WO2004038002A2

WO2004038002A2 - Modulation of dendritic cell function and other cellular responses mediated by defensin compositions

Info

Publication number: WO2004038002A2
Application number: PCT/US2003/033940
Authority: WO
Inventors: Arya Biragyn; Larry W. Kwak
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2002-10-25
Filing date: 2003-10-24
Publication date: 2004-05-06
Also published as: AU2003286692A8; AU2003286692A1; WO2004038002A3

Abstract

Methods are provided to reduce inflammation and to treat abnormal physiological conditions or diseases by modulating a robust, type 1 adaptive immune response in vivo. The present invention also provides proteins and the nucleotides encoding them, compositions, vectors (including vaccine vectors) and delivery vehicles, cells capable of expressing the proteins, and kits for practicing the invention.

Description

MODULATION OF DENDRITIC CELL FUNCTION AND OTHER CELLULAR RESPONSES MEDIATED BY DEFENSIN COMPOSITIONS

CROSS-REFERENCE TO RELATED APPLICATION

The present invention claims priority of U.S. Provisional Application 60/421,488, filed October 25, 2002, the disclosure of which is incoφorated herein by reference.

FEDERALLYSPONSORED RESEARCH

Research supporting this application was carried out by the United States of America as represented by the Secretary, Department of Health and Human Services.

FIELD OF THE INVENTION

Defensins are peptides of the immune system produced in response to infection and having a wide spectrum of activities relating to the immune response. For example, β-defensin 2 modulates dendritic cell (DC) maturation. Defensins also modulate DC maturation by counteracting the effects of suppressors inhibiting DC activation. Active, mature DC are potent antigen-presenting cells involved in the immune response. The present invention provides proteins and methods for reducing inflammation and treating diseases and abnormal physiological conditions via robust immune responses in vivo. The present invention also provides vectors (including vaccine vectors) and delivery vehicles, cells capable of expressing the proteins, and kits for practicing the invention.

BACKGROUND OF THE INVENTION Activation of innate immunity through pattern recognition receptors for ligands derived from evolutionary distant pathogens provides essential signals for initiation of the adaptive immune response (Medzhitov, R. and Janeway, C. A. Jr. Curr.Opin.Immunol 9, 4 (1997); Matzinger, P. Semin.Immunol 10, 399 (1998);

Medzhitov, R. and Janeway, C. Jr. N.EngU.Med. 343, 338 (2000)). Microbial infection activates the Toll-like receptor (TLR) signaling cascade (S. D. Wright, R. A. Ramos, P. S. Tobias, R. J. Ulevitch, J. C. Mathison, Science 249, 1431 (1990))(4), which results in expression of various proinflammatory cytokines, chemokines and large quantities of small anti-microbial peptides, such as defensins (M. J. Sweet and D. A. Hume,

J.Leukoc.Biol 60, 826 (1996)). Recently it was reported that β-defensins, epithelial antibacterial peptides with six conserved cysteine residues, might have an additional function as potential chemoattractants of immature DC (iDC) through chemokine receptor CCR6 (Yang, D. et al, Science 286, 525 (1999)).

Defensins are peptides of the innate immune system produced in response to infection, such as microbial infection of mucosal tissue and skin, and having a wide spectrum of activities relating to the immune response. Defensins are a structural class of small cationic peptides, known to have broad antimicrobial activities as the result of membrane permeabihzation mechanisms. They are characterized by their disulfide bond-stabilized β sheet structures and are classified according to the location of their highly conserved cysteine residues, typically six in number, which form the disulfide bonds.

In most non-avian vertebrates, DC arise from bone marrow-derived precursors.

Immature DC are found in the peripheral blood and cord blood and in the thymus (Rezzani et al., Brit.J.Haematol 104, 111-118 (1999)). The presence of additional immature populations has been suggested by the induction of mature DC from populations of resting cells isolated by murine peritoneal cavity lavage. DC of various stages of maturity are also found in the spleen, lymph nodes, tonsils, skin, and human intestine. Avian DC may also be found in the bursa of Fabricius, a primary immune organ unique to avians. Activated mature DC serve as antigen presenting cells (APC) as part of the adaptive immune response.

It would be desirable to have methods or substance useful for modulating the maturation/activation of DC, particularly for the treatment of a disease or other abnormal physiological condition. The immune and inflammatory responses are involved in the response to many abnormal physiological conditions and diseases, including, but not limited to, such diverse conditions as HIN-infection and other viral infections, cancer, allergy, bacterial or parasitic infection, and autoimmune diseases.

SUMMARY OF THE INVENTION

The present invention provides methods, proteins and the nucleotides encoding them, compositions, vectors (including vaccine vectors) and delivery vehicles, cells capable of expressing the proteins, and kits useful for modulating the maturation/activation of DC and their processing and presentation of antigens, particularly for the treatment of diseases or other abnormal physiological conditions, including, but not limited to, such diverse conditions as HIN-infection and other viral infections, cancer, allergy, bacterial or parasitic infection, and autoimmune diseases.

In one aspect, the present invention provides a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a defensin domain; and b. an antigen domain.

In another aspect, the present invention provides a chimeric protein having a molecular weight of less than 30,000 kilodaltons comprising: a. a defensin domain; and b. a tag peptide sequence. In another aspect, the present invention provides a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a defensin domain; and b. an antibody domain, comprising an antigen binding site.

In yet another aspect, the present invention provides a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a Toll-like receptor 4 ligand domain; and b. an antigen domain.

In another aspect, the present invention provides a chimeric protein having a molecular weight of less than 30,000 kilodaltons comprising: a. a Toll-like receptor 4 ligand domain; and b. a tag peptide sequence.

In another aspect, the present invention provides a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a Toll-like receptor 4 ligand domain; and b. an antibody domain, comprising an antigen binding site.

In additional aspects, the invention also provides nucleic acid molecules encoding these chimeric proteins, vectors (including vaccine vectors) and delivery vehicles comprising the nucleic acid molecules, cells comprising the vectors and capable of expressing the proteins, transgenic animals, and kits.

hi still another aspect, the present invention provides a method for inducing maturation of immature dendritic cells, wherein the method comprises: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a protein capable of inducing maturation, wherein the protein comprises a domain of a ligand of Toll-like receptor 4. In a further aspect, the present invention provides a method for chemoattracting immature dendritic cells, wherein the method comprises: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a protein capable of chemoattracting the dendritic cells, wherein the protein comprises a domain of a ligand of a chemokine receptor.

In yet another aspect, the present invention provides a method for treating an abnormal physiological condition or disease, wherein the method comprises inducing maturation of immature dendritic cells in vivo or in vitro by: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a protein capable of inducing maturation, wherein the protein comprises a domain of a ligand of Toll-like receptor 4.

In a further aspect, the present invention provides, a method for treating an abnormal physiological condition or disease, wherein the method comprises inducing maturation of immature dendritic cells in vivo or in vitro by: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a chimeric protein capable of inducing maturation, wherein the chimeric protein comprises a chimeric protein as described above.

In yet a further aspect, the present invention provides a method for activating the Thl immune response, wherein the method comprises inducing maturation of immature dendritic cells in vivo or in vitro by: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a chimeric protein capable of inducing maturation, wherein the chimeric protein comprises a chimeric protein as described above. In another aspect, the present invention provides a composition for activating the Thl immune response, wherein the composition comprises any one of the chimeric proteins described above.

In another aspect, the present invention provides a composition for inducing maturation of immature dendritic cells in vivo or in vitro, wherein the composition comprises any one of the chimeric proteins described above.

In yet another aspect, the present invention provides a method for suppressing the Th2 immune response, wherein the method comprises inducing maturation of immature dendritic cells in vivo or in vitro by: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a chimeric protein capable of inducing maturation, wherein the chimeric protein comprises a chimeric protein as described above.

In another aspect, the present invention provides a composition for suppressing the Th2 immune response, wherein the composition comprises any one of the chimeric proteins described above.

In yet a further aspect, the present invention provides a method of augmenting a cellular or humoral immune response using an adjuvant, wherein the adjuvant comprises a domain of a ligand of Toll-like receptor 4 or a domain of a ligand of chemokine receptor.

In another aspect, the present invention provides a method of augmenting a cellular or humoral immune response using an antigen, wherein the antigen would be delivered to an antigen-presenting cell using an adjuvant, wherein the adjuvant comprises a chimeric protein as described above. In yet another aspect, the present invention provides a method of augmenting a cellular or humoral immune response using an adjuvant, wherein the adjuvant is selected from at least one of the following: a. a chimeric protein as described above; b. a domain of a ligand of Toll-like receptor 4; c. a domain of a ligand of a chemokine receptor; d. a defensin domain; and e. a composition comprising a domain of a ligand of chemokine receptor or a domain of a ligand of a Toll-like receptor 4 and i. a portion of an antigenic material as described above; or ii. an antibody as described above.

In another aspect, the present invention provides a method of augmenting expression of a co-stimulatory molecule on an antigen-presenting cell using an adjuvant, wherein the adjuvant is selected from at least one of the following: a. a chimeric protein as described above; b. a domain of a ligand of Toll-like receptor 4; c. a domain of a ligand of a chemokine receptor; d a defensin domain; and e. a composition comprising a domain of a ligand of chemokine receptor or a domain of a ligand of a Toll-like receptor 4 and i. a portion of an antigenic material as described above; or ii. an antibody as described above.

In another aspect, the present invention provides a method of augmenting induction of an innate or adaptive immune response against a microbial compound capable of suppressing activation of dendritic cell maturation and induction of inflammation using an adjuvant, wherem the adjuvant is selected from at least one of the following: a. a chimeric protein as described above; b. a domain of a ligand of Toll-like receptor 4; c. a domain of a ligand of a chemokine receptor; d. a defensin domain; and e. a composition comprising a domain of a ligand of chemokine receptor or a domain of a ligand of a Toll-like receptor 4 and i. a portion of an antigenic material as described above; or ii. an antibody as described above.

In a further aspect, the present invention provides a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and b. either i. an antigen domain; or ii. an antibody domain, comprising an antigen binding site.

In yet another aspect, the present invention provides a method for inducing maturation of an immature dendritic cell, wherein the method comprises: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or - an antibody domain, comprising an antigen binding site.

In yet another aspect, the present invention provides a method for treating an abnormal physiological condition or disease, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or - an antibody domain, comprising an antigen binding site.

In still another aspect, the present invention provides a method of augmenting a cellular or humoral immune response using an adjuvant, wherein the adjuvant comprises a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and b. either i. an antigen domain; or ii. an antibody domain, comprising an antigen binding site.

In still another aspect, the present invention provides a method for activating the Thl immune response, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

In still another aspect, the present invention provides a method for suppressing the Th2 immune response, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

In yet another aspect, the present invention provides a method of activating an immune response using a Toll-like receptor pathway in a dendritic cell, wherein the method comprises: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

In yet another aspect, the present invention provides a method of suppressing an immune response using a Toll-like receptor pathway in a dendritic cell, wherein the method comprises: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either - an antigen domain; or an antibody domain, comprising an antigen binding site. In yet another aspect, the present invention provides a method for producing an antigen presenting cell capable of expressing or secreting a cytokine, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherem the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either - an antigen domain; or an antibody domain, comprising an antigen binding site; and c. directly or indirectly detecting the presence of the cytokine or of the mRNA encoding the cytokine.

In yet another aspect the present invention provides a method for producing an antigen presenting cell capable of expressing or secreting a chemokine, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site; and c. directly or indirectly detecting the presence of the chemokine or of the mRNA encoding the chemokine. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic of protein constructs used.

Figures 2A-2C are bar graphs of experimental results showing that murine β-defensin 2 induces maturation of bone marrow derived immature DC.

Figure 2D is a bar graph of experimental results showing that murine /3-defensin 2- matured DC produce proinflammatory cytokines IL-12, IL-lo, and IL-1/3.

Figures 3A-3C are bar graphs of experimental results showing that although murine β- defensin 2 chemoattracts iDC via CCR6, TLR-4 is the receptor for DC activation.

Figures 4A-4E depict a representative experiment of dot plots of expression of CD40 and B7.2 in CD1 lc⁺ cells subjected to various conditions.

Figure 5A is a bar graph of experimental results showing that DC treated with 5 μg/ml of various mDF2/3 containing recombinant proteins (N2mDF2_l8, N24mDF2/3 and N2 lmDF2/3) induced comparable activation of iDC, as judged by increase in proportion of CD11⁺/CD40⁺/B7.2⁺ cells.

Figures 5B-5C are bar graphs of experimental results showing that mDF2 activated iDC were isolated from both BALB/c (B) and C57/BL6 (C) strains of mice, which cannot be inhibited by treatment with 5-20 μg/ml RsDPLa (mDF2j8+RsDPLa).

Figure 5D is a bar graph of experimental results showing that mDF2 -matured DC produce proinflammatory cytokine IL-6. Conditioned media from DC incubated for 18 hours with N24mDF2/3, or mproDF2j8 with or without proteinase K (PK), or boiled mDF2/3 (mDF2/3+boil) were measured by enzyme-linked immunosorbant assay (ELISA). Figure 6A is a graph of experimental results showing that DC treated with murine β- defensin 2 elicit augmented T cell responses.

Figure 6B is a graph of experimental results showing that the effect of mDF2(3 fusion to render non-immunogenic self-tumor antigens immunogenic and elicit therapeutic antitumor immunity requires INFγ activity.

Figure 7 is a bar graph of experimental results showing that denatured mDF2|3 does not induce maturation of immature DC.

Figure 8 is a bar graph of experimental results of treatment of XS52 cells with mixtures of mDF2(S and lipopolysaccharide (LPS).

Figure 9 is a bar graph of experimental results comparing the differential effects of mDF2/3 and LPS on secretion of IL-12 (p40) from XS52 cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods, proteins and the nucleotides encoding them, compositions, vectors (including vaccine vectors) and delivery vehicles, cells capable of expressing the proteins, and kits useful for modulating the maturation/activation of DC and their processing and presentation of antigens, particularly for the treatment of diseases or other abnormal physiological conditions, including, but not limited to, such diverse conditions as HIV-infection and other viral infections, cancer, allergy, bacterial or parasitic infection, and autoimmune diseases.

As noted above, the invention provides a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a defensin domain; and b. an antigen domain. The invention also provides a chimeric protein having a molecular weight of less than 30,000 kilodaltons comprising: a. a defensin domain; and b. a tag peptide sequence.

Alternatively, the invention provides a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a defensin domain; and b. an antibody domain, comprising an antigen binding site.

Alternatively, the invention provides a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a Toll-like receptor 4 ligand domain; and b. an antigen domain.

The invention also provides a chimeric protein, wherein the antibody domain comprises at least one domain selected from the group consisting of: i. an antibody against a portion of antigenic material from a self- tumor; and ii. an antibody against a portion of antigenic material from a bacterial, viral, or parasitic antigen.

Alternatively, the invention provides a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a Toll-like receptor 4 ligand domain; and b. an antibody domain, comprising an antigen binding site.

One of ordinary skill in the art will appreciate that, in various embodiments, the chimeric proteins will have different molecular weights depending on size and composition. For example, in a prefened embodiment the molecular weight may be as low as approximately 5 kilodaltons. In a prefened embodiment, the antigen domain comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

In a prefened embodiment, the antigen domain comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a nonimmunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD 8 ; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

In a prefened embodiment, the antibody domain comprises at least one domain selected from the group consisting of: i. an antibody against a portion of antigenic material from a self- tumor; and ii. an antibody against a portion of antigenic material from a bacterial, viral, or parasitic antigen.

In a prefened embodiment, the antibody domain comprises at least one domain selected from the group consisting of: i. an antibody against a portion of an antigenic material from a nonimmunogenic tumor idiotype or to a cancer-specific polypeptide; ii. an antibody against a portion of an antigenic material from a mast cell; iii. an antibody against a portion of an antigenic material from a MHC class I or class II cell; iv. an antibody against a portion of CD4 or CD8; v. an antibody against a portion of an antigenic material from a pathogenic organism; and vi. an antibody against a portion of antigenic material from a molecule associated with an abnormal physiological response.

Preferably, the pathogenic organism is a virus, microorganism, or parasite.

In one embodiment, the chimeric protein further comprises: c. a secretory domain.

A secretory domain would be useful in a variety of settings, including gene therapy and some tissue culture applications.

Preferably, the defensin domain is a /3-defensin domain. More preferably, the β- defensin domain is a /3-defensin 2 domain.

In additional aspects, the invention provides a nucleic acid molecule encoding any one of the chimeric proteins described above. It also provides vectors, including vaccine vectors, comprising the nucleic acid molecule. Additionally, it provides delivery vehicles, such as lipid-based, viral based, or cell-based delivery vehicles, comprising the nucleic acid molecule.

In a further embodiment, the invention provides cells comprising the vectors, which are capable of expressing the chimeric proteins described above. In one embodiment, the cells are capable of secreting the chimeric protein.

In yet another embodiment, the invention provides a kit comprising a vector and a cell for receiving the vector, the vector comprising a nucleic acid, wherein the nucleic acid is operably linked to an expression control sequence and wherein the nucleic acid sequence encodes any one of the chimeric proteins described above.

As noted above, the invention provides a method for inducing maturation of immature dendritic cells, wherein the method comprises: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a protein capable of inducing maturation, wherein the protein comprises a domain of a ligand of Toll-like receptor 4.

In a prefened embodiment, the protein comprises a defensin domain or a fragment thereof, preferably a /3-defensin domain or a fragment thereof, and more preferably a /3-defensin 2 domain or a fragment thereof. The method is practiced either in vivo or in vitro.

As noted above, the invention provides a method for chemoattracting immature dendritic cells, wherein the method comprises: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a protein capable of chemoattracting the dendritic cells, wherem the protein comprises a domain of a ligand of a chemokine receptor.

In a prefened embodiment, the chemokine receptor comprises CCR6.

As noted above, the invention provides a method for treating an abnormal physiological condition or disease, wherein the method comprises inducing maturation of immature dendritic cells in vivo or in vitro by: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a protein capable of inducing maturation, wherein the protein comprises a domain of a ligand of Toll-like receptor 4.

In a prefened embodiment, the protein comprises a defensin domain or a fragment thereof, preferably a /3-defensin domain or a fragment thereof, and more preferably a 3-defensin 2 domain or a fragment thereof.

As noted above, the invention provides a method for treating an abnormal physiological condition or disease, wherein the method comprises inducing maturation of immature dendritic cells in vivo or in vitro by: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a chimeric protein capable of inducing maturation, wherein the chimeric protein comprises any one of the chimeric proteins described above.

In prefened embodiments, the abnormal physiological condition or disease comprises at least one of the following: a. cancer or growth of a non-immunogenic tumor; b. allergy; c. asthma; d. an autoimmune disease; e. an infectious disease; and f. inflammation.

As noted above, the invention provides a method for activating the Thl immune response, wherein the method comprises inducing maturation of immature dendritic cells in vivo or in vitro by: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a chimeric protein capable of inducing maturation, wherein the chimeric protein comprises any one of the chimeric proteins described above.

As noted above, the invention provides a composition for activating the Thl immune response, wherein the composition comprises any one of the chimeric proteins described above.

As noted above, the invention provides a composition for inducing maturation of immature dendritic cells in vivo or in vitro, wherein the composition comprises any one of the chimeric proteins described above.

As noted above, the invention provides a method for suppressing the Th2 immune response, wherein the method comprises inducing maturation of immature dendritic cells in vivo or in vitro by: a. providing immature dendritic cells; and b. contacting the immature dendritic cells with a chimeric protein capable of inducing maturation, wherein the chimeric protein comprises any one of the chimeric proteins described above.

In one embodiment, the method further comprises: c. activating the Thl immune response.

As noted above, the invention provides a composition for suppressing the Th2 immune response, wherein the composition comprises any one of the chimeric proteins as described above.

As noted above, the invention provides a method of augmenting a cellular or humoral immune response using an adjuvant, wherein the adjuvant comprises a domain of a ligand of Toll-like receptor 4 or a domain of a ligand of chemokine receptor. In a prefened embodiment, the protein comprises a defensin domain or a fragment thereof, preferably a |3-defensin domain or a fragment thereof, and more preferably a 3-defensin 2 domain or a fragment thereof.

In a prefened embodiment, the chemokine receptor comprises CCR6.

As noted above, the invention provides a method of augmenting a cellular or humoral immune response using an antigen, wherein the antigen would be delivered to an antigen-presenting cell using an adjuvant, wherein the adjuvant comprises a chimeric protein as described above.

As noted above, the invention provides a method of augmenting a cellular or humoral immune response using an adjuvant, wherein the adjuvant is selected from at least one of the following: a. a chimeric protein as described above; b. a domain of a ligand of Toll-like receptor 4; c. a domain of a ligand of a chemokine receptor; d. a defensin domain; and e. a composition comprising a domain of a ligand of chemokine receptor or a domain of a ligand of a Toll-like receptor 4 and i. a portion of an antigenic material as described above; or ii. an antibody as described above.

As noted above, the invention provides a method of augmenting expression of a co-stimulatory molecule on an antigen-presenting cell using an adjuvant, wherein the adjuvant is selected from at least one of the following: a. a chimeric protein as described above; b. a domain of a ligand of Toll-like receptor 4; c. a domain of a ligand of a chemokine receptor; d. a defensin domain; and e. a composition comprising a domain of a ligand of chemokine receptor or a domain of a ligand of a Toll-like receptor 4 and i. a portion of an antigenic material as described above; or ii. an antibody as described above.

Preferably, the antigen-presenting cell comprises a dendritic cell.

Preferably, the co-stimulatory molecule comprises either CD40 or B7.

As noted above, the invention provides a method of augmenting induction of an innate or adaptive immune response against a microbial compound capable of suppressing activation of dendritic cell maturation and induction of inflammation using an adjuvant, wherein the adjuvant is selected from at least one of the following: a. a chimeric protein as described above; b. a domain of a ligand of Toll-like receptor 4; c. a domain of a ligand of a chemokine receptor; d. a defensin domain; and e. a composition comprising a domain of a ligand of chemokine receptor or a domain of a ligand of a Toll-like receptor 4 and i. a portion of an antigenic material as described above; or ii. an antibody as described above.

Preferably, the chemokine receptor is CCR6.

In a prefened embodiment, the defensin domain is a defensin domain or a fragment thereof, preferably a /3-defensin domain or a fragment thereof, and more preferably a /3-defensin 2 domain or a fragment thereof.

As noted above, the invention provides a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and b. either i. an antigen domain; or ii. an antibody domain, comprising an antigen binding site.

Preferably, the Toll-like receptor ligand domain comprises a Toll-like receptor 4 ligand domain.

Preferably, the defensin domain comprises a /3-defensin domain. More preferably, the /3-defensin domain comprises a /3-defensin 2 domain.

hi a prefened embodiment, the antigen domain or the antigen binding site comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

In a prefened embodiment, the antigen domain or antigen binding site comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD8; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

Preferably, the pathogenic organism is a virus, microorganism, or parasite.

In additional aspects, the invention provides a nucleic acid molecule encoding any one of the chimeric proteins described above. It also provides vectors, including vaccine vectors, comprising the nucleic acid molecule. Additionally, it provides delivery vehicles, such as lipid-based, viral-based, or cell-based delivery vehicles, comprising the nucleic acid molecule.

hi a further aspect, the invention provides cells comprising the vectors, wherein the cells are capable of expressing the chimeric protein. In one embodiment, the cells are capable of secreting the chimeric protein.

In another aspect, the invention provides a kit comprising a vector and a cell for receiving the vector, the vector comprising a nucleic acid wherein the nucleic acid is operably linked to an expression control sequence and wherein the nucleic acid sequence encodes any one of the chimeric proteins described above.

In another aspect, the invention provides a transgenic animal comprising at least one cell as described above.

In another aspect, the invention provides a composition for inducing maturation of immature dendritic cells in vivo or ex vivo, wherem the composition comprises amny one of the chimeric proteins described above.

As noted above, the invention provides a method for inducing maturation of an immature dendritic cell, wherem the method comprises: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

Preferably, the Toll-like receptor ligand domain comprises a Toll-like receptor 4 ligand domain. Preferably, the defensin domain comprises a /3-defensin domain. More preferably, the /3-defensin domain comprises a /3-defensin 2 domain.

In a prefened embodiment, the antigen domain or the antigen binding site comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

Preferably, the pathogenic organism is a virus, microorganism, or parasite.

In one embodiment, the chimeric protein further comprises: iii. a secretory domain.

As noted above, the invention provides a method for treating an abnormal physiological condition or disease, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

Preferably, the defensin domain comprises a 3-defensin domain. More preferably, the /3-defensin domain comprises a /3-defensin 2 domain.

In a prefened embodiment, the antigen domain or antigen binding site comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD8 ; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

Preferably, the pathogenic organism is a virus, microorganism, or parasite.

In one embodiment, the chimeric protein further comprises: iii. a secretory domain. In a prefened embodiment, the abnormal physiological condition or disease comprises at least one of the following: a. cancer or growth of a non-immunogenic tumor; b. allergy; c. asthma; d. an autoimmune disease; e. an infectious disease; and f. inflammation.

As noted above, the invention provides a method of augmenting a cellular or humoral immune response using an adjuvant, wherein the adjuvant comprises a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and b. either i. an antigen domain; or ii. an antibody domain, comprising an antigen binding site.

In a prefened embodiment, the antigen domain or the antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

In a prefened embodiment, the antigen domain or antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD8 ; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

Preferably, the pathogenic organism is a virus, microorganism, or parasite.

In one embodiment, the chimeric protein of the adjuvant further comprises: c. a secretory domain.

In another aspect, the invention provides a method of augmenting a cellular or humoral immune response using an antigen, wherein the antigen would be delivered to an antigen-presenting cell using an adjuvant, wherein the adjuvant comprises any one of the chimeric proteins described above.

In a prefened embodiment, the antigen-presenting cell comprises a dendritic cell.

Preferably, the defensin domain comprises a /3-defensin domain. More preferably, the 3-defensin domain comprises a /3-defensin 2 domain.

In a prefened embodiment, the antigen domain or the antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen. In a prefened embodiment, the antigen domain or antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD8; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

Preferably, the pathogenic organism is a virus, microorganism, or parasite.

In another aspect, the invention provides a method of augmenting expression of a co-stimulatory molecule on an antigen-presenting cell using an adjuvant wherein the adjuvant comprises any one of the chimeric proteins described above.

In a prefened embodiment, the co-stimulatory molecule comprises either CD40 or B7.

Preferably, the defensin domain comprises a /3-defensin domain. More preferably, the 3-defensin domain comprises a /3-defensin 2 domain. In a prefened embodiment, the antigen domain or the antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

In a prefened embodiment, the antigen domain or antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD8; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

Preferably, the pathogenic organism is a virus, microorganism, or parasite.

As noted above, the invention provides a method for activating the Thl immune response, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site. Preferably, the Toll-like receptor ligand domain comprises a Toll-like receptor 4 ligand domain.

As noted above, the invention provides a method for suppressing the Th2 immune response, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

As noted above, the invention provides a method of activating an immune response using a Toll-like receptor pathway in a dendritic cell, wherein the method comprises: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

Preferably, the Toll-like receptor pathway comprises a Toll like receptor 4 pathway and the Toll-like receptor ligand domain comprises a Toll-like receptor 4 ligand domain.

As noted above, the invention provides a method of suppressing an immune response using a Toll-like receptor pathway in a dendritic cell, wherein the method comprises: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either - an antigen domain; or an antibody domain, comprising an antigen binding site.

As noted above, the invention provides a method for producing an antigen presenting cell capable of expressing or secreting a cytokine, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or - an antibody domain, comprising an antigen binding site; and c. directly or indirectly detecting the presence of the cytokine or of the mRNA encoding the cytokine.

Preferably, the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

Preferably, the cytokine is selected from the group consisting of ILlα, ILl/3, TNF, IL6, IL12, and IFNγ.

As noted above, the invention provides a method for producing an antigen presenting cell capable of expressing or secreting a chemokine, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherem the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either - an antigen domain; or an antibody domain, comprising an antigen binding site; and c. directly or indirectly detecting the presence of the chemokine or of the mRNA encoding the chemokine.

Preferably, the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain. Preferably, the chemokine is selected from the group consisting of IL8, RANTES, MDC, IP10, and MlPlα

Preferably, the invention is used for the treatment of vertebrates; for the treatment of vertebrate cells, cell lines, tissues, or organs; for research purposes relating thereto; or for any other purposes encompassed by the description above. More preferably, the invention is used for the treatment of mammals; for the treatment of mammal cells, cell lines, tissues, or organs; for research puφoses relating thereto; or for any other purposes encompassed by the description above. Still more preferably, the invention is used for the treatment of mammals; for the treatment of mammal cells, cell lines, tissues, or organs; for research purposes relating thereto; or for any other puφoses encompassed by the description above.

A "chimeric DNA" is at least two identifiable segments of DNA the segments being in an association not found in nature. Allelic variations or naturally occurring mutational events do not give rise to a chimeric DNA as defined herein.

A "chimeric protein" is a protein with at least two identifiable segments, the segments being in an association not found in nature. In one embodiment, a chimeric protein may arise, for example, from expression of a chimeric DNA capable of being expressed as a protein and having at least two segments of DNA operably linked to enable expression of at least a portion of each segment as a single protein. Other embodiments will suggest themselves to one of ordinary skill in the pertinent art.

As used herein, the terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length, which may have any three-dimensional structure, and may perform any function, known or unknown. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs, including, but not limited to, single-, double-stranded and triple helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA, recombinant polynucleotides, branched polynucleotides, aptamers, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules (e.g., comprising modified bases, sugars, and/or internucleotide linkers).

A "peptide" is a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds or by other bonds (e.g., as esters, ethers, and the like).

An "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both D or L optical isomers, and amino acid analogs and peptidomimetics. "Amino acids" also includes imino acids. An "oligopeptide" refers to a short peptide chain of three or more amino acids. If the peptide chain is long (e.g., greater than about 10 amino acids), the peptide is a "polypeptide" or a "protein." While the term "protein" encompasses the term "polypeptide", a "polypeptide" may be a less than full-length protein.

A "tag peptide sequence" is a short peptide or polypeptide chain of 3 or more amino acids, which is attached to a protein of interest. In a prefened embodiment, a polypeptide, protein, or chimeric protein comprises a tag peptide sequence, which is used for purification, detection, or some other function, such as by specific binding to an antibody. The antibody may be in solution or bound to a surface (e.g., a bead, filter, or other material). The tag peptide sequence should not interfere with the function of the rest of the polypeptide, protein, or chimeric protein. An example of a tag peptide sequence useful in the present invention is a short c-Myc tag with six His residues fused at the carboxyl-terminus. Other examples will be well-known to those of ordinary skill in the pertinent art.

As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and/or translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include, but is not required to include, splicing of the mRNA transcribed from the genomic DNA, capping of the 5' end of the mRNA, polyadenylation of the 3' end of the mRNA, or other processing modifications or events.

As used herein, "under transcriptional control" or "operably linked" refers to expression (e.g., transcription or translation) of a polynucleotide sequence which is controlled by an appropriate juxtaposition of an expression control element and a coding sequence. In one aspect, a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription of that DNA sequence. In another aspect, a DNA (or RNA) sequence having an open reading frame (ORF) is "operably linked" to another DNA (or RNA) sequence also having an ORF, when the ORFs are within the same reading frame and are not interrupted by a stop codon. The ORFs may be separated by a "linker" or "linking sequence," which may encode amino acids to modulate the function of the polypeptide (e.g., a cleavage site, a binding site, an antigen, or a signal sequence). Alternatively, the linker sequence may serve primarily to place the flanking ORFs within the same reading frame. A "chimeric protein" of this type may also be termed a "fusion protein." A "fusion protein" may be useful in a "reporter assay", such as a CAT assay or luciferase assay system, in which the activity of the portion of the peptide encoded by nucleotides from one of the sources is used to measure a property, such as transcriptional activation, nucleotide or protein binding, etc., of the portion of the peptide encoded by nucleotides from another source.

As used herein, "signal sequence," or "secretory sequence" denotes the endoplasmic reticulum translocation sequence. This sequence encodes a "signal peptide," "secretory peptide," or "secretory domain" that communicates to a cell to direct a polypeptide to which it is linked (e.g., via a chemical bond) to an endoplasmic reticulum vesicular compartment, to enter an exocytic/endocytic organelle, to be delivered either to a cellular vesicular compartment, the cell surface or to secrete the polypeptide. This signal sequence may be excised by the cell during the maturation of a protein. Secretory sequences and domains of various species are well known in the art. A "domain" is a region of a protein or polypeptide having a significant tertiary structure.

"Conservatively modified variants" of domain sequences also can be provided within the scope of the invention. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. Alternatively, one or more amino acids may be substituted with an amino acid having a similar structure, activity, charge, or other property. Conservative substitution tables providing functionally similar amino acids are well-known in the art (see, e.g., Proc.Natl.Acad.Sci. USA 89: 10915-10919 (1992)).

As used herein, "in vivo" nucleic acid delivery, nucleic acid transfer, nucleic acid therapy and the like, refer to the introduction of a vector comprising an exogenous polynucleotide directly into the body of a "host organism," such as a human or non- human mammal, whereby the exogenous polynucleotide is introduced to a cell of such organism in vivo. Similarly, "in vitro" nucleic acid delivery, etc., refer to the introduction of a vector comprising an exogenous polynucleotide directly into the "host cell" or "host cell line." The cell or cell line may be prokaryotic or eukaryotic. It may occur in nature or be naturally or artificially altered by mutation, disease, etc. In prefened embodiments, the vector encodes a protein or polypeptide capable of being expressed in the host organism, cell, or cell line.

The term "in situ" refers to a type of in vivo nucleic acid delivery in which the nucleic acid is brought into proximity with a target cell (e.g., the nucleic acid is not administered systemically). For example, in situ delivery methods include, but are not limited to, injecting a nucleic acid directly at a site (e.g., into a tissue, such as an organ tissue or a tumor), contacting the nucleic acid with cell(s) or tissue through an open surgical field, or delivering the nucleic acid to a site using a medical access device such as a catheter.

A "host organism" is an organism or living entity, which may be prokaryotic or eukaryotic, unicellular or multicellular, and which is desired to be, or has been, a recipient of exogenous nucleic acid molecules, polynucleotides, and/or proteins. Preferably, the "host organism" is a bacterium, a yeast, or a eukaroytic multicellular living entity (preferably an animal, more preferably a mammal, still more preferably a human). "Mammals" include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

As used herein, a "target cell" or "recipient cell" refers to an individual cell or cell which is desired to be, or has been, a recipient of exogenous nucleic acid molecules, polynucleotides and/or proteins. The term is also intended to include progeny of a single cell.

A "host cell" encompasses a prokaryotic or eukaryotic single-cell organism, a target cell, or a recipient cell.

The terms "cancer" and "neoplasm" are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. The term "tumor," in either singular or plural form, includes both "cancer" and "neoplasm" and also includes non-malignant, but abenant, growths of cells. The distinction between cancer/neoplasm tumor cells and non-malignant tumor cells may be determined using various tests, especially histological examination.

A cell has been "transformed," "transduced," or "transfected" by exogenous or heterologous nucleic acids when such nucleic acids have been introduced inside the cell. Transforming DNA may or may not be integrated (covalently linked) with chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element, such as a plasmid or a non-integrated viral vector. In a eukaryotic cell, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations (e.g., at least about 10).

An "effective amount" is an amount sufficient to affect beneficial or desired results. An effective amount may be administered one or more times to achieve the beneficial or desired result.

As used herein, a "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, conect and/or normalize an abnormal physiological response. In one aspect, a "therapeutically effective amount" is an amount sufficient to reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant feature of pathology, such as the size of a tumor mass, antibody production, cytokine production (e.g., for Th2 response), fever or white cell count. Additionally, the therapeutically effective amount is an amount sufficient to increase by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant feature of pathology, such as cytokine production (e.g., for Thl response).

An "antibody" is protein that binds specifically to a particular substance, known as an "antigen" (described infra). An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies (e.g., multispecific antibodies). In nature, antibodies are generally produced by lymphocytes in response to immune challenge, such as by infection or immunization. An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and those portions of an immunoglobulin molecule that contains the paratope, including Fab, Fab', F(ab')₂ and F(v) portions. A small single-chain F(v) comprising the variable (V) region of a light chain may be used, particularly when tissue penetration is desired.

An "antigen" is any substance that reacts specifically with antibodies or T lymphocytes (T cells). An "antigen-binding site" is the part of an immunoglobulin molecule that specifically binds an antigen. Additionally, an antigen-binding site includes any such site on any antigen-binding molecule, including, but not limited to, an MHC molecule or T cell receptor. "Antigen processmg" refers to the degradation of an antigen into fragments (e.g., the degradation of a protein into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by "antigen-presenting cells" to specific T cells.

The term "antigenic material" covers any substance that will elicit an innate or adaptive immune response. As used herein, "a portion of an antigenic material" covers any antigenic material or fragment thereof, which is capable of eliciting an innate or adaptive immune response, even if the fragment is an incomplete representation or subset of the antigenic material as a whole. In one embodiment, it includes the minimal antigen sequence required to elicit a specific immune response (preferably approximately 8-15 amino acid residues in length) when bound to an MHC recognized by a T cell.

An "epitope" or "antigenic determinant" is a structure, usually made up of a short peptide sequence or oligosaccharide, that is specifically recognized or specifically bound by a component of the immune system. It is the site on an antigen recognized by an antibody. For example, as described supra, a T cell epitope is at least a portion of a short peptide derived from a protein antigen during antigen processing by an antigen- presenting cell. T-cell epitopes have generally been shown to be linear oligopeptides. Two epitopes conespond to each other if they can be specifically bound by the same antibody. Two epitopes conespond to each other if both are capable of binding to the same B cell receptor or to the same T cell receptor, and binding of one antibody to its epitope substantially prevents binding by the other epitope.

The tenn "antigen presenting cell" (APC) includes any cell which presents on its surface an antigen in association with a major histocompatibility complex molecule, preferably a class II molecule, or portion thereof. Examples of suitable APCs are discussed in detail below and include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells, hybrid APCs, and foster antigen presenting cells. Methods of making hybrid APCs are described and known in the art. The primary APCs for T lymphocytes are dendritic cells, macrophages, and B lymphocytes, while the primary APCs for B lymphocytes are follicular dendritic cells. "Antigen presentation" is the display of ligands (i.e., antigenic peptide fragments bound to MHC molecules (following antigen processing) on the surface of an APC for presentation to, and recognition by, a T cell receptor (T lymphocyte receptor).

"Dendritic cells" (DC) are potent antigen-presenting cells, capable of triggering a robust adaptive immune response in vivo. It has been shown that activated, mature DC provide the signals required for T cell activation and proliferation. These signals can be categorized into two types. The first type, which gives specificity to the immune response, is mediated through interaction between the T-cell receptor/CD3 ("TCR/CD3") complex and an antigenic peptide presented by a major histocompatibility complex ("MHC" defined above) class I or II protein on the surface of APCs. The second type of signal, called a co-stimulatory signal, is neither antigen-specific nor MHC- restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals. This two-fold signaling can, therefore, result in a vigorous immune response. As noted supra, in most non-avian vertebrates, DC arise from bone manow-derived precursors. Immature DC are found in the peripheral blood and cord blood and in the thymus. Additional immature populations may be present elsewhere. DC of various stages of maturity are also found in the spleen, lymph nodes, tonsils, and human intestine. Avian DC may also be found in the bursa of Fabricius, a primary immune organ unique to avians. In a prefened embodiment, the dendritic cells of the present invention are mammalian, preferably human, mouse, or rat. "Langerhans cells" (LC) are skin-specific members of the DC family and have an APC function. "XS52 cells" are LC-like cells established from a murine DC line.

A "co-stimulatory molecule" encompasses any single molecule or combination of molecules which, when acting together with a peptide MHC complex bound by a T cell receptor on the surface of a T cell, provides a co-stimulatory effect which achieves activation of the T cell that binds the peptide. Examples of co-stimulatory molecules include, but are not limited to, CD40, 4-1BB, and CD24a.

Mature dendritic cells are less able to capture new proteins for presentation but are much better at stimulating resting T cells to grow and differentiate. Mature dendritic cells can be identified by their change in moφhology; by their nonadherence; and by the presence of various markers. Such markers include, but are not limited to, cell surface markers such as B7.2, CD40, CDl lc⁺, and MHC class II. Alternatively, maturation can be identified by observing or measuring the production of cytokines, such as pro- inflammatory cytokines. Mature dendritic cells can be collected and analyzed using typical cytofluorography and cell sorting techniques and devices, such as a fluorescence- activated cell sorter (FACS). Antibodies specific to cell surface antigens of mature dendritic cells are commercially available.

"Immune effector cells" refers to cells capable of binding an antigen and which mediate an immune response. These cells include, but are not limited to, T cells (T lymphocytes), B cells (B lymphocytes), monocytes, macrophages, natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates.

"T cells" or "T lymphocytes" are a subset of lymphocytes originating in the thymus and having heterodimeric receptors associated with proteins of the CD3 complex (e.g., a rearranged T cell receptor, the heterodimeric protein on the T cell surfaces responsible for antigen/MHC specificity of the cells). T cell responses may be detected by assays for their effects on other cells (e.g., target cell killing, macrophasge, activation, B-cell activation) or for the cytokines they produce.

"CD4" is a cell surface protein important for recognition by the T cell receptor of antigenic peptides bound to MHC class II molecules on the surface of an APC. Upon activation, naive CD4 T cells differentiate into one of at least two cell types, Thl cells and TH2 cells, each type being characterized by the cytokines it produces. "Thl cells" are primarily involved in activating macrophages with respect to cellular immunity and the inflammatory response, whereas "Th2 cells" or "helper T cells" are primarily involved in stimulating B cells to produce antibodies (humoral immunity). CD4 is the receptor for the human immunodeficiency virus (HIV). Effector molecules for Thl cells include, but are not limited to, interferon γ (IFN-γ), GM-CSF, tumor necrosis factor (TNF-α), CD40 ligand, Fas ligand, interleukin-3 (IL-3), TNF-/3, and IL-2. Effector molecules for Th2 cells include, but are not limited to, IL-4, IL-5, CD40 ligand, IL-3, GS-CSF, IL-10, TGF-/3, and eotaxin. Activation of the Thl type cytokine response can suppress the Th2 type cytokine response.

"CD8" is a cell surface protein important for recognition by the T cell receptor of antigenic peptides bound to MHC class I molecules. CD8 T cells usually become "cytotoxic T cells" or "killer T cells" and activate macrophages. Effector molecules include, but are not limited to, perform, granzymes, Fas ligand, IFN-γ, TNF-c-, and TNF- β.

"B cells" or "B lymphocytes" are a subset of lymphocytes that develop in the bone manow (in non-avian vertebrates) or in the bursa of Fabricius (in avians). When the cell-surface antigen receptor (B cell receptor) is activated by antigen, B cells differentiate into cells producing antibody molecules of the same antigen-specificity as this receptor via reanangement and expression of immunoglobulin genes. B cell responses may be detected by assays for the antibodies they produce.

An "isolated" or "purified" population of cells is substantially free of cells and materials with which it is associated in nature. By substantially free or substantially purified APCs is meant at least 50% of the population are APCs, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90%o free of non-APCs cells with which they are associated in nature.

A "genetic modification" refers to any addition, deletion or disruption to a cell's normal nucleotides. Any method which can achieve the genetic modification of APCs are within the spirit and scope of this invention. Art recognized methods include viral mediated gene transfer, liposome mediated transfer, transformation, transfection and transduction.

An "immunogen" is a substance capable of eliciting an immune response. Each immunoglobulin molecule can potentially bind a variety of antibodies directed at its unique features, or "idiotype," which is comprised of a series of "idiotopes." An "idiotope" is a single antigenic determinant on a variable region of an antibody or T cell receptor. It is the set of idiotopes on an antibody which comprise the idiotype that makes that antibody unique. The "dominant idiotype" is the idiotype found on the major fraction of antibodies generated in response to an antigen.

A "mast cell" is a large bone manow-derived cell found in connective tissues throughout the body. Mast cells contain large storagegranules of various mediator molecules, including histamine, and also synthesize mediators upon activation. Degranulation may be induced by various stimuli, including cross-linking of IgE bound to cell surface Fee receptors. Immediate hypersensitive response, such as acute allergic response, mediated by IgE and mast cell degranulation in response to an antigen can result in life-threatening vasodilation and smooth-muscle contraction, resulting in circulatory collapse and suffocation due to tracheal swelling. This response is known as "anaphylaxis" or "anaphylactic shock."

A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.

A "delivery vehicle" may be lipid-base, viral-based, or cell-based. Delivery may be orally, by injections of various types, by absoφtion through the skin or other membrane, etc. Various forms of delivery are well-known in the art. Devices, such as needles, cannulae, catheters, patches, and chambers may be used. Delivery includes, but is not limited to, delivery of a nucleotide sequence, including by infection; delivery of a protein, including a modified protein; or delivery of some other composition.

A "pathogenic organism" includes a virus, microorganism, or a parasite. A pathogenic organism is capable of triggering an abnormal physiological condition or disease or an abnormal physiological response. A pathogenic organism may be infectious.

Examples of an "abnonrial physiological condition or disease" and an "abnormal physiological response" include, but are by no means limited to, cancer or growth of a non-immunogenic tumor, allergy, asthma, an autoimmune disease, an infectious disease, and inflammation. Cancer and non-immunogenic tumor cells are often characterized by abnormal protein expression, including expression of proteins encoded by mutated nucleotide sequences, abnormal levels of protein expression, or inappropriate expression of proteins. Allergies and asthma (especially allergy-related asthma) are often characterized by abenant accumulation of mast cells, bone manow-derived cells which degranulate to release histamines and which synthesize histamines in response to abenant activation by a number of stimuli (e.g., IgE) in response to allergens.

Autoimmune diseases are directed against "self antigens and are characterized by abnormal levels of MHC class II cells and autoreactive T cells (especially CD4⁺ and CD8⁺ T cells). Infection by an infectious disease triggers an immune response. Inflammation, which may be due to an infection, an injury, or an autoimmune disorder, triggers a response similar to the immune response. These conditions are characterized by up-regulation of some proteins and down-regulation of others (see Table 1 and discussion thereof). An "adjuvant" is any substance capable of enhancing the immune response to an antigen with which it is mixed. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. as well as BCG (bacilli Calmette-Guerin) and Corynabacterium parvum, which are often used in humans. In embodiments of the present invention, adjuvants include defensins (preferably /3-defensins, more preferably /3-defensin 2), ligands of Toll- like receptor 4, and ligands of CCR6 and other chemokine receptors.

A "chemokine" is a small cytokine involved in the migration and activation of cells, including phagocytes and lymphocytes, and plays a role in inflammatory responses. Examples of chemokines include, but are not limited to, IL8, RANTES, MDC, IP10, MIPlα, and MIP \

A "cytokine" is a protein made by a cell that affect the behavior of other cells through a "cytokine receptor" on the surface of the cells the cytokine effects. Cytokines manufactured by lymphocytes are sometimes termed "lymphokines." Examples of cytokines include, but are not limited to, ILlα, ILl/3, TNF, IL6, IL12 (p40), and IFNγ.

Chemokines and cytokines can bind to "receptors," which range in specificity from broad recognition (i.e., binding many types of chemokines, cytokines, or other molecules) to highly specific recognition (e.g., binding a small group of related molecules, binding only closely related molecules or only one type of molecule).

Examples of "chemokine receptors" include, but are not limited to, CCR2, CCR5, CCR6, and CCR7. Examples of "surface receptors" of interest to the present invention include, but are not limited to, mannose receptor (e.g., C type 1), macrophage scavenger receptor (e.g., scavenger R2), and prolactin receptor.

Expression or secretion of a chemokine, cytokine, receptor, marker, or other protein of interest may be measured, either directly or indirectly, using a wide range of methods known to those of ordinary skill in the art. Methods include protein assays, immunoprecipitation methods, Western blotting and other types of direct or indirect immunoblotting, spectophotometry or ultraviolet (UN) methods. As noted above, mature DC can be identified by observing or measuring the production of pro-inflammatoryo cytokines. Mature DC can be collected and analyzed using typical cytofluorography and cell sorting techniques and devices, such as a fluorescence-activated cell sorter (FACS). Antibodies specific to cytokines and chemokines, as well as to cell surface antigens and other markers of mature DC, are commercially available. Depending on the method used, detection may take place using a tagged or labeled protein, a reporter plasmid, a radiolabel (e.g., using a radioactive isotope, such as ³⁵S-Met or ³⁵S-Cys), a chemical label or stain, a fluorescent label, an immunolabel, or by other detection methods known in the art. The protein may be detected in vivo, in situ, or in vitro. In a prefened embodiment, the detection will be quantitative or capable of quantitation in order to measure levels of the protein. For example, the protein may be detected in blood, in a sample of isolated blood cells (e.g., leukocytes), in lymph, in saliva, or in other types of biological samples (including cell samples (e.g., bone manow, lymph nodes). These methods are particularly useful for medical applications of the present invention. In addition, a protein may be detected in situ, such as by detection (e.g., staining, labeling) in a cell sample or in cells from a cell line. Protein detection may take place in a transgenic animal, such as in an animal transgenic for a reporter (e.g., a reporter plasmid or sequence) or an animal expressing a tagged protein which can then be detected. Such detection may take place in vivo or in situ.

Alternatively, the level of the conesponding mRΝA for a given cytokine, chemokine, receptor, marker or other protein of interest may be detected or measured, either directly or indirectly, via a variety of methods known to those of ordinary skill in the art. These methods include, but are not limited to, Northern blotting, hybridization detection (e.g., with oligonucleotides or longer nucleic acid sequences, which may be radiolabeled, chemical labeled, immunolabeled, or fluorescence labeled), or polymerase chain reaction (PCR). PCR methods may be qualitative or, more preferably, quantitative (e.g., quantitative PCR). The mRNA may be detected in vivo, in situ, or in vitro. For example, the protein may be detected in blood, in a sample of isolated blood cells (e.g., leukocytes), in lymph, in saliva, or in other types of biological samples (including cell samples (e.g., bone manow, lymph nodes). Nucleic acids used for hybridization or for PCR may be specific or degenerate, hi addition, they may conespond to the species of animal from which the sample is taken, or the sequence may conespond to a different species (e.g., use of a mouse sequence to probe a rat, human, or chicken sample).

The "major histocompatibility complex" (MHC) is a gene cluster on a vertebrate chromosome, which encodes a set of membrane glycoproteins called the "MHC molecules" or "major histocompatibility antigens." Cells expressing these proteins are "MHC cells" and are divided into two classes. MHC Class I cells present peptides generated in the cytosol to CD8 T cells. MHC Class II cells present peptides degraded in intracellular vesicles to CD4 T cells.

"Humoral immunity" is antibody-mediated specific immunity made in a "humoral immune response" to infection or immunization. "Cellular immunity" or "cell- mediated immunity" is any adaptive immune response in which antigen-specific T cells play a major part in the "cell-mediate immune response." It includes all adaptive immunity that cannot be transfened to a naive recipient with a serum antibody.

A "transgenic animal" is created when gene manipulation is used to modify the germ cells of animals permanently. Typically, foreign genes are placed in the genome by "transgenesis," generating a transgenic organism.

"Defensins" are peptides of the immune system produced in response to infection and having a wide spectrum of activities relating to the immune response. Defensins are a structural class of small cationic peptides, known to have broad antimicrobial activities as the result of membrane permeabihzation mechanisms. They are characterized by their disulfide bond-stabilized β sheet structures and are classified according to the location of their highly conserved cysteine residues, typically six in number, which form the disulfide bonds. There are various types of defensins, including α-defensins, β- defensins, and other types. Defensins and defensin-like proteins have been sequenced in a wide range of organisms, including humans, rats, mice, and other mammals; avians; insects; and plants. A search of the GenBank database reveals many defensin and defensin-like sequences, including, but not limited to, human sequences (e.g., NM 153325, NM 080389; NM 153324; NM 153323; NM 153289; AF540981; AF540980; AF540979; AF540978; AF540977; AF040153; NM 005218; NM 004942; AF295370; and many others) and mouse sequences (e.g., XM 14519; XM 141520; XM 146242; XM 130651; XM 146196; AJ344114; NM 145157; BC024380; NM 139221; NM 139220; NM 139219; NM 139225; and many others).

In one embodiment, a defensin domain of the present invention has a functional activity, which includes, but is not limited to, directly or indirectly up-regulating the Thl pathway or down-regulating the Th2 pathway. Examples of assays are described infra. Other assays will suggest themselves to one of ordinary skill in the pertinent art.

EXAMPLES

In the course of studies utilizing β-defensins and chemokines to target delivery of non-immunogenic antigens to iDC in vivo as vaccines, it was unexpectedly observed that the resulting immune responses differed substantially depending on the type of chemo- attractant moiety used. Particularly, murine /3-defensin 2 (mDF2/3)-based vaccines elicited modest levels of antigen-specific antibodies, but very potent cell mediated responses and antitumor immunity.

Murine /3-defensin 2 (mDF2/3), which has the potential to amplify adaptive immunity by chemoattracting immature DC through CCR6 to the inflammatory sites, acts directly on iDC as an endogenous ligand for Toll like receptor 4 (TLR-4), inducing up-regulation of co-stimulatory molecules and DC maturation. These events, in turn, trigger robust, type 1 polarized adaptive immune responses in vivo.

To test whether jS-defensins had any direct effect on DC function, bone marrow derived iDC were incubated for 18 hours with various fusion proteins consisting of murine _/3-defensin 2 or 3 (mDF2j3 and mDF3/3, respectively, Fig.l) linked to a non- immunogenic lymphoma antigen (idiotype sFv) (as described in A. Biragyn et al., Natwe Biotechnology 17, 253 (1999)). The maturation status of DC was determined by the expression of cell surface markers such as B7.2, CD40, CDl lc , and MHC class II, as well as by the production of pro-inflammatory cytokines. h nature, /3-defensins are produced in a functionally inactive pro-defensin form which is activated by cleavage of the pro-sequence. Therefore, recombinant murine pro-/3-defensin fusion proteins were produced as controls (mproDF2/3, Fig.l and Fig.2). Other controls were recombinant tumor antigen alone (sFv), or fused with chemokine MCP-3 (MCP3, Fig.l and Fig.2). All proteins were 95% pure, and contained less than 0.5 U endotoxin.

Materials and Methods

Production of recombinant proteins. All proteins were expressed in Escherichia coli and purified from inclusion bodies using the same procedures described previously (Fig.l). Briefly, inclusion bodies were denatured with 6 M GuHCL-buffer and refolded in a solution of 0.5 M L-Arg-HCL at 10°C for 72 hours. Refolded proteins were dialyzed against 100 mM urea and 20 mM Tris-HCL, pH7.4, and purified first on Heparin- Sepharose chromatography (Pharmacia- Amersham), followed by Ni-NTA chromatography (Qiagen). N21mDF2β was further purified by cation-exchange chromatography (Pharmacia-Amersham) and reversed -phase HPLC. The purity of mDF2β was determined by electrophoresis in 4-20% TG gels (Novex), Western blotting was performed with anti c-Myc tag 9E10 mAb. The ammo-terminus of the representative protein N21mDF2β was sequenced, resulting in MELDHCHTNG (SEQ ID NO: 1), which conesponds to the mature sequence of mDF2β, except for Met, which was added during construction of the bacterial expression vector. All samples were more than 95 %> pure, and residual endotoxin was removed by repeated purification on Acticlean colums (Sterogene Bioseparations, Inc., Carlsbad, CA). The final endotoxin content of all samples was below 0.5 U/μg of protein, as assayed by Limulus amebocyte lysate kit (BioWhittaker, Walkersville, MD). Production of DC. DC were prepared from bone manow of 4- to 6-month-old mice by using published protocols. Briefly, erythrocytes were lysed with ACL lysis buffer (BioWhittaker, Walkersville, MD). CD8⁺, CD4⁺, B220⁺ and I-A⁺ cells were removed using a mixture of mAbs and rabbit complement (mAbs: TIB-146 anti-B220; TIB-150 anti-CD8; TIB-207 anti-CD4; TD3-229 anti-I-Ab from ATCC, Manassas, NA; and low-toxicity rabbit complement from Cedarlane Laboratories Ltd., Hornby, Ontario, Canada). Cells were plated in individual 96-well tissue culture plates in DC medium (RPMI 1640 containing 5%> heat-inactivated FBS, 1% penicillin, streptomycin, 1%> L- glutamine and 5 x 10-5 2-ME) supplemented with 10 ng ml each of recombinant mouse IL-4 and mouse GM-CSF (Peprotech, Rocky Hill, ΝJ). After 4-5 days of culture, half of the medium was gently removed from the wells and replaced with an equal volume of fusion protein-containing DC medium and incubated for 18 hours. Νonadherent and weakly adherent cells were analyzed by FACS, and supernatants were assayed for cytokines IL-12, IL-1, IL-6 and TΝF-α by enzyme-linked immunosorbant assay (ELISA) using standard methods known in the art (see, e.g., Fig.2D). Cells were stained using mAbs (CDl lc- APC, B7.2-PE, CD40-FITC or isotype matched control mAbs, Pharmingen) in buffer with mouse IgG 25 μg/tube. Samples were analyzed on FACSCalibur (Beckton Dickinson) using CellQuest software. DC generated from various mouse strains retained the immature phenotype. The typical DC preparation contained 30-60% CDl lc⁺, 28-62% CD86⁺ (B7.2), 15-38% CD40⁺ and 25-64% class II expressing cells.

DC activation. DC were cultivated in 96-well plates and treated by addition of 2x samples, prepared in DC medium and sterile filtered through 0.2 μ HT Tuffryn membrane syringe filter (Pall Coφ., Ann Arbor, MI). Specific inhibitors of lipopolysaccharide (LPS), such as polymixin B (5-10 μg ml) or RsDPLa (5-20 μg/ml), were mixed with the samples and incubated with DC for 18 hours (see, e.g., Fig.2A). To test the effects of boiling, mDF2β (100 μg ml) or LPS (10 μg/ml) were boiled for 15 min. (see, e.g., Fig.2B). Similarly, protein samples (100 μg ml) or LPS (10 μg/ml) were treated with 10 μg/ml proteinase K for 45 min at 37°C in DPBS (containing Ca^""^" and Mg⁺⁺) (see, e.g., Fig.2B). Loss of activity of mDF2β after boiling or proteinase K treatment was verified by the inability to chemoattract mCCR6 expressing HEK293 cells.

Effect of signal transduction inhibitors was studied by incubating DC with 5 μg ml mDF2β or 10 ng/ml LPS alone or with pharmacologic signal transduction inhibitors PD98059 (20 μM), LY294002 (10 μM), SB203580 (2 μM),and PP2 (1 μM), all from Calbiochem, and TPCK (20 μM, Sigma- Aldrich) in culture medium for 18 hours, and a proportion of the triple-positive cells for CDllc⁺/I-A^Hlg /B7.2^"1" or CDl lc⁺/CD40⁺/B7.2⁺ were assayed by FACS (see, e.g., Fig.2C). Inhibition % = 100 (%) x [(data from cells treated with inhibitors)/(data from cells treated without inhibitors)].

Microarray analysis of mRNA expression in DC. Total RNA was isolated from 20 xlO⁶ untreated DC or DC treated with 5 μg/ml mDF2β or 10 ng/ml LPS for 6 hours and 24 hours using T Izol reagent (GIBCO-BRL, Gaithersburg, MD). Gene profiling analysis was performed following the Microanay Center protocol, CCR/ NCI, Bethesda, MD. Briefly, gene microchips containing 10,000 cDNA of murine genes and ESTs were hybridized with total RNA (15 μg/microarray) isolated from DC labeled with Cy3 (untreated DC) or Cy5 (DC treated with either mDF2β or LPS) (see Table 1). The data were analyzed with software provided by the Microanay Center, CCR/ NCI (http://ncianay.nci.nih.gov/cgi-bin/restricted/production/cgi-bin/MAAccessTools.pl). Stimulation index represents the ratio of Cy3/Cy5.

TLR-4 activation assay of transduced cells. Transient DNA transfection was performed by using the calcium phosphate technique with 1 x 10 HEK293 cells for 3 hours in 96-well plates with 12 ng IgkB-luciferase, 12 ng pSN-β-galactosidase, 2.5 ng pcDΝA-hTLR4, 6 ng pEF-boss-hMD2, and control plasmid. Then, 24 hours after transfection, the cells were stimulated with the indicated concentrations of defensins, controls or LPS for 24 hours. LPS inhibitor polymyxin B (10 μg/ml) was included in defensin and control stimulations to reduce possible effects by LPS (which was below 0.5 EU/μg protein). The transfected cell lines were cultivated for 48 hours and harvested, and cell lysates were assayed for firefly and Renilla luciferase activity by using the Dual Luciferase Reporter Assay System (Promega) and for β-galactosidase (Tropix) on a Lumat LB9501 (Berthold) (see, e.g., Fig.3C). Firefly luciferase activity of individual transfections was normalized against Renilla luciferase activity. Data reflect the luciferase RLU divided by the control β-galactosidase RLU.

In vitro chemotaxis assay. The migration of DC (50 μl, 10 cells/ml) was assessed using a 48-well microchemotaxis chamber (Neuro Probe, Cabin John, MA) with a 5-μm polycarbonate filter (Osmonics, Livermore, CA) as described (see Fig.4). Cells were incubated at 37°C in humidified air with 5% CO₂ for 1.5 hours. DC migrating across the filter were counted using a Bioquant semiautomatic counting system. The results (as the mean ± SE of triplicate samples) are presented as chemotactic index (C. I.) defined as the fold increase in the number of migrating cells in the presence of test factors over the spontaneous cell migration (in the absence of test factors). Human MIP-3c- and MIP-3β were from PeproTech (Rocky Hill, NJ).

Cell lines and mice. The BALB/c A20 lymphoma (Fig.5) was from the American Type Culture Collection (ATCC, Rockville, MD) and expresses IgGk. Murine CCR6 expressing HEK293 cells (HEK293/CCR6) were gift of Dr. J. Farber (NIAID/NIH).

Therapy of established tumor with DNA vaccine. Six- to 9-week-old female BALB/c mice (Charles River Laboratories, Frederick, MD) or IFN-γ KO mice of BALB/c background (Jackson) were challenged with 2.5 x 10⁵ syngeneic A20 tumor cells. At days 1, 4, 8, and 18 these mice (10 per group) were immunized with Helios Gene Gun System (Bio-Rad, Hercules, CA) with 1-2 μg plasmid DNA, and mice were followed for tumor progression. Differences in survival between groups were determined by noiiparametric logrank test (BMDP statistical software, Los Angeles) (Fig.6B). Animal care was provided in accordance with the procedures outlined in a Guide for the Care and Use of Laboratory Animals. Treatment of iDC with nιDF2β, denatured mDF2β, and controls. Immature DC were treated with 5 μg ml of refolded and active mDF2/3 (mDF2b) or urea-denatured mDF2/3 (mDF2b den). Control iDC were mock treated (CM) or treated with recombinant murine /3-defensin 3 (mDF3/3; mDF3b) protein. All samples contained 5 μg/ml of LPS-inhibitor polymixin B (PxB). After overnight incubation, cells were harvested and stained for surface expression of CDllc⁺ B7.2⁺/CD40⁺ (see Fig.7).

Treatment ofXS52 cells with mixtures ofmDF2β and LPS. XS52 cells were treated with 5 μg/ml of mDF2/3, alone (mDF2b) or with the following concentrations of LPS: 1 ng/ml (mDF2b+Ll), 10 ng/ml (mDF2b+L10) or 100 ng/ ml (mDF2b+L100), respectively. Similarly, control recombinant protein sFv315 (5 μg/ml) was treated without or with LPS at 1 ng/ml (sFv315+Ll), 10 ng/ml (sFv315+L10) or 100 ng/ml (sFv315+L100). In addition, some cells were treated with 2.5 μg/ml mDF2/3 (mDF2b2.5+L10), 1 μg/ml mDF2/3 (mDF2bl+L10), and 0.1 μg/ml mDF2/3 (mDF2b0.1+L10), respectively, mixed with 10 ng ml LPS. Additional control cells were treated with 5 μg/ml of LPS inhibitor polymixin B (PxB) alone, or together with 5 μg/ml mDF2/3 (mDF2b+PxB), or LPS at 100 ng/ml (LlOO+PxB), respectively. After 40 hours of incubation, cells were harvested and stained for expression of CDl lc⁺/B7.2⁺/CD40⁺ (see Fig.8).

Treatment ofXS52 cells with mDF2β or LPS to determine effects on secretion of IL-12. XS52 cells were treated with 5 μg ml mDF2/3 (with 5 μg/ml polymixin B) (mDF2b+PxB) or 10 ng/ml LPS with or without polymixin B (LPS+PxB and LPS, respectively). Control groups were treated with murine pro-/3-defensin 2 (mproDF2/3) with or without polymixin B (mproDF2b+PxB and mproDF2b, respectively) or with polymixin B alone (PxB). The presence of IL12 (p40) was measured in conditioned media by ELISA (see Fig.9) using standard methods known in the art. Results

Figure 1 is a schematic of protein constructs used. All recombinant proteins contained a short c-Myc tag and six His residues (Tag) fused at the carboxyl-temiinus. Several different variants of mDF2β were tested; mDF2β alone (N21mDF2β), or fusion proteins N2mDF2β or N24mDF2β of mDF2β with murine single-chain antibodies (sFv38 or sFv315, nonimmunogenic tumor idiotypes cloned from murine 38C13 or MOPC315 B cell tumors, respectively). Control proteins consisted of sFv alone (sFv315), or sFv fused with functionally active murine β-defensin 3 (mDF3β), or MCP-3 (MCP3), or with a naturally inactive murine pro-β-defensin 2 (mproDF2β). The chemoattractant moiety was separated from sFv with an 11-amino acid spacer peptide (SP).

Figures 2A-2D show that murine /3-defensin 2 induces maturation of bone manow derived immature DC.

In Figure 2 A, the proportion of positive cells for CDl 1⁺/CD40⁺/B7.2⁺ after incubation of iDC with recombinant /3-defensin 2 (5 μg/ml) for 18 hours (see Suppl. Fig.l) was increased from 14.51±1.88% to 49±8.15%. Control DC were incubated in culture medium (CM), with 5 μg/ml sFv alone or fused with pro-/3-defensin (mproDF2/3), murine /3-defensin 3 (mDF3/3), MCP-3 (MCP3), or 10 ng/ml LPS. The proportion of CD11⁺/CD40⁺ B7.2⁺ in CM treated group was in average 14.51+1.88%. To confirm specificity, iDC were incubated with supernatants from the mDF2/3, or mproDF2/3 samples pretreated with 9E10 mAb, specific for myc tag, coupled with protein A- sepharose beads (mDF2|3*, or mproDF23*, pretreated with mAb, repeated twice). *** P<0.001 is for comparison of pooled data between treated with mAb and untreated mDF2/3. Pooled data were from five independent experiments.

Figure 2B shows that the effects of mDF2|3 were abrogated by pretreatment of samples with proteinase K (PK), or by boiling for 15 min prior to DC incubation (boil). *** PO.005 is for comparison of pooled data between mDF2/3 and mDF2/3+boil. Representative data were from three independent experiments.

Figure 2C shows that specific inhibitors of LPS such as polymixin B at 5 and 25 μg/ml (mDF2/3+PM 5 and mDF2/3+PM 25, respectively) do not inhibit mDF2/3-induced maturation of iDC treated for 18 h. The experiment was repeated 3 times. A protein pulsing experiment was performed as follows: DC were washed in DPBS after lh incubation with mDF2/3 in CM (mDF2/3 lh CM), or in serum free medium (mDF2/3 lh). *** P<0.004 is for comparison of pooled data between mDF2/3 and mproDF2/3.

Figure 2D shows that mDF2/3-matured DC produce proinflammatory cytokines IL12, ILlc. and ILl/3. Conditioned media from DC incubated for 18h with mDF2/3, or mproDF2/3 with or without proteinase K (PK), or boiled mDF2/3 (mDF2/3+boiι) were measured by ELISA. Control groups were treated with 10 ng/ml LPS (boiled or not boiled) with or without PK pretreatment. Representative data were from three independent experiments. DC were isolated from BALB/c mice. A representative recombinant protein N24mDF2/3 (Fig.l) was used as a source of mDF2/3.

Figures 3A-3C show that although murine _/3-defensin 2 chemoattracts iDC via CCR6, TLR-4 is the receptor for DC activation.

In Figure 3A, both mDF2/3 and LPS, but not MCP-3 fusion protein (MCP3) induce maturation of iDC from CCR6 KO mice. CCR6 KO phenotype was verified by PCR analysis. Solid bar, CDl lc⁺ B7.2⁺/CD40⁺; open bars, CDl lc⁺/B7.2⁺/I-A^high. Data are representative of two independent experiments.

Figure 3B shows that iDC from the mice with TLR-4 mutation or TLR-4 locus deletion failed to mature by treatment with mDF2/3 or LPS (C3H/HeJ and C5710ScNr, respectively), compared with DC from wild-type (w.t.) mice (C3H/HeN). DC were treated with LPS 10 ng/ml or 5 μg/ml recombinant proteins. Open bar, C3H/HeN; hatched bar, C3H/HeJ; and cross-hatched bar, C5710ScNr. The experiment was repeated three times.

Figure 3C shows activation of the luciferase reporter gene with mDF2/3. Data are representative of two independent experiments. Cells were transiently co-transfected with murine TLR-4 and MD2 and treated with mDF2_J8 (mDF2/3 5 and mDF2/325), or control recombinant protein sFv315 at 5 or 25 μg/ml. All samples were in culture medium (CM) containing 10 μg/ml polymixin B. Control group was treated with 10 ng/ml LPS in CM without polymixin B. A representative recombinant protein N24mDF2/3 (Fig.l) was used as a source of mDF2/3.

Figure 4 depicts a representative experiment of dot plot of expression of CD40 and B7.2 in CDl lc⁺ cells. Proportion of triple-positive cells for CDl 1 /CD40⁺/B7.2⁺ is shown (%). Cells were stained after 18 hours of incubation in culture medium alone (no treatment), with 5 μg/ml N2mDF2β or mproDF2β, or with 1 n^ml or 10 ng/ml LPS, respectively.

Figure 5 A shows that DC treated with 5 μg/ml of various mDF2β containing recombinant proteins (N2mDF2β, N24mDF2β and N21mDF2β, see Fig. 1) induced comparable activation of iDC, as judged by increase in proportion of CDl 1⁺/CD40⁺/B7.2⁺ cells. The control DC were left untreated (CM) or incubated with 5 μg/ml sFv alone or fused with pro-β -defensin (mproDF2β), murine β-defensin 3 (mDF3β), MCP-3 (MCP3), or 10 ng/ml LPS. To confirm specificity, iDC were incubated with supematants from the N2mDF2β, N24mDF2β, or mproDF2β samples pretreated with 9E10 mAb, specific for Myc tag, coupled with protein A-Sepharose beads (N2mDF2β*, N24mDF2β* or mρroDF2β*, pretreated with mAb, repeated twice). ***P < 0.001 is for comparison of pooled data between treated with mAb and untreated mDF2β. Pooled data are from five independent experiments. Figures 5B-5C show that mDF2β activated iDC were isolated from both BALB/c (B) and C57/BL6 (C) strains of mice, which cannot be inhibited by treatment with 5-20 μg/ml RsDPLa (mDF2β+RsDPLa). In contrast, the RsDPLa treatment completely abrogated LPS-induced DC maturation (LPS+RsDPLa). The experiment was repeated three times.

Figure 5D confirms that mDF2β -matured DC produce proinflammatory cytokine IL-6. Conditioned media from DC incubated for 18 hours with N24mDF2β, or mproDF2β with or without proteinase K (PK), or boiled mDF2β (mDF2β+boil) were measured by ELISA. Control groups were treated with 10 ng/ml LPS (boiled or not boiled) with or without PK pretreatment. Representative data were from three independent experiments.

Figure 6A shows that DC treated with murine _/3-defensin 2 elicit augmented T cell responses. CDl lc iDC from BALB/c mice were inadiated at 3000 rad after overnight incubation with 5 μg/ml of mDF2_J8 or 10 ng/ml LPS, and washed three times with cold DPBS to remove soluble stimulants. Ie5 untreated splenocytes from C57BL6 mice were cultured alone (splen. alone) or mixed with titrated amounts of inadiated DC. Proliferation of splenocytes was measured by uptake of H thymidine after four days. P-value is comparison between mDF2/3 and MCP3 treated samples. Data are representative of two experiments.

Figure 6B shows that the effect of mDF2/3 fusion to render non-immunogenic self-tumor antigens immunogenic and elicit therapeutic antitumor immunity requires INFγ activity. IFNγ gene knock out (INFγ KO) or w.t. BALB/c mice were inoculated i.p. with 2.5xl0⁵ syngeneic A20 lymphoma cells on day 0. Then, on days 1, 4, 8 and 18 mice were immunized with 2 μg DNA constructs expressing sFv20, A20 tumor derived idiotype, fused with mDF2/3 (mDF2). Control groups were treated with PBS, or with constructs expressing an inelevant idiotype sFv38, derived from the 38C13 lymphoma, fused to MIP3o- (control DNA). Logrank p- value is for comparison with control DNA immunization. Results shown are representative of three experiments with ten mice per group.

Figure 7 shows the results of treatment of iDC with mDF2/3, denatured mDF2/3, and controls in the presence of the LPS inhibitor polymixin B (PxB). Immature DC were treated with 5 μg/ml of refolded and active mDF2/3 (mDF2b) or urea-denatured mDF2/3 (mDF2b den). Control iDC were mock treated (CM) or treated with recombinant murine /3-defensin 3 (mDF3/3; mDF3b) protein. All samples contained 5 μg/ml of polymixin B (PxB). After overnight incubation, cells were harvested and stained for surface expression of CDllc⁺/B7.2⁺/CD40⁺, shown as a percentage of total cells.

Figure 8 shows the results of treatment of XS52 cells with mixtures of mDF2/3 and LPS. XS52 cells were treated with 5 μg/ml of mDF2₁8, alone (mDF2b) or with the following concentrations of LPS: 1 ng/ml (mDF2b+Ll), 10 ng/ml (mDF2b+L10) or 100 ng/ ml (mDF2b+L100), respectively. Similarly, control recombinant protein sFv315 (5 μg/ml) was treated without or with LPS at 1 ng/ml (sFv315+Ll), 10 ng/ml (sFv315+L10) or 100 ng/ml (sFv315+L100). In addition, some cells were treated with 2.5 μg/ml mDF2/3 (mDF2b2.5+L10), 1 μg/ml mDF2/3 (mDF2bl+L10), and 0.1 μg/ml mDF2_j3 (mDF2b0.1+L10), respectively, mixed with 10 ng/ml LPS. Additional control cells were treated with 5 μg/ml of LPS inhibitor polymixin B (PxB) alone, or together with 5 μg/ml mDF2/3 (mDF2b+PxB), or LPS at 100 ng/ml (LlOO+PxB), respectively. After 40 hours of incubation, cells were harvested and stained for expression of CDl lc⁺/B7.2⁺/CD40⁺, shown as a percentage of total cells.

Figure 9 shows the results of treatment of XS52 cells with mDF2/3 or LPS to determine effects on secretion of IL-12. XS52 cells were treated with 5 μg/ml mDF2/3 (with 5 μg/ml polymixin B) (mDF2b+PxB) or 10 ng/ml LPS with or without polymixin B (LPS+PxB and LPS, respectively). Control groups were treated with murine pro-/3- defensin 2 (mproDF2/3) with or without polymixin B (mproDF2b+PxB and mproDF2b, respectively) or with polymixin B alone (PxB). The presence of IL12 (p40) was measured in pg/ml in conditioned media by ELISA using standard methods known in the art.

The proportion of CD 11 c⁺ cells expressing both CD40 and B7.2 was not changed by the treatment of iDC with pro-/3-defensin 2 fusion protein, MCP-3, or sFv alone compared with complete medium (Fig.2 A). Similarly, expression of MHC class II was not increased in CDl lc⁺ cells by any of those agents. In contrast, iDC treated with as little as 5 μg/ml mDF2/3 fusion protein expressed significantly higher levels of MHC class II and B7.2⁺/CD40⁺ cells (Fig.2A and Fig.4). Furthermore, two other recombinant fusion proteins of mDF2/3, either with a short c-myc peptide tag sequence (N21mDF2/3), or fused with a different sFv (N24mDF2/3), also induced DC maturation (Fig.5 A). This effect was abrogated completely by preabsoφtion of mDF2/3 proteins with anti c-myc specific mAb (pretreated with mAb, Fig.2A and Fig.5A).

Lipopolysaccharide (LPS) is known to induce DC maturation, and consistent with this, was able to induce activation of CDl lc⁺ B7.2⁺/CD40⁺ cells (Fig.3A). It is unlikely that the effects of mDF2/3 were due to contaminating LPS, since the endotoxin content of the proteins was well below the threshold level of 1 ng/ml LPS (Fig.4). Moreover, DC maturation was abrogated completely by treatment of mDF2/3, but not LPS, with proteinase K (mDF2_/3+PK, Fig.2b), or by boiling for 15 min (mDF2/3+boil), suggesting that the component responsible for inducing DC activation was a protein. Efficiency of protein denaturation by boiling was confirmed by the inability of boiled mDF2/3 to chemoattract CCR6 expressing HEK293 cells. Furthennore, the activity of LPS on DC activation could be completely blocked by microbial peptides, such as polymixin B (LPS+PM, Fig.3C) (A. Wiese et al., J.Membr.Biol. 162, 127 (1998)), or by nontoxic Diphosphoryl lipid A from the nontoxic LPS of Rhodobacter sphaeroides (LPS+RsDPLA, Fig.5B, C), both of which are able to compete with LPS for binding to LBP and CD14, respectively (B.W. Jarvis et al., Infect.Immun. 65, 3011 (1997)). fri contrast, neither of these inhibitors affected mDF2/3-induced maturation (Fig.2C and Fig.5B, C). Additionally, the DC maturation could be induced by brief (1 hour) treatment with mDF2/3 (mDF2b lh CM), even in serum free medium (mDF2b lh, Fig.2c), suggesting that mDF2/3 could directly activate iDC in the absence of serum accessory proteins, such as LPS binding protein, which is needed for LPS activity. Finally, DC maturation required fully functional β-defensin 2, as mproDF2/3 or denatured mDF2/3 (mDF2b+boil, Fig.2b), which do not chemoattract DC (via CCR6), failed to induce maturation.

With reference to Figure 7, the results show that mDF23, but not denatured mDF2/3, induced iDC maturation, and that the observed maturation could not be attributed to LPS induction, due to the presence of LPS-inhibitor PxB in all samples (Fig.7).

With reference to Figure 8, for XS52 (Langerhans-like) cell samples containing 5 μg/ml mDF23, the presence or amount of LPS exhibited no additive effect, in contrast to the increasing percentages of mature cells observed with concomitantly increasing LPS concentrations in the sFv control samples (Fig.8). Moreover, decreasing the concentration of mDF2/3 while maintaining the level of LPS resulted in concomitantly decreasing percentages of cell maturation. Finally, among the controls treated with LPS inhibitor PxB, the sample treated with both PxB and 5 μg/ml mDF2/3 exhibited a percentage of cell maturation equivalent to the samples treated with 5 μg/ml mDF2/3 alone or with various amounts of LPS, while the sample treated with both PxB and 100 ng/ml LPS (the highest concentration tested) exhibited a percentage of cell maturation no higher than the PxB control. Taken together these results show that cell maturation in the nιDF23 samples occurs in response to the treatment with mDF2/3, independently of other factors, such as the presence or amount of LPS or, conversely, the presence of LPS-inhibitor PxB.

Figure 9 shows the differential effects of mDF2_/3 and LPS on secretion of IL12 from XS52 cells (Fig.9). No secretion of IL12 is observed in the cells treated with LPS inhibitor polymixin B (PxB) alone. Likewise the cells treated with the naturally- occurring, inactive murine pro-/3-defensin 2 (mproDF2/3) also fail to secrete IL12, independent of the presence or absence of PxB, the latter indicating an absence of any contaminating LPS activity. IL12 secretion is observed, however, in the cells treated with mDF2/3 in combination with PxB. The cells treated with LPS alone secrete IL12, but this activity is inhibited by the presence of PxB in the cells treated with both LPS and PxB.

CCR6 is unlikely to be the signaling receptor of mDF2_/3-induced DC maturation, because DC isolated from CCR6 deficient mice (CCR6 KO) were still capable of being activated by treatment with either mDF2β or LPS, but not with control MCP-3 fusion protein (Fig.3 A). The CCR6 KO phenotype was verified by PCR and by the inability of splenocytes from these mice to migrate in response to MIP3α. In addition, a homologous anti-microbial peptide, murine /3-defensin 3 (mDF3/3) which is also capable of acting as a chemoattractant for iDC via CCR6 (A. Biragyn et al., J.Immunol. 167, 6644 (2001)), failed to induce maturation of DC (Fig.2A).

Treatment of iDC with mDF2/3 and LPS generated similar expression profiles for pro-inflammatory chemokines and cytokines, including RANTES, MDC, IP- 10, MIPlo ILl 3, TNFc. and IL12, as well as the expression of receptors, such as CCR7, which is also associated with the maturation state of DC (Table 1). mRNA for cell surface receptors associated with the iDC, such as CCR2 and CCR5, mannose receptor and macrophage scavenger receptor 2, were all down regulated (Table 1).

Table 1. mRNA expression profiles of DC incubated with either mDF2/3 or LPS for 6 and 24 hours. Representative data from mRNA expression anays of genes are shown. Numerical values (stimulation index) indicate specific mean fluorescence intensity after subtraction of background fluorescence from untreated DC.

Gene \ Treatment mDF2/36h LPS 6h mDF2β24h LPS 24h

Chemokine:

RANTES 1.7 3.9 8.8 9.5

MDC 2.4 1.7 2.2 2.8

IP-10 1.7 1.8 1.6 2.6

MlP-lα 3.3 2.8 1 1.2 IP-ljS 2.6 2.9 0.9 1.2

Chemokine receptor:

CCR2 0.5 0.3 0.2 0.2

CCR5 1 0.7 0.3 0.4

CCR7 1.8 2.3 2 2.6

Proinflammatory cytokine:

IL-1_/3 7.2 6.2 4.7 6.9

TNFc. 3.5 2.5 1.2 1.2

IL12 (p40) 2.4 5 2.1 3.1

Costimmulatory molecule:

CD40 1.9 2.4 1.4 1.4

4-1BB 1.5 1.5 2.7 3.1

CD24a 0.4 0.3 0.2 0.1

Surface receptors:

Mannose receptor, C type 1 0.4 0.3 0.2 0.3

Macrophage scavenger 0.4 0.3 0.4 0.3 receptor

Prolactin receptor 0.3 0.4 0.8 0.8

Other genes:

IGF binding protein 4 0.4 0.5 1.4 1.3

Serum amyloid A3 7.3 8.2 3 3.4

Superoxide dismutase 2

(Mitochondrial) 3.8 6.3 2 2

Nitric oxide synthase 2 1.4 1.6 4.7 6.2

Caspase 12 6.4 1.2 1.1 1.1

Metallothionein 1 1.4 1.4 2.4 3.4

Metallothionein U 1.5 1.4 4.4 6.2

SOCS-3 2.3 3.8 1.9 2.3

Transcription factor 2 1.6 2.7 3.4 5.8 lκB-β 2.3 2.7 1.5 1.7

VEGF-A 1.1 1.1 1.9 2.7

Thrombospondin 1 1.1 1.1 2.2 4.8

Pre-B cell colony enhancing factor 1.8 2.5 1.5 2.2

Furthermore, both mDF2/3- and LPS-induced DC maturation were similarly inhibited by co-incubation with various pharmacologic inhibitors of signal transduction molecules, suggesting that mDF2/3 and LPS share signal transduction pathways and possibly the same receptor; namely, Toll-like receptor 4 (TLR-4). Neither mDF2/3 nor LPS induced maturation of DC isolated from either TLR-4 mutant or TLR-4 locus deleted mice (C3H/HeJ and C57BL10ScNcr strains, respectively), while inducing maturation of DC from control C3H/HeN mice (Fig.3B). Finally, mDF2/3, but not control antigen (sFv315), activated TLR-4 expressed by HEK293 cells transiently transfected with murine TLR-4 and MD2 plasmids (Fig.3C). Overall, these data strongly indicate that mDF23 is an endogenous signaling ligand for TLR-4.

Functionally, mDF2/3-activated DC exhibited Thl -polarized responses, such as the production of proinflammatory cytokines IL12, IL-lα, ILl/3 and IL-6 (Fig.2D and

Fig.5D). In addition, the proliferative response of splenocytes from C57BL/6 mice in a mixed lymphocyte reaction was significantly increased by pre-treatment of DC from BALB/c mice with mDF2/3 or LPS, but not with control mρroDF2/3, mDF3/3 or MCP3 proteins (Fig.6A), suggesting that mDF2/3 augmented primary T cell immune responses by activating DC.

It was demonstrated that mDF23-based vaccines elicited therapeutic, T-cell dependent, antitumor immunity in vivo (mDF2_/β, Fig.6B). However, IFNγKO mice immunized with these mDF2/3 fusion constructs failed to reject tumors (mDF2/3 IFNγ, Fig.6B), suggesting that IFNγ is required and providing an important association between mDF2/3 and type 1 immunity in vivo. Furthermore, the vaccine required that tumor antigen was physically linked with fully functional mDF2β, while unlinked free peptide mixture or fusion antigens with an inactive pro-/3-defensin 2 did not elicit any antitumor immunity. Thus, linkage of tumor antigens with mDF2β enabled not only efficient APC targeting, but presumably also activated DC maturation in vivo. Importance of DC maturation in induction of adaptive immune responses is also suggested by a similar observation that linkage with agonistic anti-DEC205 mAb antigen facilitated efficient antigen uptake and processing by DC, yet this induced tolerance unless DC were first activated by CD40 engagement.

Incubation of iDCs with mDF2/3 or LPS, but not mDF3/3, induced the differentiation into mature cells which expressed CDl lc, CD40, and B7.2 cell surface molecules, as described above. These mature DC became capable of presenting antigen, as evidenced by the fact that they were able to augment a mixed luecocyte (proliferative) response. Furthermore, mDF2/3, like LPS, induced dendritic cells to produce cytokines, such as IL1, TNF, IL6, and IL12, and chemokines, such as IL8, MDC, IP10, MIPlc. and to up-regulated the expression of the CCR7 receptor (Table 2). However, maturation of DC was associated with a decrease in the expression of chemokine receptors such as CCR2, CCR5, and CCR6 and the mannose and scavenger 2 receptors. The possibility that LPS contamination of the recombinant protein accounted for its activating effect on iDCs was ruled out (Table 2), as limulus assays failed to detect any LPS in the mDF23 preparation. The activity of mDF2/3, unlike that of LPS, was destroyed by digestion with proteinase K and boiling for 15 minutes. In contrast, the activity of LPS, but not of mDF2/3, was blocked by polymixin B, diphosphoryl lipid A from Rhodobacterium, and by incubating iDCs in serum-free medium lacking LBP for one hour.

The activating effect of LPS and mDF2/3 on iDCs from CCR6 knockout mice was also compared. Unexpectedly, the mDF2/3, as well as LPS, was able to induce maturation of iDCs in the absence of CCR6, suggesting that another receptor interaction is responsible for this effect. The capacity of LPS and mDF2/3 to stimulate iDCs from mice with mutant TLR4 genes was then compared. Immature DCs obtained from defective C3H/HeJ and C57BL1 OScNcr mice with TLR4 gene defects were unresponsive to both LPS and mDF2/3. mDF2/3 also induced receptor gene expression of HEK293 cells transiently co-transfected with murine TLR4 and MD2 genes. These results suggest that mDF2/3 uses TLR4 to induce Thl immune responses independent of its CCR6 chemotactic effect. Consequently these data identify mDF2_/8 as a unique endogenous ligand for TLR4.

The results are summarized in Table 2.

Table 2. Comparison of effects on immature dendritic cells (iDCs) of murine /3-defensin 2 (mDF2/3) and lipopolysaccharide (LPS).

IFN, interferon; IL, interleukin; TLR, Toll-like receptor; TNF, tumor necrosis factor.

These findings show that murine _/3-defensin 2, which has hitherto been considered a peptide with direct antimicrobial effects, modulates adaptive immune response not only by recruiting iDC to the site of inflammation through chemokine receptor CCR6, but also by activating DC maturation signaling tlirough a microbial pattern recognition receptor, TLR-4. These results indicate that mDF2/3 is an endogeneous ligand of TLR-4 signaling, for example, for heat shock antigens HSP60 and HSP70 expressed during stress/necrosis. mDF2_/8 may be involved in the potentiation of subthreshold amounts of LPS.

Defensins, such as mDF2/3, are produced to counteract the effects of suppressors of DC maturation (e.g., as in counteracting bacteria that directly or indirectly (such as through LPS) suppress activation and/or maturation of DC, thereby suppressing the immune response). In this way, defensins, such as mDF2/3, act both directly and indirectly (by counteracting suppressors) to modulate Thl response.

Additionally, it has been observed that defensins can kill cells, including cells involved in the formation of blood vessels, thereby blocking angiogenesis. The inhibition of angiogenesis is a means of treating cancers and other abnormal physiological conditions and diseases characterized by angiogenesis or vascular hypeφroliferation. Administration would be performed by direct injection in situ or by other methods, such as a patch, an infusion chamber, etc.

Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

The foregoing examples demonstrate experiments performed and contemplated by the present inventors in making and canying out the invention. It is believed that these examples include a disclosure of techniques which serve to both apprise the art of the practice of the invention and to demonstrate its usefulness. It will be appreciated by those of skill in the art that the techniques and embodiments disclosed herein are prefened embodiments only that in general numerous equivalent methods and techniques may be employed to achieve the same result. All of the references identified hereinabove, are hereby expressly incoφorated herein by reference to the extent that they describe, set forth, provide a basis for or enable compositions and/or methods which may be important to the practice of one or more embodiments of the present inventions.

REFERENCES

In addition to references cited within the text of the specification, the sources listed below are incoφorated herein by reference and form apart of the disclosure of this application. The references cited within the text of the specification are likewise incoφorated herein by reference and form a part of the disclosure of this application.

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Claims

CLAIMSWhat is claimed is:

1. A chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and b. either i. an antigen domain; or ii. an antibody domain, comprising an antigen binding site.

2. The chimeric protein of claim 1, wherein the Toll-like receptor ligand domain comprises a Toll-like receptor 4 ligand domain.

3. The chimeric protein of claim 1 , wherein the defensin domain comprises a β- defensin domain.

4. The chimeric protein of claim 3, wherein the _/3-defensin domain comprises a β- defensin 2 domain.

5. The chimeric protein of claim 1, wherein the antigen domain or the antigen binding site comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

6. The chimeric protein of claim 1 , wherein the antigen domain or antigen binding site comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; i v. a portion of CD4 or CD 8 ; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

7. The chimeric protein of claim 6, wherein the pathogenic organism is a virus, microorganism, or parasite.

8. The chimeric protein of claim 1, further comprising: c. a secretory domain.

9. A nucleic acid molecule encoding the chimeric protein according to claim 1.

10. A vector comprising the nucleic acid molecule according to claim 9.

11. The vector of claim 10, wherein the vector is a vaccine vector.

12. A delivery vehicle comprising the nucleic acid molecule of claim 9.

13. The delivery vehicle of claim 12, wherein the delivery vehicle is lipid-based, viral-based, or cell-based.

14. A cell comprising the vector of claim 10.

15. The cell of claim 14, wherein the cell is capable of expressing the chimeric protein.

16. The cell of claim 15, wherein the cell is additionally capable of secreting the chimeric protein.

17. A kit comprising a vector and a cell for receiving the vector, the vector comprising a nucleic acid wherein the nucleic acid is operably linked to an expression control sequence and wherein the nucleic acid sequence encodes the chimeric protein of claim 1.

18. A transgenic animal comprising at least one cell according to claim 14.

19. A composition for inducing maturation of immature dendritic cells in vivo or ex vivo, wherein the composition comprises a chimeric protein of claim 1.

20. A method for inducing maturation of an immature dendritic cell, wherein the method comprises: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of mducmg maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

21. The method of claim 20, wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

22. The method of claim 20, wherein the defensin domain comprises a /3-defensin domain.

23. The method of claim 22, wherein the /3-defensin domain comprises a /3-defensin 2 domain.

24. The method of claim 20, wherein the antigen domain or the antigen binding site comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

25. The method of claim 20, wherem the antigen domain or antigen binding site comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD8 ; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

26. The method of claim 25, wherein the pathogenic organism is a virus, microorganism, or parasite.

27. The method of claim 20, wherein the chimeric protein further comprises: iii. a secretory domain.

28. A method for treating an abnormal physiological condition or disease, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

29. The method of claim 28, wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

30. The method of claim 28, wherein the defensin domain comprises a /3-defensin domain.

31. The method of claim 30, wherein the /3-defensin domain comprises a /3-defensin 2 domain.

32. The method of claim 28, wherein the antigen domain or the antigen binding site comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

33. The method of claim 28, wherein the antigen domain or antigen binding site comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD8 ; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

34. The method of claim 33, wherein the pathogenic organism is a virus, microorganism, or parasite.

35. The method of claim 28, wherein the chimeric protein further comprises: iii. a secretory domain.

36. The method of claim 28, wherein the abnormal physiological condition or disease comprises at least one of the following: a. cancer or growth of a non-immunogenic rumor; b. allergy; c. asthma; d. an autoimmune disease; e. an infectious disease; and f. inflammation.

37. A method of augmenting a cellular or humoral immune response using an adjuvant, wherein the adjuvant comprises a chimeric protein having a molecular weight of less than 100,000 kilodaltons comprising: a. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and b. either i. an antigen domain; or ii. an antibody domain, comprising an antigen binding site.

38. The method of claim 37, wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

39. The method of claim 37, wherein the defensin domain comprises a /3-defensin domain.

40. The method of claim 39, wherein the /3-defensin domain comprises a /3-defensin 2 domain.

41. The method of claim 37, wherein the antigen domain or the antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

42. The method of claim 37, wherein the antigen domain or antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD8; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

43. The method of claim 42, wherein the pathogenic organism is a virus, microorganism, or parasite.

44. The method of claim 37, wherein the chimeric protein of the adjuvant further comprises: c. a secretory domain.

45. A method of augmenting a cellular or humoral immune response using an antigen, wherein the antigen would be delivered to an antigen-presenting cell using an adjuvant, wherein the adjuvant comprises the chimeric protein according to claim 1.

46. The method of claim 45, wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

47. The method of claim 45, wherein the antigen-presenting cell comprises a dendritic cell.

48. The method of claim 45, wherein the defensin domain comprises a /3-defensin domain.

49. The method of claim 48, wherein the /3-defensin domain comprises a /3-defensin 2 domain.

50. The method of claim 45, wherein the antigen domain or the antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

51. The method of claim 45, wherein the antigen domain or antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD8 ; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

52. The method of claim 51 , wherein the pathogenic organism is a virus, microorganism, or parasite.

53. The method of claim 45, wherein the chimeric protein of the adjuvant further comprises: c. a secretory domain.

54. A method of augmenting expression of a co-stimulatory molecule on an antigen- presenting cell using an adjuvant wherein the adjuvant comprises the chimeric protein of claim 1.

55. The method of claim 54, wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

56. The method of claim 54, wherein the antigen-presenting cell comprises a dendritic cell.

57. The method of claim 54, wherein the co-stimulatory molecule comprises either CD40 or B7.

58. The method of claim 54, wherein the defensin domain comprises a /3-defensin domain.

59. The method of claim 58, wherein the (3-defensin domain comprises a /3-defensin 2 domain.

60. The method of claim 54, wherein the antigen domain or the antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of antigenic material from a self-tumor; and ii. a portion of antigenic material from a bacterial, viral, or parasitic antigen.

61. The method of claim 54, wherein the antigen domain or antigen binding site of the adjuvant comprises at least one domain selected from the group consisting of: i. a portion of an antigenic material from a non-immunogenic tumor idiotype or to a cancer-specific polypeptide; ii. a portion of an antigenic material from a mast cell; iii. a portion of an antigenic material from a MHC class I or class II cell; iv. a portion of CD4 or CD 8 ; v. a portion of an antigenic material from a pathogenic organism; and vi. a portion of antigenic material from a molecule associated with an abnormal physiological response.

62. The method of claim 61 , wherein the pathogenic organism is a virus, microorganism, or parasite.

63. The method of claim 54, wherein the chimeric protein of the adjuvant further comprises: c. a secretory domain.

64. A method for activating the Thl immune response, wherem the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either - an antigen domain; or an antibody domain, comprising an antigen binding site.

65. The method of claim 64, wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

66. The method of claim 64, wherein the defensin domain comprises a j8-defensin domain.

67. The method of claim 66, wherein the /3-defensin domain comprises a /3-defensin 2 domain.

68. A method for suppressing the Th2 immune response, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

69. The method of claim 68, wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

70. The method of claim 68, wherein the defensin domain comprises a /3-defensin domain.

71. The method of claim 70, wherein the _/8-defensin domain comprises a /3-defensin 2 domain.

72. A method of activating an immune response using a Toll-like receptor pathway in a dendritic cell, wherein the method comprises: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

73. The method of claim 72, wherein the Toll-like receptor pathway comprises a Toll-like receptor 4 pathway and wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

74. A method of suppressing an immune response using a Toll-like receptor 4 pathway in a dendritic cell, wherein the method comprises: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor 4 ligand domain, wherein the Toll-like receptor 4 ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site.

75. The method of claim 74, wherein the Toll-like receptor pathway comprises a Toll-like receptor 4 pathway and wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

76. A method for producing an antigen presenting cell capable of expressing or secreting a cytokine, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either , an antigen domain; or an antibody domain, comprising an antigen binding site; and c. directly or indirectly detecting the presence of the cytokine or of the mRNA encoding the cytokine.

77. The method of claim 76, wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

78. The method of claim 76, wherein the cytokine is selected from the group consisting of ILl , ILl_/3, TNF, IL6, IL12, and IFNγ.

79. A method for producing an antigen presenting cell capable of expressing or secreting a chemokme, wherein the method comprises inducing maturation of an immature dendritic cell in vivo or ex vivo by: a. providing an immature dendritic cell; and b. contacting the immature dendritic cell with a protein capable of inducing maturation, wherein the protein is a chimeric protein comprising: i. a Toll-like receptor ligand domain, wherein the Toll-like receptor ligand domain comprises a defensin domain; and ii. either an antigen domain; or an antibody domain, comprising an antigen binding site; and c . directly or indirectly detecting the presence of the chemokine or of the mRNA encoding the chemokine.

80. The method of claim 79, wherein the Toll-like receptor ligand domain of the chimeric protein comprises a Toll-like receptor 4 ligand domain.

81. The method of claim 79, wherein the chemokine is selected from the group consisting of IL8, RANTES, MDC, IP10, and MIPlo.