The present invention provides adjuvants, vaccines and related methods that are useful in eliciting immune responses, particularly immune responses against tumor antigens. We discovered that flagellin is capable of inhibiting tolerance when it is administered in conjunction with a tolerogenic antigen. This effect is likely mediated by the ability of flagellin to induce IL-12 while keeping IL-10 levels low. Furthermore, flagellin can be provided in an extended-releasing manner by using a flagellin-expressing cell. Preferably, the flagellin-expressing cell is treated such that it is no longer capable of replicating, yet retaining the ability to express flagellin, such as by lethal irradiation.

Description of the Invention

SUMMARY

The present invention provides adjuvants, vaccines and related methods that are useful in eliciting immune responses, particularly immune responses against tumor antigens. We discovered that flagellin is capable of inhibiting tolerance when it is administered in conjunction with a tolerogenic antigen. This effect is likely mediated by the ability of flagellin to induce IL-12 while keeping IL-10 levels low. Furthermore, flagellin can be provided in an extended-releasing manner by using a flagellin-expressing cell. Preferably, the flagellin-expressing cell is treated such that it is no longer capable of replicating, yet retains the ability to express flagellin for a period of time, such as by lethal irradiation.

Accordingly, one aspect of the present invention provides a composition comprising a flagellin-expressing cell, wherein the cell has been lethally irradiated. This composition is useful as an adjuvant, and as an inhibitor of immune tolerance.

The flagellin-expressing cell may be any eukaryotic cell, preferably a vertebrate cell, such as a mammalian or avian cell. The flagellin-expressing cell may lack MHC class I molecules, MHC class II molecules, or both. In some embodiments, the flagellin-expressing cell is a B78-H1 cell or K562 cell that has been transfected with a flagellin gene.

The flagellin may be any flagellin. For example, the flagellin may be the flagellin of Escherichia, Salmonella, Proteus, Pseudomonas, Bacillus, Campylobacter, Vibrio, Treponema, Legionella, Clostridia, or Caulobacter spp.

The composition may optionally comprise an antigen that is associated with a tumor cell. The tumor cell may be from a tumor selected from the group consisting of leukemia, lymphoma, lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, pancreatic cancer, breast cancer and central and peripheral nervous system cancer. The use of antigens from other tumor cells, either benign or malignant, is also contemplated. The antigen may be any portion of the tumor cell, or the tumor cell itself. The tumor cell may be lethally irradiated.

The flagellin-expressing cell and the tumor cell may be from the same species, such as human, or from different species. The flagellin-expressing cell and the tumor cell may be derived from the same individual. In some embodiments, the flagellin-expressing cell may be the tumor cell that has been harvested from the subject and transfected with a flagellin gene.

Another aspect of the present invention provides a method for preparing an adjuvant, comprising lethally irradiating a flagellin-expressing cell.

A further aspect of the invention provides a method for preparing a vaccine against a tumor, comprising lethally irradiating a flagellin-expressing cell, and combining an antigen that is associated with the tumor. The antigen may be a tumor cell from the tumor, and the tumor cell may be optionally lethally irradiated.

Also provided is a method for treating a tumor in a subject, comprising administering to the subject an antigen that is associated with the tumor and an adjuvant to elicit an immune response against the tumor, wherein the adjuvant comprises a flagellin-expressing cell. The adjuvant may be administered at the same time or different time as the antigen. The adjuvant and/or the antigen may be administered only once or multiple times. The flagellin-expressing cell is preferably lethally irradiated before being administered. The adjuvant and the antigen may be administered in a composition prepared by combining the adjuvant and the antigen, and lethally irradiating the composition.

Another aspect of the invention provides a method for inducing the production of IL-12 in a subject, comprising administering to the subject a flagellin-expressing cell.

Yet another aspect of the invention provides a method of inhibiting tolerance to a tumor in a subject, comprising administering to the subject an antigen that is associated with the tumor and a flagellin-expressing cell.

Still another aspect of the invention provides a method for screening for a candidate compound that inhibits immune tolerance, said method comprising: (a) providing an antigen-presenting cell; (b) contacting the antigen-presenting cell with a test compound; (c) detecting IL-10 and IL-12 produced by the antigen-producing cell, wherein induction of IL-12 without a cognate induction of IL-10 by the test compound indicates that the test compound is a candidate compound. Once candidate compounds are identified, they can be subject to further tests to determine their efficacy in inhibiting immune tolerance.

In any method provided by the present invention, as with any composition of the invention, the flagellin-expressing cell may be any eukaryotic cell, preferably a vertebrate cell, such as a mammalian or avian cell. The flagellin-expressing cell may lack MHC class I molecules, MHC class II molecules, or both. In some embodiments, the flagellin-expressing cell is a B78-H1 cell or K562 cell that has been transfected with a flagellin gene. The flagellin may be any flagellin. For example, the flagellin may be the flagellin of Escherichia, Salmonella, Proteus, Pseudomonas, Bacillus, Campylobacter, Vibrio, Treponema, Legionella, Clostridia, or Caulobacter spp.

The antigen may be any portion of a tumor cell, or the tumor cell itself. The tumor cell may be lethally irradiated. Any tumor cell of interest may be employed. For example, the tumor cell may be from a tumor selected from the group consisting of leukemia, lymphoma, lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, pancreatic cancer, breast cancer and central and peripheral nervous system cancer.

The flagellin-expressing cell and the tumor cell may be from the same species, such as human, or different species. The flagellin-expressing cell and the tumor cell may be derived from the same individual. In some embodiments, the flagellin-expressing cell may be the tumor cell that has been transfected with a flagellin gene.

DETAILED DESCRIPTION

The present invention provides adjuvants, vaccines and related methods that are useful in eliciting immune responses, particularly immune responses against tumor antigens. We discovered that flagellin is capable of inhibiting tolerance when it is administered in conjunction with a tolerogenic antigen. This effect is likely mediated by the ability of flagellin to induce IL-12 while keeping IL-10 levels low. Furthermore, flagellin can be provided in an extended-releasing manner by using a flagellin-expressing cell. Preferably, the flagellin-expressing cell is treated such that it is no longer capable of replicating, yet retaining the ability to express flagellin, such as by lethal irradiation.

Methods and Compositions

Pathogens express molecules known as pathogen associated molecular patterns (PAMPs), which serve as danger signals to APCs. Some of these signals are unique products of microbial metabolism, such as LPS or lipoproteins, or highly conserved features of particular microorganisms, such as CpG DNA motifs or bacterial flagellin. These molecules are ligands of the toll-like receptors (TLRs) of APCs. Toll-like receptors are evolutionary conserved, germline encoded receptors, expressed primarily on macrophages and dendritic cells that recognize PAMPs. Recognition of PAMPs by TLRs leads to the activation of NFkB transcription factors and members of the mitogen-activated protein kinase (MAP kinase) family, and ultimately results in the activation of these APCs.

Flagellin, the ligand of TLR5, is the basic element of bacterial flagella. Bacterial motility depends on the flagellum, an extracellular propeller consisting of several thousands of flagellin units. The presence of flagella is strongly related with the infectivity of some pathogenic bacteria. In addition to giving these bacteria the ability to move in the aqueous environment, the flagellum also aids to the attachment to host cells, thereby contributing to the virulence of pathogenic microorganisms. Consistent with its role as TLR5 ligand, flagellin has recently been shown to work as a potent adjuvant of T cell function in vitro and in vivo (McSorley S J et al., Bacterial flagellin is an effective adjuvant for CD4+ T cells in vivo, J Immunol 2002 Oct. 1; 169(7):3914-9).

We hypothesized that flagellin can provide a "danger" signal to activate APCs in tumor antigen presentation, thereby inhibiting T cell tolerance to the tumor antigen. To this end, we first tested the adjuvant function of our flagelllin preparation, and confirmed that it was capable of increasing the production of IFN.gamma. and IL-2 (Example 1). Next we determined whether in vivo treatment using flagellin would prevent antigen specific T cell tolerance induced by high doses of antigens. As shown by Example 2, T cell responses were almost completely abrogated in the animals treated with a tolerogenic dose of the antigen. In contrast, the mice injected with the same dose of antigen and flagellin yielded significant levels of T cell response. These results indicate that flagellin has preserved the capacity of T cells to respond to tolerogenic levels of antigens. Consistent with a role of flagellin to activate APCs and inhibit tolerance, peritoneal macrophages treated with flagellin displayed higher levels of B7.1, CD40 and MHC class I molecules as compared with untreated macrophages (Example 3).

Anti-inflammatory cytokines like IL-10-influence the T cell decision toward an anergy response. Inflammatory cytokines such as IL-12 play the opposite role, inducing T cell differentiation into a helper T cell that will drive an adaptive immune response against the antigen presented by the APC. We further discovered that flagellin can induce the production of IL-12 while keeping IL-10 levels low (Example 4). Thus, it is contemplated that flagellin changes the balance between anti-inflammatory cytokines and inflammatory cytokines to inhibit tolerance.

Flagellin-induced IL-12 production depends on the presence of the receptor for flagellin, TLR5 (Example 5). In contrast to flagellin, LPS (TLR4 ligand) and CpG (TLR9 ligand), stimulated both IL-12 and IL-10 (Examples 4 and 6). Furthermore, flagellin is capable of inhibiting the effect of LPS and CpG in inducing IL-10 (Example 7). Therefore, although PAMPs are known as danger signals to APCs, not all of them can tip the balance between anti-inflammatory cytokines and inflammatory cytokines to inhibit tolerance. Flagellin not only leads to tolerance prevention, but also directs the actions of other PAMPs in this regard. The effect of flagellin was further confirmed by in vivo experiments (Example 8). Accordingly, the present invention provides a method for inhibiting tolerance in a subject to an antigen by providing a flagellin and the antigen to the subject.

We explored a new formulation to deliver flagellin by using a flagellin-expressing cell, which can provide flagellin to the subject in a continuous mode. Therefore, we constructed a flagellin-expressing cell and showed that the lysates of this cell can enhance IL-12 production (Example 9). It is contemplated that the flagellin-expressing cell can optionally be treated before administration in a manner that would prevent the cell from replicating, while retaining the ability of the cell to express flagellin. A lethal irradiation is an example of such a treatment. Thus, the flagellin is preferably provided by administering a flagellin-expressing cell to the subject. In particular, the flagellin-expressing cell can be first lethally irradiated so that is would not replicate in the body of the subject, yet it can continue to express flagellin, e.g., for a few days.

The flagellin-expressing cell may be any eukaryotic cell, preferably a vertebrate cell, such as a mammalian or avian cell. The cell may be a normal cell, a tumor cell, a fetal cell, an adult cell, from an established cell line, or from a primary culture. The cell is preferably from the same species as the subject receiving the cell. In some embodiments, the cell may be from the subject. Exemplary sources of the cell include, but are not limited to, human, rat, mouse, rabbit, rodent, dog, cat, horse, cattle, sheep, goat, pig, chicken, or non-human primate.

An advantage of using a flagellin-expressing cell from the same species as the subject is to avoid massive immune responses against the cross-species cell (xeno-responses). Even if a flagellin-expressing cell from the same species is used, immune responses against a different individual of the same species (allo-responses) may still divert the immune system and quickly eliminate the flagellin-expressing cell. One way of avoiding this problem is to use a flagellin-expressing cell that lacks MHC class I molecules, MHC class II molecules, or both. For example, B78-H1 cells and K562 cells are murine and human cells, respectively, that lack the MHC class I and class II molecules.

Flagellin sequences are readily obtainable based on knowledge in the art. In fact, the flagellin sequences from numerous bacterial species, as well as structural analyses, have been published. Any polypeptide with flagellin function, namely one that binds a naturally occurring TLR5 and triggers at least one of the biological functions of the TLR5 in antigen-presenting cells upon such binding, can be used in the present invention. These include polypeptides comprising any of the naturally occurring bacterial flagellin proteins, and polypeptides that are substantially identical with any of the naturally occurring bacterial flagellin proteins at the amino acid sequence level, wherein the polypeptides are capable of binding a naturally occurring TLR5.

Methods of transfecting cells are also well known in the art. These methods include, but are not limited to, transfection, microinjection, scrape-loading, and receptor-mediated uptake by the cell. Transfection may be transient or stable. Exemplary current methods of transfection include calcium phosphate precipitation, electroporation, lipofection, and peptide-mediated transfection. Ballistic DNA delivery and transduction (i.e., the introduction of foreign DNA by virus or virus vector infection) can also be employed.

For example, a flagellin can be delivered to cells by means of an expression vector. Suitable expression vectors comprise a promoter that is active in the cells in which the flagellin is to be expressed. Expression vectors useful for practicing the invention may also include selectable markers, cell-type or cell-cycle-specific enhancers or repressors, polylinkers, start codons, ribosome binding sites, internal ribosome entry sites, introns, stop codons, polyadenylation signals, or other features that facilitate cloning and vector stability, mRNA stability and localization in the cell, and translation efficiency, or combinations thereof. Expression vectors include viral expression vectors. Selection of these features is largely based on the cells to be transfected, and the expression characteristics desired. A large number of commercially available vectors are available for expressing polypeptides in cells.

For localization in the cell, it is contemplated that the flagellin may be engineered to contain the motif YXXO (SEQ ID NO:1: Y is tyrosine, X is any amino acid, and O is an amino acid with a bulky hydrophobic side chain). This motif has been shown as responsible for targeting the lysosomal membrane protein LAMPI (Lysosomal associated membrane protein one) to the lysosomal compartment. It is also present in other proteins localized on the lysosome membrane, such as LAP (lysosomal acid phosphatase) and LAMP2, and is able to target by itself the CD44 protein, a cellular membrane receptor, to the lysosomal compartment.

The antigen of the present invention may be any antigen of interest. In particular, the antigen may be an antigen that is associated with a tumor. For example, the antigen may be a tumor cell to which an immune response is desired, or it may be part of the tumor cell, such as any extract of the tumor cell; any fraction of the tumor cell; one or more surface proteins, nuclear proteins, glycoproteins, lipids, or nucleic acids of the tumor cell; cytoplasmic membrane of the tumor cell; or any combination of the above. The antigen may be naturally occurring, recombinantly produced, or synthesized. A preferred antigen comprises tumor cells from the tumor to which an immune response is desired. For example, a biopsy sample from a tumor, harvested from a subject bearing the tumor, can be processed and used as an antigen in a vaccine for treating the tumor. Preferably, tumor cells are lethally irradiated before being administered as an antigen.

The flagellin-expressing cell and/or the antigen can be administered by a variety of routes to stimulate an immune response. For example, they can be delivered intranasally, subcutaneously, intradermally, intralymphatically, intramuscularly, intratumorally, orally, intravascularly, intraperitoneally and intracerebrally. Nasal delivery routes may be useful for inducing both mucosal and systemic immune responses. Those skilled in the art will know how to select appropriate delivery routes for particular formulations of flagellin-expressing cell and/or antigen.

Compositions of this invention can be administered to a subject as a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the active ingredient (e.g., flagellin-expressing cell and/or antigen). The choice of pharmaceutically acceptable carrier depends on the route of administration of the pharmaceutical composition and the particular physical and chemical characteristics of the flagellin-expressing cell/antigen. Pharmaceutically acceptable carriers are well known in the art and include sterile aqueous solvents such as physiologically buffered saline, and other solvents or vehicles such as glycols, glycerol, oils such as olive oil and injectable organic esters. A pharmaceutically acceptable carrier can further contain physiologically acceptable compounds that stabilize the active ingredient, increase its solubility, or increase its absorption. Such physiologically acceptable compounds include carbohydrates such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; and low molecular weight proteins. Other suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences (Gennaro, A. R. ed. (2000) Remington's Pharmaceutical Sciences, 20th edition. Williams & Wilkins Pa., U.S.A.).

Another aspect of the present invention provides a method for preparing an adjuvant, wherein the adjuvant comprises a flagellin-expressing cell. The flagellin-expressing cell is preferably lethally irradiated. The adjuvant can be used to enhance the immune response to an antigen, particularly an antigen that is associated with a tumor. The resulting adjuvants are also provided in the present invention.

A further aspect of the invention provides a method for preparing a vaccine against a tumor, comprising lethally irradiating a composition that comprises a flagellin-expressing cell, and combining the composition with an antigen that is associated with the tumor.

Also provided is a method for treating a tumor in a subject, comprising administering to the subject an antigen that is associated with the tumor and an adjuvant to elicit an immune response against the tumor, wherein the adjuvant comprises a flagellin-expressing cell. The adjuvant may be administered at the same time or different time as the antigen. Either the adjuvant or the antigen may be administered only once or multiple times. The flagellin-expressing cell is preferably lethally irradiated before being administered. The adjuvant and the antigen may be administered in a composition prepared by combining the adjuvant and the antigen, and lethally irradiating the composition.

Another aspect of the invention provides a method for inducing the production of IL-12 in a subject, comprising administering to the subject a flagellin-expressing cell. It is contemplated that the flagellin-expressing cell does not induce, or induces only a low level of, IL-10. Furthermore, the flagellin-expressing cell has the effect of reducing IL-10 production that is induced by other stimuli in the subject.

Yet another aspect of the invention provides a method for inhibiting tolerance to a tumor in a subject, comprising administering to the subject an antigen that is associated with the tumor and a flagellin-expressing cell.

Still another aspect of the invention provides a method of screening for a candidate compound that inhibits immune tolerance, said method comprising: (a) providing an antigen-presenting cell; (b) contacting the antigen-presenting cell with a test compound; and (c) detecting IL-10 and IL-12 produced by the antigen-producing cell, wherein induction of IL-12 without a cognate induction of IL-10 by the test compound indicates that the test compound is a candidate compound. Once candidate compounds are identified, they can be subject to further tests to determine their efficacy in inhibiting immune tolerance.

The methods and compositions of the invention described herein may be combined with any other treatment or therapy for treating cancer, for example, chemotherapy, radiation therapy, surgery, and combinations thereof.
 

Claim 1 of 21 Claims

1. A composition comprising a flagellin-expressing eukaryotic cell, wherein the cell has been lethally irradiated.
 

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