading control.
• C TG- elicited macrophages were harvested and cell lysates were incubated with antibodies to TLR4 or mouse/goat IgG as described in Materials and Methods; resulting precipitates were separated by SDS-PAGE and probed by Western blot analysis for the presence of Prxl, CD14 and MD2.
• FIG. 1 Kinetics of TLR4/Prxl Interaction.
• A TG-elicited macrophages were stimulated with 200 nM FITC-Prxl or PE-conjugated anti-TLR4 (PE-TLR4). Samples were harvested at the indicated times samples and cell populations were analyzed by Amnis technology. Representative examples of immunostained cells and a merged image of the two stains for each time point are shown.
• B The average similarity coefficient of all cells for each time point is shown; error bars represent standard deviation.
• Prxl Binding to TLR4 is Structure Dependent (A) TG-elicited macrophages isolated from TLR4 +/+ (white bars) or TLR4 ⁇ ⁇ macrophages (filled bars) and incubated with media (None), Prxl, PrxlC52S, or PrxlC83S at 200nM for 24h and supernatants were harvested and analyzed for the presence of TNF-a and IL-6. (B) TG-elicited macrophages isolated from TLR4 +/+ (white bars) or TLR4 ⁇ ⁇ macrophages (filled bars) and incubated with 2000 nM of FITC-labeled proteins for 20 minutes, followed by analysis by flow cytometry.
• A TG-elicited macrophages isolated from TLR4 +/+ (white bars) or TLR4 ⁇ ⁇ macrophages (filled bars) and incubated with 2000 nM of FITC-labeled proteins for 20 minutes, followed by analysis
• Viable cells were selected for analysis by elimination of 7-AAD high populations. Results were normalized for any differences in FITC-labeling and reported in MFI/FITC per nM protein; error bars represent standard deviation. Asterisks indicate a P value ⁇ 0.01. (C) TG- elicited macrophages were incubated with FITC-BSA (squares), Prxl (dark circles),
• PrxlC52S (gray circles), and Prxl C83S (open circles) at various concentrations for 20 min and analyzed by flow cytometry. Results are normalized for differences in FITC-labeling and reported in MFI/FITC per nM protein. Each curve is representative of three individual trials.
• FIG. 8 Expression of shRNA specific for Prxl in PC-3M cells leads to a decrease in Prxl expression.
• A Cell lysate isolated from PC-3M cells (right panel) engineered to express control (Scramble) shRNA or Prxl specific shRNA (shPrxl) was separated by gel electrophoresis, blotted and probed with antibodies specific for Prxl .
• B Expression of shRNA specific for Prxl leads to decreased Prxl levels.
• PC3-M cell lines engineered to express either control shRNA (Scramble) or shRNA specific for PrxT were harvested and analyzed for expression of Prxl or Prx2 by Western analysis.
• Figure 9 provides a graphical demonstrating an adjuvant effect of Prxl against a tumor in an animal model.
• the present invention is based on the unexpected discovery that Peroxiredoxin 1 (Prxl) is a ligand for Toll-like receptor 4 (TLR4), and that this function of Prxl enables it to act as an adjuvant.
• the invention provides compositions and methods for enhancing an immune response against an antigen in an individual.
• the composition comprises isolated Prxl and an antigen.
• the amino acid sequence of Prxl and DNA and RNA sequences encoding it are well known in the art.
• the isolated Prxl protein is provided as a decamer.
• the isolated Prxl protein can comprise or consist of a 191 amino acid sequence, wherein the protein comprising or consisting of the 191 amino acid sequence has peroxiredoxin activity.
• the Prxl comprises the amino acid sequence shown for NCBI Reference Sequence: NP 859047.1 in the Aug 23, 2009 entry which is incorporated herein by reference. It is considered that any splice variant and/or Prxl isomer can be used in the invention.
• the method of the invention comprises administering the composition to an individual so that an immune response against the antigen is stimulated.
• the stimulated immune response can have a therapeutic or prophylactic effect and can include a cell- mediated and/or humoral response, or a combination thereof.
• the stimulated immune response can be greater than the immune response stimulated by the antigen alone.
• Prxl is an anti-oxidant and chaperone molecule that is found in most cell types and is secreted from transformed and activated cells.
• TLR4 is a member of the Toll-like receptor (TLR) family. Interaction of TLRs with their ligands initiates the release of inflammatory mediators such as pro-inflammatory cytokines and the maturation/activation of cells of the immune response. Both inflammation and immune cell maturation/activation are required for induction of antigen specific immunity, including anti-tumor immunity. Agents capable of triggering inflammation and immune cell maturation/activation are referred to in the art as adjuvants.
• Prxl interacts with TLR4 on the surface of immune cells responsible for induction of antigen specific immunity, such as dendritic cells and macrophages.
• TLR4 immune cells responsible for induction of antigen specific immunity, such as dendritic cells and macrophages.
• Prxl interaction with TLR4 leads to production of maturation/activation of dendritic cells and macrophages and secretion of pro-inflammatory cytokines.
• Prxl is an immune adjuvant.
• addition of Prxl to an anti-tumor vaccine increase the potency of the vaccine in animals.
• TLR agonists have been used to improve antitumor immunity, including the TLR9 agonist CpG motif and the TLR7 agonist Imiquimod.
• CpG motifs are DNA oligodeoxynucleotide sequences (ODNs) that contain an unmethylated cytosine-guanosine and flanking nucleotides that are capable of binding to TLR9.
• Imiquimod [l-(2-methylpropyl)-lH-imidazo[4,5-c] quinolin-4 amine; AlderaTM; R-837, S26308] is a synthetic TLR7 agonist that can induce the maturation and migration of DCs28, the expression of IFN-a, TNF-a, IL-la, IL-6, IL-12 and IL-8, and enhance activation of CD8+ T cells.
• NCI National Cancer Insitute
• Prxl exhibits many of the same activities as CpG and imiquimod, however, the receptor to which it binds, TLR4 is more broadly expressed than the TLRs that interact with CpG and imiquimod. Thus, Prxl has potential to be a more effective anti-cancer vaccine adjuvant.
• the present invention can be used to stimulate an immune response to any antigen, and thus the antigen will function as an immunogen.
• the antigens include but are not limited to protein, polypeptide or peptide antigens.
• the antigen may be well characterized, or may be unknown, other than by a known or suspected presence in, for example, a lysate from a particular cell type or any other sample of a biological tissue that contains or could contain the antigen.
• the antigen to which the present invention stimulates an immune response is a tumor antigen.
• Tumor antigens can be obtained by conventional techniques, such as by preparation of tumor cell lysates by repeatedly freezing and thawing tumor cells/tissues obtained from either fresh tumor biopsy tissues or from tumor cells generated in vitro by tissue culture.
• the tumor lysate can be obtained by centrifugation and harvesting the supernatant fluid.
• the tumor cell lysates can be used immediately or frozen and stored until ready for use.
• the antigen can be used in a purified form or in partially purified or unpurified form as cell lysate.
• the antigen may be expressed by recombinant DNA techniques in any of a wide variety of expression systems.
• isolated Prxl proteins may be provided for use in the invention such that discreet, isolated Prxl proteins are complexed with one antigen, or with different antigens.
• Such complexes can be formed using various conditions, such as by differing Prxl to antigen ratios, using a variety of buffers, incubation times, and temperatures.
• the Prxl protein and the antigen are present in the composition of the invention as a complex, and may be either covalently or non-covalently associated with each other.
• the Prxl protein and the antigen may be joined to each other by chemical bonding, such as by covalent bonds, ionic bonds, hydrogen bonds, and/or van der Waals bonds, or combinations thereof.
• Methods for forming protein/antigen complexes with or without covalent bonding are known in the art and can be employed to form complexes between isolated Prxl protein and one or more antigens.
• the complexes of the invention may comprise an isolated Prxl protein and an antigen, or may consist essentially of a Prxl protein and an antigen, or may consist of isolated Prxl protein and an antigen.
• isolated it is meant that the Prxl protein is separated from its natural environment. An isolated protein does not necessarily have to be a purified protein. However, isolated Prxl may nevertheless be purified to any desired degree of purification for use in the present invention. Isolated Prxl protein includes Prxl protein produced by recombinant methods. Prxl multimers, such as decamers, are also considered to be isolated Prxl proteins according to the invention.
• isolated Prxl protein includes Prxl protein that is linked to an antigen via peptide bonds, such as in a fusion protein.
• DNA sequences encoding the Prxl protein and the antigen can be constructed using conventional techniques and expressed in a suitable cell type using any appropriate expression vector.
• the fusion protein can then be expressed in the cells and isolated using any method known to those skilled in the art.
• the Prxl /antigen fusion protein may be separated by a linker sequence.
• compositions of the invention suitable for administration to an individual can be be prepared by mixing the isolated Prxl and/or the antigen with any suitable pharmaceutically acceptable carriers, excipients and/or stabilizers.
• suitable pharmaceutically acceptable carriers excipients and/or stabilizers.
• the individual in whom an immune response is stimulated according to the method of the invention is an individual who is at risk for, is suspected of having, or has been diagnosed with a cancer.
• the antigen with which the Prxl protein is an antigen expressed by any type of cancer cell specific examples of which include but are not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, pseudomyxoma peritonei, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinom
• the individual is at risk for, is suspected of having, or has been diagnosed with an infectious agent.
• the antigens used in the invention can be those expressed by infections agents. Examples of such infectious agents include, but are not limited to viruses, bacteria, fungi and other parasites.
• viruses include, but are not limited to, hepatitis type B or type C, influenza, varicella, adenovirus, herpes simplex virus type I or type II, rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I or type II.
• bacteria include, but are not limited to, M. tuberculosis, mycobacterium, mycoplasma, neisseria and legionella.
• examples of other parasites include, but are not limited to, rickettsia and chlamydia.
• compositions of the invention can be administered using any suitable route of administration.
• Some non-limiting examples include oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal.
• Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration.
• compositions of the invention can be performed in conjunction with conventional therapies that are intended to treat a disease or disorder associated with the antigen.
• the composition could be administered prior to, concurrently, or subsequent to conventional anti-cancer therapies.
• therapies can include but are not limited to chemotherapies, surgical interventions, and radiation therapy.
• an appropriate dosage and treatment regimen provides the composition in an amount effective to stimulate an immune response that provides a therapeutic and/or prophylactic benefit.
• a response can be monitored by an improved clinical outcome, e.g., inhibition in tumor growth and/or metastasis, improved resistance to infection, improved immune cell activation, and/or other parameters that will be apparent to those skilled in the art, dependant upon the condition being treated.
• Routes and frequency of administration of the therapeutic compositions disclosed herein, as well as dosage will vary from individual to individual, and may be readily established using standard techniques.
• the invention provides a composition comprising an isolated population of antigen presenting cells (APCs) and a complex comprising isolated Prxl and an antigen.
• the Prxl /antigen complex can be used to prime APCs, such as dendritic cells, and primed APCs can be administered to an individual to achieve an enhanced immune response against the individual.
• APCs antigen presenting cells
• the composition comprising an isolated population of APCs and a complex comprising isolated Prxl and an antigen can be such that all, substantially all, or less than all of the cells in the composition are dendritic cells.
• the cells in the composition can comprise 100% dendritic cells, or between 100% and 10%> (including all integers there between) dendritic cells.
• composition comprising an isolated population of APCs and a complex comprising isolated Prxl and an antigen is administered to an individual to stimulate a prophylactic or therapeutic immune response in the individual.
• the APCs administered to the individual may be allogenic or syngeneic.
• This Example provides a description of the materials and methods used in
• Lipopolysaccharide LPS, Escherichia coli serotype 026 :B6
• polymyxin B sulfate salt BSA
• ovalbumin OVA
• 7-Amino-Actinomycin D (7-AAD) and thioglycollate brewer modified media was purchased from (Becton Dickinson, La Jolla, CA).
• Capture and detection antibodies for IL-6 and TNF-a used in Luminex assays, as well as protein standards, were purchased from Invitrogen (Carlsbad, CA).
• Antibodies specific for CD1 lb, Gr-1, F4/80, and all isotypes were purchased from PharMingen (Mountain View, CA).
• Antibodies against TLR2, TLR4, and NFKB subunits were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
• Blocking antibodies against MD2 and CD 14 were purchased from Santa Cruz Biotechnology.
• the phycoerythrin (PE) conjugated anti-TLR4 antibody was purchased from eBioscience (San Diego, CA).
• Antibodies specific for Prxl were obtained from Lab Frontier (Seoul, South Korea); this antibody is specific for Prxl and detects only a single band in Western analysis of cells that express Prxl (Figure 8A).
• C57BL/6NCr (TLR4 +/+ and TLR2 +/+ ), C57BL/10ScNJ (TLR4 7 ), B6.129-Tlr2 tmlKir/J (TLR2 "/_ ), C3H/HeNCr (TLR4 +/+ ), and C3H/HeNJ (TLR4 "/_ ) pathogen-free mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed in microisolator cages in laminar flow units under ambient light. The mice were maintained in a pathogen-free facility at Roswell Park Cancer Institute (Buffalo, NY). The Institutional Animal Care and Use Committee approved both animal care and experiments.
• the cultured mouse macrophage cell line (RAW264.7) was maintained in Dulbeco's Modified Eagle Media (DMEM) containing 10% defined fetal bovine serum and lOOU/ml penicillin and 100 ug/ml streptomycin at 37°C and 5.0% C0 2 .
• DMEM Dulbeco's Modified Eagle Media
• RAW264.7 cells were transfected with the pcDNA3.1 plasmid containing either control or MyD88 dominant negative (DN) encoding oligonucleotides using FuGENE 6 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The transfected cells were then selected using G418 for cells expressing the control or MyD88 DN. Cells were then stimulated with buffer, Prxl, or LPS for 24h and culture media was harvested for IL-6 cytokine analysis by ELISA.
• DMEM Dulbeco's Modified Eagle Media
• Peritoneal elicited macrophage cells from mice were obtained by an intraperitoneal injection of 1.0 ml of 3.0 % (w/v) thioglycoUate media (TG). Four days after injection, mice were sacrificed and macrophages were obtained by peritoneal lavage. Macrophages were enriched by adherence selection for 1 h in complete media (DMEM supplemented with 10% defined FBS, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin) and were characterized through FACS analysis for expression of CD1 lb, Grl and F4/80 using standard techniques; cells that were CD1 lb + Grl " F4/80 + were identified as macrophages. Immature bone marrow derived dendritic cells were generated by culture of bone marrow derived cells in GM-CSF using standard techniques. Dendritic cells were identified by the expression of CD1 lc.
• the unbound proteins from the DEAE column containing Prxl, PrxlC52S, or PrxlC83S were pooled and loaded onto a Superdex 200 (16/60, GE Healthcare, USA), and equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl.
• the fractions containing Prxl, Prxl C52S, or PrxlC83S were pooled and stored at -80°C.
• Prxl, PrxlC52S, and PrxlC83S were found to contain 14.14 ⁇ 0.050 EU/ml, 14.07 ⁇ 0.67 EU/ml, and 14.17 ⁇ 0.025 EU/ml respectively.
• Adherent TG-elicited macrophage cells were washed 5-10 times with PBS, to remove any non-adherent cells. Once washed, complete media containing purified Prxl, PrxlC52S, PrxlC83S, or LPS at the specified concentrations were added in the presence or absence of Prxl, MD-2 and CD 14 blocking or control antibodies. In the indicated experiments Prxl proteins or LPS were incubated with polymyxin B or were boiled for 20 minutes prior to addition. After 24 h the supernatant was collected and analyzed by cytokine specific ELISA or the Luminex multiplex assay system. Serum samples were collected as indicated above and IL-6 levels were determined by ELISA. TNF-a and IL-6 ELISA kits were purchased from BD Bioscience (Franklin Lakes, NJ) and assays were completed according to manufacturer's instructions.
• Luminex analyses were performed by the Institute Flow Cytometry Facility in 96-well microtiter plates (Multiscreen HV plates, Millipore, Billerica, MA) with PVDF membranes using a Tecan Genesis liquid handling robot (Research Triangle Park, NC) for all dilutions, reagent additions and manipulations of the microtiter plate.
• Bead sets, coated with capture antibody were diluted in assay diluents, pooled and approximately 1000 beads from each set were added per well.
• Recombinant protein standards were titrated from 9,000 to 1.4 pg/ml using 3-fold dilutions in diluent. Samples and standards were added to wells containing beads.
• the plates were incubated at ambient temperature for 120 min on a rocker, and then washed twice with diluent using a vacuum manifold to aspirate. Biotinylated detection antibodies to each cytokine were next added and the plates were incubated 60 min and washed as before. Finally, PE conjugated streptavidin was added to each well and the plates were incubated 30 min and washed. The beads were resuspended in 100 ⁇ wash buffer and analyzed on a Luminex 100 (Luminex Corp., Austin, TX). Each sample was measured in duplicate, and blank values were subtracted from all readings.
• Luminex 100 Luminex Corp., Austin, TX
• BSA, Prxl, PrxlC52S, and PrxlC83S proteins were conjugated to FITC using a FITC conjugation kit (Sigma, St. Louis, MO). A twenty- fold excess of FITC and individual proteins were dissolved into a 0.1M sodium bicarbonate/carbonate buffer (pH adjusted to
• Prxl , Prxl C52S, and Prxl C83S were 31.00 ⁇ 1.92, 38.52 ⁇ 2.39, 74.49 ⁇ 2.64, and 44.44 ⁇ 2.64 respectively.
• FITC-conjugated BSA, Prxl, PrxlC52S, and PrxlC83S were diluted in 1.0 % BSA in PBS to the specified concentrations and a total reaction volume of 100 ⁇ . These mixtures were incubated with 1.OxlO 6 cells/mL for 20 min on ice to prevent internalization. Cells were washed twice with 1% BSA in PBS and cells were incubated to demonstrate viable from nonviable cells with 7-AAD, less than 30 min before FACsCalibur analysis. Data was acquired from a minimum of 20,000 cells, stored in collateral list mode, and analyzed using the WinList processing program (Verity Software House, Inc., Topsham, ME).
• FITC -conjugated BSA was used as a negative binding control and for mutant studies variations in FITC labeling were normalized by FITC labeling per nM proteins.
• Immunoprecipitation was carried out with 500 ⁇ g of cell lysates and 4 ⁇ g of anti-
• TLR4 or anti-TLPv2 overnight at 4°C. After the addition of 25 of Protein G-agarose (Santa Cruz Biotechnology), the lysates were incubated for an additional 4 h. To validate specific protein interactions, goat IgG (Santa Cruz Biotechnology) or mouse IgG (Santa Cruz Biotechnology).
• Colocalization experiments were performed by the addition of 200 nM FITC-labeled Prxl and PE-conjugated anti-TLR4 to the media of TG-elicited macrophages and kept at 37°C for the indicated times before being transferred to ice, fixed and analyzed.
• Perm Wash buffer (0.1% Triton X-100, 3% FBS, 0.1% sodium azide in phosphate- buffered saline) containing 10 ⁇ g/ml anti-NF B p65 antibody (Santa Cruz Biotechnology) for 20 min at room temperature.
• the cells were then washed with Perm Wash buffer and resuspended in Perm Wash buffer containing 7.5 ⁇ g/ml FITC conjugated F(ab ' ) 2 donkey anti- rabbit IgG for 15 min at room temperature.
• Cells were washed twice in Perm Wash buffer and re-suspended in 1% paraformaldehyde containing 5 ⁇ DRAQ5 nuclear stain
• IDEAS® software package A hierarchical gating strategy was employed using image-based features of object contrast (gradient RMS) and area versus aspect ratio to select for in-focus, single cells.
• Co-localization and nuclear translocation was determined in each individual cell using the IDEAS ® similarity feature which is a log transformed Pearson's correlation coefficient of the intensities of the spatially correlated pixels within the whole cell, of the Prxl and TLR4 images or NFKB and DRAQ5 images, respectively
• the similarity score is a measure of the degree to which two images are linearly correlated.
• Electrophoretic mobility shift assay (EMS A)
• EMS A was performed using conventional techniques. Briefly, 10 ⁇ g of nuclear protein was incubated with y- 32 P-labeled double-stranded NFKB oligonucleotide in 20 ⁇ , of binding solution containing 10 mM HEPES (pH 7.9), 80 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM EDTA, 100 ⁇ g/mL poly(deoxyinosinic-deoxycytidylic acid). The DNA-protein complexes were resolved on a 6% polyacrylamide gel under non-denaturing conditions at 200 V for 2 h at 4°C. Gels were dried and then subjected to autoradiography.
• Example 2 provides a description of results obtained using the materials and methods described in Example 1. Prxl stimulation of cytokine secretion from DCs and TG-macrophages and maturation of DCs is dependent upon TLR4
• TG-elicited murine macrophages were used to assess the ability of Prxl to stimulate cytokine secretion. Macrophage phenotype was assessed by analysis of peritoneal exudate cell populations for CD l ib, Grl, and F4/80 expression. The isolated populations were greater than 99% CDl lb + and of the CDl lb + cell population a majority were Grl " , F4/80 + ( Figure 1A). Stimulation of TG-elicited macrophages with Prxl resulted in the dose dependent secretion of TNF-a and IL-6 that was significantly greater than that observed in unstimulated cells at all doses (P ⁇ 0.01; Figure IB).
• iBMDCs immature bone marrow derived DCs
• iBMDCs immature bone marrow derived DCs
• iBMDCs were incubated with increasing concentrations of Prxl for 24h and examined for cell surface expression of co-stimulatory molecules and secretion of TNF-a.
• Addition of Prxl led to significant dose dependent increase in cell surface expression of the co-stimulatory molecule, CD86 ( Figure 2A) and TNF-a secretion ( Figure 2B) at all doses tested (P ⁇ 0.01 when compared to control).
• TG-elicited macrophages were incubated for 24h in the presence of supernatant collected from Prxl -secreting tumor cells or supernatant collected from tumor cells engineered to express shRNA specific for Prxl . Expression of shRNA resulted in reduced expression of Prxl, but not Prx2 Figure 8B).
• iBMDCs and TG-elicited macrophages were isolated from C57BL/6NCr (TLR4 +/+ ) and C57BL/10ScNJ (TLR4 "/_ ) mice and stimulated with Prxl, LPS or Pam 3 Cys, a TLR2 agonist.
• Prxl The ability of Prxl to induce TLR4 dependent inflammation in vivo was tested by i.p. injection of recombinant Prxl into either C57BL/6NCr (TLR4 +/+ ) or C57BL/10ScNJ (TLR4 "/_ ) mice. Blood was collected 2h post injection and the extent of systemic inflammation was determined by assessing the level of systemic IL-6 (Figure 3C). Injection of Prxl resulted in a significant increase in systemic IL-6 levels (P ⁇ 0.0002) in C57BL/6NCr (TLR4 +/+ ) mice, but had no significant effect on systemic IL-6 levels in C57BL/10ScNJ (TLR4 ⁇ ⁇ ) mice.
• the kinetics of the Prxl and TLR4 interaction was determined using image stream analysis (Amnis) to examine co-localization of the two molecules.
• TG-elicited macrophages were incubated with FITC-labeled Prxl and PE-conjugated anti-TLR4 antibodies.
• the merged images of representative cells indicate that Prxl and TLR4 localize together on the membrane of the macrophage within 5 minutes and that by 30 min, TLR4 and a portion of the Prxl molecules have been internalized (Figure 5 A).
• the histograms to the right of the merged images are a statistical analysis of the similarity of FITC-Prxl and PE-anti-TLR4 in 5,000 cells on a pixel-by-pixel basis.
• Prxl acts as both a peroxidase and a protein chaperone (Wood, et al. (2003) Trends
• PrxlC52S mutant lacks peroxidase activity but retains the decamer structure needed for chaperone activity
• PrxlC83S exists mainly as a dimer, has reduced chaperone activity and intact peroxidase activity. Cytokine secretion following PrxlC52S stimulation of TG-elicited macrophages was not significantly distinct from that observed following stimulation with Prxl ( Figure 6A); however, TG-elicited macrophages stimulated with PrxlC83S displayed a significant reduction in cytokine secretion (P ⁇ 0.01).
• Prxl binding to TG-elicited macrophages was dependent upon the presence of TLR4 as binding of Prxl and the enzymatic null mutant (PrxlC52S) was significantly decreased in the absence of TLR4 (Figure 6B).
• PrxlC83S binding was minimal to either TLR4 expressing or non-expressing macrophages, confirming that Prxl interaction with TLR4 is peroxidase independent and structure dependent.
• Saturation binding (Figure 6C) and competition analyses (Figure 6D) were used to determine the K d , and Ki values for Prxl binding to the surface of TG-elicited macrophages.
• the KJ for Prxl binding to TG-elicited macrophages was 1.6 mM and the Ki was 4.1 mM (Table 1).
• Prxl stimulation of cytokine secretion is MyD88-dependent and leads to TLR4-dependent translocation of NFKB to the nucleus
• Prxl was used to stimulate cytokine expression from RAW264.7 cells expressing dominant negative (DN) MyD88 protein.
• IL-6 secretion following Prxl stimulation is dependent on MyD88 function ( Figure 7A), indicating that Prxl activates the MyD88 signaling cascade, which can lead to activation of NFKB.
• NFKB translocation following Prxl stimulation was analyzed in macrophages isolated from C3H/HeNCr and C3H/HeNJ mice.
• C3H/HeNJ mice have a mutation in the TLR4 ligand binding domain that prevents ligand binding.
• TG-elicited macrophages from C3H/HeNCr and C3H/HeNJ mice were incubated with 200 nM Prxl at 37°C for the indicated times, transferred to ice and incubated with antibodies against NFKB p65; the nuclear stain DRAQ5 was added 15 minutes prior to image stream analysis.
• Prxl incubation with macrophages isolated from C3H/HeNCr mice triggered NFKB translocation within 5 min and nuclear localization was apparent for up to 60 min ( Figure 7B).
• Prxl stimulates TLR4-dependent secretion of TNF-a and IL-6 from TG-elicited macrophages and DCs.
• Cytokine secretion was the result of TLR4 stimulation of the MyD88-dependent signaling cascade and resulted in activation and translocation of NFKB.
• Prxl is an intercellular protein that is secreted from tumor cells and activated T cells. The ability of Prxl to interact with TLR4 and stimulate the release of pro-inflammatory cytokines suggests that it may also act as an endogenous damage-associated molecular pattern molecule (DAMP).
• DAMP damage-associated molecular pattern molecule
• HSP72 and HMGB1 which have also been classified as endogenous DAMPs, have been shown to interact with TLR4.
• Saturation and competition studies indicate that Prxl has a Ka of -1.3 mM and a K; of ⁇ 4.1 mM; extrapolation of data presented by Binder et al. (Binder, et al. 2000. J. Immunol. 165:2582-2587) implies that HSP72 has a 3 ⁇ 4 of 2.1-4.4 mM and a K ; of 10-21.8 mM, suggesting that Prxl interaction with TLR4 is stronger than that of HSP72. Binding affinities are not available for HMGB 1.
• TLR4 as a receptor for a recombinant protein may be complicated by the potential of the presence of LPS within a recombinant protein preparation.
• two controls were included in all of the performed studies.
• recombinant proteins were combined with polymixin B prior to their addition to immune cells.
• Polymixin B is a powerful inactivator of LPS; pre- incubation of recombinant Prxl with polymixin B had no effect on the ability of Prxl to stimulate cytokine expression ( Figure 1).
• pre-incubation of LPS with the same concentration of polymixin B significantly inhibited its ability to stimulate cytokine release.
• Prxl, HSP72 and HMGB1 not appear to have significant structural similarity nor do these molecules appear to share homology with LPS. Prxl, HSP72 and HMGB1 are molecular chaperones and the lack of structural homology between HSP72/HMGB 1 and other TLR4 ligands has led some to speculate that the chaperone cargo rather than the chaperone is being recognized by TLR4. In support of this hypothesis, recent studies have shown that HMGB1 binding to TLR9 is a result of TLR9 recognition of HMGB1/DNA complexes. Extracellular Prxl is present as a decamer, which is associated with Prxl chaperone activity (Wood, et al. 2002.
• PrxlC83S mutant which lacks chaperone activity and exists primarily as a dimer (Wood, et al. 2002. Biochemistry 41 :5493-5504) did not appear to bind to TLR4 ( Figure 4B); however the purified mutant protein was able to stimulate cytokine secretion from
• Prxl stimulation of cytokine secretion was dependent on TLR4 and MyD88 ( Figure 3, 4 and 5); however, FITC-labeled Prxl did bind to macrophages isolated from TLR4 ⁇ ⁇
• PbA the malaria homo log of Prxl requires MD2 to bind to TLR4; our studies indicate that Prxl stimulation of cytokine secretion is optimal in the presence of serum and that antibodies to CD 14 and MD2 block cytokine secretion from Prxl stimulated cells. Furthermore, immunoprecipated complexes of TLR4 and Prxl contain MD2 and CD 14, suggesting that these proteins contribute to the binding of Prxl to TLR4.

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