WO2000000158A2

WO2000000158A2 - Alkylamines and their precursors as specific modulators of human gamma-delta t cell function

Info

Publication number: WO2000000158A2
Application number: PCT/US1999/014303
Authority: WO
Inventors: Jack F. Bukowski; Michael B. Brenner
Original assignee: The Brigham And Women's Hospital, Inc.
Priority date: 1998-06-30
Filing date: 1999-06-24
Publication date: 2000-01-06
Also published as: CA2335791A1; AU5205999A; EP1090106A2; JP2002519007A; WO2000000158A3; WO2000000158A9

Abstract

Methods and compositions for inducing the proliferation of Vη2Vδ2 T cells are provided. The compositions contain alkyklamine agents, such as alkylamines and alkylamine precursors. The agents also are useful for activating the ηδ T cell receptor.

Description

ALKYLAMINES AND THEIR PRECURSORS AS SPECIFIC MODULATORS OF HUMAN GAMMA-DELTA T CELL FUNCTION

Government Support

The work described herein was supported, in part, by Grant Nos. Al 01330-3 and CA/AI 5 47724-11 from the National Institutes of Heath. The government may retain certain rights in these inventions.

Field Of The Invention This invention relates to methods and compositions for stimulating a γδ T cell-mediated immune response against tumors and against infectious diseases, such as bacterial and viral 0 infections. Such γδ stimulation may also be used to decrease autoimmunity or to enhance immune tolerance to allergens. The methods involve administering an alkylamine or an alkylamine precursor to a subject or to cells isolated from a subject to stimulate activation, proliferation, cytokine release, cytotoxic activity or apoptosis of Vγ2Vδ2 T cells.

Background of the Invention 5 T cells are thymus derived cells in the immune system which mediate the cellular immune response. T lymphocytes (T cells) include two subgroups: αβ T cells and γδ T cells. The αβ T cells have αβ receptors that recognize antigenic peptides that are bound to the major histocompatibility complex (MHC) I or II molecules; these represent approximately 90-98% of T cells. The γδ T cells have γδ receptors; these represent 3-5% of T cells. 0 γδ T cells play an important role in the defense against bacterial and viral infections as well as in autoimmunity. γδ T cells are expanded in humans with infectious diseases such as tuberculosis, salmonellosis, brucellosis, ehlichiosis, tularemia, malaria, leishmaniasis, mononucleosis, and in HIV (early stages). These cells are expanded in the synovium of patients with rheumatoid arthritis, in the CSF and CNS plaques of patients with multiple sclerosis, and 5 in the lungs of patients with sarcoidosis. In contrast to αβ T cells, which recognize peptide antigens in the context of MHC molecules, the predominant subset of γδ T cells in human peripheral blood, termed Vγ2Vδ2 T cells, recognize unprocessed nonpeptide phosphate antigens in the absence of professional' antigen presenting cells (APC). Contrary to the abundant information available regarding the nature of the interaction between the αβ T cell 0 receptor with the MHC-bound peptide antigens, the little information regarding the nature of the interaction between the γδ T cell receptors and their ligands.

The antigens responsible for expanding γδ T cells have been most extensively studied from mycobacteria, where they were identified as prenyl pyrophosphates. One such natural antigen is w -pentenylpyrophosphate, which is secreted by mycobacteria. TUBag 1 and 2 are another group of closely related antigenic molecules that have been isolated from the cytoplasm of mycobacteria. These also are pyrophosphate-containing molecules whose complete structures have not been determined.

Other naturally occurring but less potent antigens include 2,3 diphosphoglycerate, glycerol-3 -phosphate, ribose-1 -phosphate, and xylose- 1 -phosphate. In addition to these naturally occurring antigens, there are several alkyl and alkenyl phosphate and pyrophosphate analogs that have been synthesized and that reportedly are recognized by Vγ2Vδ2 T cells. (See, e.g., U.S. 5,639,653, issued to Bloom et al., for a complete description of these phosphate- or pyrophosphate-containing compounds.)

Because γδ T cells protect against a variety of bacterial and viral diseases and have been implicated in modulating autoimmunity and immune tolerance, there exists a need to selectively stimulate this subpopulation of T cells to enhance an immune response to these diseases and conditions, as well as to better understand the molecular processes underlying γδ T cell mediated immune responses. In view of the foregoing, there also exists a need to develop improved drug therapies to replace or supplement the existing methods for stimulating a Vγ2Vδ2 T cell-mediated immune response and, preferably, to develop novel drugs to reduce bacterial or viral infections and autoimmune conditions at their earliest stages, and to enhance immune tolerance.

Summary Of The Invention The invention is based on the discovery that alkylamines, which are the major products of certain bacteria that are associated with sepsis, chorioamnionitis, preterm labor, enteritis, gingivitis and also are found in plant foods and human body fluids, cause proliferation of Vγ2Vδ2 T cells in a T cell receptor (TCR)-specifιc manner. The alkylamine antigens are the first phosphate-free antigens described for Vγ2Vδ2 T cells and, thus, represent a distinct chemical class of ligand for Vγ2Vδ2 T cells. The discovery that the alkylamines stimulate γδ T cell expansion is surprising and unexpected since all previously described antigens for Vγ2Vδ2 T cells are characterized by a critical phosphate or pyrophosphate moiety. Thus, contrary to expectations, the working examples provide evidence that the same Vγ2Vδ2 T cell receptor which recognizes the previously characterized negatively charged alkylphosphate compounds can be activated by the positively charged alkylamine molecules as well. Based on these discoveries, Applicant describes herein a newly discovered function for alkylamines, namely, the ability to stimulate activation and/or proliferation of Vγ2Vδ2 T cells. Accordingly, the instant invention is directed to compositions of alkylamines and novel alkylamine precursors, as well as to methods that are based upon the discovery of this newly-discovered function.

The methods of the invention involve administering an alkylamine agent to a subject or to cells isolated from a subject to stimulate activation and/or proliferation of the Vγ2Vδ2 T cells. Activation of Vγ2Vδ2 T cells is a process that is mediated by the γδ T cell receptor. In general, activation of T cell receptors is effected by binding of the receptors to their respective antigens. Although not intending the invention to be limited to any particular theory or mechanism, the evidence provided in the examples suggests that activation of the γδ T cell receptor also may be initiated by binding of the alkylamines of the invention to the γδ T cell receptor.

Depending upon the intra- and extra-cellular environment, activation of the Vγ2Vδ2 T cells can initiate a number of cellular changes including, but not limited to, proliferation of Vγ2Vδ2 T cells, stimulation or inhibition of cytokine production by the Vγ2Vδ2 T cells, stimulation or inhibition of cytotoxic activity by Vγ2Vδ2 T cells, and stimulation of the Vγ2Vδ2 T cells to undergo apoptosis and die. Thus, the invention also embraces methods and compositions for these particular aspects of activating Vγ2Vδ2 T cells. Each of these activities has been well documented in, for example, α/β T cells, and can be assessed using standard procedures (e.g., ELISA assays to measure cytokine release, apoptosis assays). For example, following activation by antigen, CD4 T lymphocytes produce one of two distinctive cytokine profiles which has led to their classification as T helper 1 (T_H1) and T helper 2 (T_H2) cells. The different cytokines produced by these cells lead to differences in immune function. T_H1 cells primarily secrete interferon-gamma (IFN-gamma), but also interleukin-2 (IL-2) and tumor necrosis factor-beta (TNF-beta, Lymphotoxin), and induce cellular immunity. T_H2 cells primarily secrete interleukin-4 (IL-4), but also IL-5 and IL-10, and downregulate cellular immunity while playing a major role in the induction of antibody responses mediated by plasma cells. The cytokine TNF-α can be produced during both T_H1 and T_H2 immune responses, although levels are higher in T_H1 responses and this cytokine is known for cytolytic effects that contribute to the efficacy of cellular immunity. Most of the original work on T_H1 and T_H2 lymphocyte subsets was performed with mouse helper T cell clones, however, it is now recognized that human CD4 T cells secrete similar cytokine profiles and can also be classified into T_H1 and T_H2 subsets (Powrie, F. et al., 1993, Immunol. Today 14:270; Romagnani, S. et al, 1992, Int. Arch. Allergy Immunol. 4:279; Romagnani, S. et al., 1991, Immunol. Today 8:256). Although not intending to limit the invention to any particular theory or mechanism, in view of the discoveries disclosed herein, it is believed that the alkylamines of the invention are capable of stimulating cytokine production by Vγ2Vδ2 T cells in a manner analogous to that described in the literature for α/β T cells. Thus, the ability of the alkylamines of the invention to induce Vγ2Vδ2 T cell activation can be determined using routine experimentation and standard procedures to measure well established cellular characteristics. According to one aspect of the invention, a method for stimulating activation of

Vγ2Vδ2 T cells is provided. The method for stimulating activation involves contacting the cells with a Vγ2Vδ2 T cell activation stimulating amount of an alkylamine agent, in vivo or ex vivo, to stimulate activation of the Vγ2Vδ2 T cells. As used herein, "contacting the cells" with the alkylamine agent means placing the cells in sufficient proximity to the alkylamine agent for a time and under conditions sufficient for the alkylamine to stimulate the activation of the Vγ2Vδ2 T cells. Activation is assessed using routine procedures. Contacting may be performed in vivo or ex vivo.

According to another aspect of the invention, a method for stimulating the proliferation of Vγ2Vδ2 T cells is provided. The method involves contacting the cells with a Vγ2Vδ2 T cell proliferation stimulating amount of an alkylamine agent, in vivo or ex vivo, to stimulate proliferation of the Vγ2Vδ2 T cells. A proliferation stimulating amount of an alkylamine agent can be the same as an activation stimulating amount of the agent; such amounts are determined by performing routine tests to determine whether the amount has induced proliferation (e.g., by counting cell numbers) or activation (e.g., by measuring cytokine production by the cells).

As used herein, "contacting the cells" with the alkylamine agent means placing the cells in sufficient proximity to the alkylamine agent for a time and under conditions sufficient for the alkylamine to stimulate the proliferation of the Vγ2Vδ2 T cells and/or to activate the γδ T cell receptor. Contacting may be performed in vivo or ex vivo. As used herein, "stimulating the proliferation of Vγ2Vδ2 T cells" means increasing the number of Vγ2Vδ2 T cells by a detectable, statistically significant amount or by an amount that results in a phenotypic change in the subject or in the cell that is contacted with the alkylamine agent. By stimulating the proliferation of Vγ2Vδ2 T cells, it is believed that an enhanced immune response is generated against microbes and tumor cells that express antigens recognized by the Vγ2Vδ2 T cells. In autoimmune conditions in which Vγ2Vδ2 T cells may limit tissue damage, thereby effectively treating the disease or condition, increased proliferation of such Vγ2Vδ2 T cells would be desirable. For ease of discussion, such diseases or conditions are collectively referred to as "diseases" or "conditions" throughout this document. Exemplary diseases that can be treated (prevent, inhibit the progression, or reduce the symptoms) by stimulating the proliferation of Vγ2Vδ2 T cells include infectious disease, e.g., bacterial or viral infections, such as gingivitis-causing bacteria, E. coli infections, listeria infections, tuberculosis, salmonellosis, plasmodium infections, bacteriodes infections, porphyromonas infections, klebsiella infections, Yersinia infections, clostridium infections, brucellosis, ehlichiosis, tularemia, malaria, leishmaniasis, mononucleosis, Epstein-Barr viral infections (EBV), HIV infections, and herpes simplex virus infections. Other diseases that can be treated include allergies to food or inhaled allergens, parasitic infections, leukemia, lymphoma, leprosy, malaria, rheumatoid arthritis, ulcerative colitis, anemia, systemic lupus erythematosus, Lyme disease, viral hepatitis, and Crohn's disease. Although not intending to limit the invention to a particular mechanism of action, it is believed that Vγ2Vδ2 T cells may act by down-regulating autoreactive α/β T cells that are known to cause disease by reacting against autoantigens or allergens. Alkylamine recognition by Vγ2Vδ2 T cells may thus result in greater down- regulation of α/β T cells and less tissue damage or allergic reaction.

For in vivo applications, the alkylamine agent administered to a subject is an isolated alkylamine agent (an "alkylamine of formula I or III" or an "alkylamine precursor of formula II") in an amount effective to stimulate the proliferation of Vγ2Vδ2 T cells in vivo. As used herein, a subject refers to a mammal preferably a primate and, more preferably, a human. It is noted that the preferred subjects treated according to the methods set forth above are otherwise free of symptoms calling for alkylamine agent treatment, either by administration of the alkylamine or by an alkylamine precursor. Preferably, the alkylamine agent is administered to the subject in conjunction with other methods for treating the condition, such as an infectious condition (e.g., antibiotic therapy). Thus, the compositions and methods of the invention are useful for replacing existing drug therapies, as well as for improving the effectiveness of existing therapies for treating conditions that are characterized by inadequate numbers or function of Vγ2Vδ2 T cells. In general, such conditions are associated with infectious disease - o - or autoimmune conditions such as those identified herein or by assessing the number of Vγ2Vδ2 T cells in peripheral blood mononuclear cells (PBMC) using standard procedures. For example, γδ T cells are expanded in certain autoimmune disorders (e.g., synovium of patients with rheumatoid arthritis, in the CSF and CNS plaques of patients with multiple sclerosis, and in the lungs of patients with sarcoidosis).

In a particularly preferred embodiment, the alkylamine agent is delivered directly to the site at which there is an infection. For example, treating bacterial- or viral-mediated gingivitis, can be accomplished by formulating the alkylamine agent into a medicament that is suitable for oral delivery and that remains in the proximity of the gingival tissue (e.g., a chewing gum, a floss, a gingival tissue implant). In this manner, the compositions can be targeted to particular sites within the gums to stimulate Vγ2Vδ2 T cell proliferation at these sites. According to yet another preferred embodiment, the alkylamine agent is delivered directed to the female reproductive tract for treating the progression of opportunistic infections. For example, the alkylamine agents of the invention can be formulated for, e.g., vaginal delivery. Optionally, the alkylamine agent is delivered in combination with an anti-infective agent, such as an antibiotic or anti-viral agent for treating the infective agent.

According to yet another aspect of the invention, a method for activating a γδ T cell receptor is provided. The method involves contacting the receptor with an alkylamine of formula I, as defined below, under conditions to permit alkylamine-mediated activation of the receptor. As used herein, a γδ receptor is a term of art which refers to the T cell receptor which recognizes antigenic molecules, and which is present on γδ T cells. Thus, the method of the invention can be performed ex vivo (e.g., on γδ T cell receptors that are expressed on isolated cells) or in vivo.

The alkylamine agents that are useful for practicing the claimed invention are alkylamines or alkylamine precursors. Preferably, the alkylamine agents are in isolated form, i.e., substantially free of contamination that would preclude pharmaceutical use. No prior use for the alkylamines or alkylamine precursors of the invention for treating infectious disease, allergies, or autoimmune conditions, that can be treated by increasing the proliferation of Vγ2Vδ2 T cells or for activating a γδ T cell receptor has been proposed. As used herein an "alkylamine" refers to a compound of formula I or III. The preferred alkylamines are compounds of formula I: R-NH2 (formula I), wherein R is a straight- or branched chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive. The preferred alkylamines are ethyl-, n-propyl-, -iso-propyl-, n-butyl, iso-butyl-, sec-butyl-, and iso-amylamines.

R optionally contains substituted functional groups including, e.g., hydroxy, amino, and carboxyl. Although a primary amine is shown in formula I, secondary or tertiary amines of formula III and alkylamine precursors of formula II which, upon hydrolysis, form primary and secondary amines also are embraced within the instant invention.

There are several other amine molecules that also are found as naturally occurring bacterial products or constituents of plant foods and human body fluids which are slightly less structurally related to the alkylamine antigens described above. Chief among these is putrescine (1,4-butanediamine) which is n-butylamine with an additional amine group on the fourth carbon. Putrescine is essential for mammalian cell proliferation and differentiation. It is a precursor for GABA, the major neuroinhibitory substance in the vertebrate CNS. It causes gastrointestinal mucosal cell growth in vitro and in vivo, and is a metabolic product of certain bacterial species including Listeria, Clostridia, Bacteroides, Escherichia, Salmonella, Shigella, and Proteus. Since putrescine has a 4 carbon chain and differs from the antigenic molecule n- butylamine by only the addition of an amine group, there is a high likelihood that it will be recognized by Vγ2Vδ2 T cells. The higher polyamine spermidine is a product of the addition to putrescine of an n-propylamine moiety derived from the decarboxylation of S- adenosylmethionine. It is also important in cell proliferation and is found in bacteria. Thus, spermidine will also be tested as a potential antigen for Vγ2Vδ2 T cells.

According to yet another aspect of the invention, novel alkylamine precursors are provided. As used herein, an "alkylamine precursor" refers to a compound of formula II,

O I

Rl-C-NH-R (formula II),

wherein R is a straight- or branched chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive, and wherein Rl is a straight- or branched chain alkyl or alkenyl group containing from one to twenty carbon atoms, inclusive. Thus, R may contain 1, 2, 3, 4, 5, or 6 carbon atoms and Rl may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Preferably, Rl contains from one to ten carbon atoms, inclusive. The R and Rl groups are, independently, straight-chained or branched-chain and may be saturated or unsaturated. R and Rl, optionally and independently, contain substituted functional groups including, e.g., hydroxy, amino and carboxy. The preferred alkylamine precursors are theanine, N⁵- isopropylglutamine, N⁴-ethylasparagine, and N⁵-seobutylglutamine. Upon cleavage, e.g., in vivo, the precursors of formula II form a mild acid (RlCOO^("}) and an alkylamine of formula I or III.

As used in reference to Rl , an "alkyl or alkenyl group" refers to an alkyl or alkenyl chain containing from one to twenty carbon atoms, inclusive. Preferably, the alkyl or alkenyl group is present in a naturally-occurring mono-or dicarboxylic acid. Exemplary monocarboxylic acids include those in which the alkyl group is propyl, butyl, and amyl. Exemplary dicarboxylic acids include the amino acids and, in particular, glutamatic acid or aspartic acid. The preferred alkylamine precursors are selected from the group consisting of theanine, N⁵- w -propylglutamine, N4-ethylasparagine, and N⁵-£eobutylglutamine.

According to another aspect of the invention, novel compositions for use in accordance with the methods of the invention are provided. Novel pharmaceutical compositions contain an alkylamine of formula I or III and/or an alkylamine precursor of formula II, together with a pharmaceutically acceptable carrier. The alkylamine of formula I or III and the alkylamine precursor of formula II are as defined herein. In certain preferred embodiments, the pharmaceutical compositions are formulated as oral formulations and, more preferably, are formulated for administration to a gingival tissue (e.g., a chewing gum, tooth-paste additive, a gingival tissue implant). Alternatively, the novel compositions of the invention can be formulated as a food additive or vitamin-type food supplement to reduce the likelihood of food- transmitted infection and/or increase immune resistance to infectious disease or tumors by stimulating the proliferation of Vγ2Vδ2 T cells. Additionally, the novel compositions of the invention can be formulated for delivery to the female reproductive tract, e.g., for treating the progression of opportunistic infections. For example, the alkylamine agents of the invention can be formulated for vaginal delivery (e.g., jelly, tampons, and so forth).

According to yet another aspect of the invention, a method for making a medicament is provided. The method involves placing an alkylamine of formula I or III and/or an alkylamine precursor of formula II in a pharmaceutically acceptable carrier. The alkylamine of formula I or III and the alkylamine precursor of formula II are as defined herein. For example, these compounds may be given orally, intravenously, intrathecally, intranasally, intramuscularly, or as an inhalant, and the nature of the pharmaceutically acceptable carrier is changed appropriately according to the mode of administration.

According to yet another aspect of the invention, a method for making a food additive or vitamin-type supplement is provided. The method involves placing an alkylamine of formula I or III and/or an alkylamine precursor of formula II in a pharmaceutically acceptable carrier and formulating the composition as a food additive or vitamin-type supplement. The alkylamine of formula I or III and the alkylamine precursor of formula II are as defined herein.

The invention also contemplates the use of the alkylamines of formula I or III and/or the alkylamine precursors of formula II in experimental model systems to determine the role that Vγ2Vδ2 T cell proliferation plays in combating infectious disease, as well as to determine the role that these molecules play in activating the γδ T cell receptor. Thus, the invention is also directed to novel screening assays to identify molecules (e.g., by testing combinatorial libraries) that interrupt the functional activity of the alkylamine agents of the invention.

These and other aspects of the invention will be described in greater detail below. Throughout this disclosure, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains unless defined otherwise.

Brief Description Of The Drawings Figure la is a reaction pathway for the metabolism of L-theanine to ethylamine and glutamate. Figure lb is a drawing of the structures of ethyl pyrophosphate and z^'sø-pentenyl pyrophosphate.

Figure 2 is a series of line and bar graphs showing the effect of ethylamine on the induction of IL-2 release from Vγ2Vδ2 and VγlVδl cells. Ethylamine caused IL-2 release in a polyclonal Vγ2Vδ2 T cell line (a), Vγ2Vδ2 T cell clones (b), but not αβ T cell clones (c) nor a VγlVδl T cell transfectant (d, open circles). The Vγ2Vδ2 T cells clones 12G12, DG.SPF6 and CP.1.15 were derived from the PBMC of healthy donors by stimulation with a mycobacterial extract, whereas DGSF.13 was derived from the synovial fluid of a patient with rheumatoid arthritis. The αβ T cell clones were isolated from the PBMC of healthy donors by limiting dilution and expanded with.PHA. The Vγ2Vδ2 T cell line was made by positive selection of anti-TCRδl⁺ cells from the PBMC of a healthy donor using magnetic beads. These T cells were expanded in PHA and 1 nM IL-2 and then subjected to a negative selection procedure using monoclonal antibodies OKT4 and BMA-031 treatment followed by magnetic beads charged with goat anti-mouse Ig. The final cells were >99% Vγ2Vδ2 TCR⁺ by flow cytometric analysis. The Vγ2Vδ2 T cell transfectant was made by transfecting TCR- J.RT3- T3.5 cells with cDNA made from DG.SF13, a Vγ2Vδ2 TCR+ phosphate antigen reactive γδ T cell clone obtained by stimulation of synovial fluid mononuclear cells with a mycobacterial extract. The VγlVδl T cell transfectant was obtained by transfecting TCR-J.RT3-T3.5 cells with cDNA made from F7, a VγlVδl TCR+ γδ T cell clone. Stimulation of T cell clones and transfectants was performed in 96 well flat bottom plates with 1 x 10⁵ responder cells per well in 0.2 ml ⁴⁷. In some experiments, 5 x 10⁴ mitomycin-treated or glutaraldehy de-fixed B lymphoblastoid cells (LCL) or SH-5YSY neuroblastoma cells per well were used as feeders or antigen presenting cells (APC), but these APC were not necessary to obtain IL-2 release from the transfected Jurkat cells. Half log dilutions of ethylamine starting at 100 mM (a, d) or a fixed dose of 30mM (b, c), or as a positive control, the calcium ionophore, ionomycin, (at 1 mg/ml) were added in the presence of 10 ng/ml PMA as a costimulator ⁵⁶. After 24 hr, supernatants were harvested, and tested at a final dilution of 1/8 for their ability to stimulate the growth of the IL-2-dependent HT-2 cell line. Proliferation assays were performed in triplicate using 5 x 10³ HT-2 cells per flat bottom well of a 96-well plate. After 18 hours, the cells were pulsed with ³H thymidine (1 mCi/well), harvested at 24 hours, and counted by liquid scintillation on a Betaplate system. The standard deviation of the triplicate determination was less then 10% of the mean. Figure 3 is a bar graph showing the effect of acid hydrolyzed tea extracts or L-theanine on Vγ2Vδ2 T cells. Hydrolyzed green and black tea (a) and hydrolyzed L-theanine (b) caused expansion of γδ T cells from PBMC. A concentrated green tea extract was made by mixing 15 g green tea leaves with 500 ml boiling water and steeping for 10 min. The mixture was centrifuged at 200 X g for 20 min and filtered to remove remaining large particles, yielding 400 ml of concentrated tea extract. This was lyophilized to yield 4 g solid material. Two grams of this extract, 2 g of Nestea iced tea (100% tea; Nestle, Lausanne, Switzerland), and 500 mg of L-theanine (Sigma) were refluxed individually in 5N HCI for 2 hr and brought to pH 7.4 with NaOH. These substances were then tested for their ability to expand γδ T cells from PBMC. Hydrolyzed tea extracts were used at a 1 :30 dilution, and hydrolyzed L-theanine was used at lOmM.

Figure 4 is a bar graph showing the effect of alkylamines on Vγ2Vδ2 T cell expansion. PBMC were mixed with 400 mM concentrations of either alkylamines or EPP and after 7 days γδ T cells were quantitated by flow cytometry. The total number of cells in the cultures remained constant over the 7 day period. Data are expressed as the percentage of CD3⁺ cells with γδ TCR. Significant expansion of γδ T cells occurred using alkylamine concentrations as low as 50 mM. All γδ T cells were Vγ2Vδ2 as assessed using V chain specific antibodies. The ascites-derived monoclonal antibodies against T cell antigens used were as follows: control antibody (P3), pan γδ TCR (anti-TCRδl), Vδl/Vδl (dTCSl), Vδ2 (BB3), Vγ2 (7A5), and CD3 (OKT3). The specificity of these antibodies is reviewed in ⁵⁷. FITC-conjugated (Fab')₂ goat anti-mouse IgG was purchased from Tago, (Burlingame, CA). Ethyl pyrophosphate (EPP) was synthesized as described ²³. Alkylamines were purchased from Sigma Chemical Company (St. Louis, MO).

Figure 5a is a line graph showing the effect of Proteus morganii bacterial supematants and their extracts on Vγ2Vδ2 PBMC T cell expansion. Supematants and their extracts caused expansion of Vγ2Vδ2 T cells from PBMC. Supernatant from a broth culture of Proteus morganii (circles), an extract from this supernatant enriched for amines (triangles), and uninoculated bacterial culture media (squares) were mixed with PBMC. On day 3, 0.3 nM IL-2 was added to all cultures. On day 12, cells were analyzed by flow cytometry with anti-γδ TCR monoclonal antibodies to enumerate γδ T cell numbers. The total number of cells in the cultures remained constant over the 12 day period. Data are expressed as the percentage of CD3+ cells with γδ TCR. Greater than 99% of γδ T cells were Vγ2Vδ2+ T cells as assessed using V chain specific antibodies. Proteus morganii, strain 235, (National Collection of Types Cultures, London) was grown in LB broth at 37 C overnight. The culture supernatant was obtained by centrifugation at 2000 g for 10 minutes. To enrich for alkylamines, 10 ml of supernatant was saturated with NaCl and brought to pH 1.5 with concentrated sulfuric acid. After centrifugation at 2000 g for 10 minutes, the supernatant was extracted thrice with 7 ml diethyl ether and the aqueous extract was brought to pH 12 with 10 N NaOH. This fraction was then extracted thrice with 7 ml chloroform followed by an extraction of the organic phase with 3 ml 5 N HCI. The aqueous phase was then dried in an oven at 95 C, reconstituted with 1 ml H₂O, brought to pH 7.4, and passed through a 0.45 micron filter.

Figure 5b is a bar graph showing the effect of alkaline phosphatase treatment on the ability of bacterial supematants to expand γδ T cells. Alkaline phosphatase treatment of supematants from bacterial broth cultures of P or phyromonas gingivalis and Porphyromonas intermedius has no effect on their ability to expand γδ T cells. Bacterial supematants or EPP were treated with alkaline phosphatase ²³ and cultured with PBMC at a dilution of 1 :6 to test their ability to expand γδ T cells. As a control, alkaline phosphatase treatment reduced the EPP-mediated expansion of γδ T cells in PBMC from 9.6 to 6.1% of CD3⁺ cells in experiment 1, and from 26.9 to 6.0% in experiment 2.

Figure 6a is a line graph showing the ability of bacterial supematants to induce IL-2 release from T cells. Alkaline phosphatase treatment of extracts of Proteus morganii broth 5 culture supematants had no effect on their antigenic ability. Monoethylphosphate (circles), iso- butylamine (squares) or extracts of Proteus supematants (triangles) were treated with alkaline phosphatase (open symbols), or mock-treated (closed symbols) and used as antigens to stimulate IL-2 release from the Vγ2Vδ2 TCR transfectant DBS43. Half-log dilutions of alkylamine antigen stock solutions were added to 10⁵ responder TCR transfectants in the presence of 10 nM

10 phorbol myristate acetate as a co-stimulator. After 24 hours, supematants were harvested and tested at a final dilution of 1/8 for their ability to stimulate the growth of the IL-2 dependent HT-2 cell line. Alkaline phosphatase treatment was carried out as follows: 500 μl of either undiluted bacterial supernatant, or a bacterial supernatant extracted to enrich for amines (as described above) or a 10 mM solution of purified alkylamines (e.g., 10 mM iso-butylamine or

15 10 mM monoethylphosphate) were mock-treated or treated with 5 units of shrimp alkaline phosphatase (Sigma) for 2 hours at 37 °C and used as antigens.

Figure 6b is a line graph showing the ability of purified alkylamines to induce IL-2 release from a Vγ2Vδ2 TCR transfectant, DBS43. DBS43 requires approximately 100-fold more alkylamine antigen concentration for a detectable IL-2 response as compared to γδ T cell

20 expansion from PBMC. There was no detectable IL-2 release from a VγlVδl TCR transfectant (data not shown).

Figure 7 is a list of phosphate and alkylamine antigens and their corresponding structures.

Figure 8 is a series of bar graphs showing the effect of APC on ethylamine recognition

25 by Vγ2Vδ2 T cells. APC had no effect on TCR-dependent recognition of ethylamine by a Vγ2Vδ2 TCR transfectant. SH-5YSY neuroblastoma cells (right panels) or EBV-transformed lymphoblastoid cells (data not shown) were treated with mitomycin C and added as APC to a Vγ2Vδ2 TCR transfectant and the response to ethylamine was measured by IL-2 release and compared to the IL-2 response seen without APC (left panels). Whereas APC enhanced the

30 response to ethylpyrophosphate by about 100-fold (lower panels), there was no detectable effect of APC on the response to ethylamine (upper panels).

Figure 9 illustrates an exemplary synthetic reaction for forming the alkylamine precursors of the invention.

Figure 10 illustrates the hydrolysis of an alkylamine precursor of formula II. Detailed Description Of The Invention

The invention is based on the discovery that alkylamines cause proliferation of Vγ2Vδ2 T cells in a T cell receptor (TCR)-specific manner. The alkylamine antigens are the first phosphate-free antigens described for Vγ2Vδ2 T cells and, thus, represent a distinct chemical class of ligand for Vγ2Vδ2 T cells. The discovery that the alkylamines stimulate γδ T cell expansion is surprising and unexpected since all previously described antigens for Vγ2Vδ2 T cells are characterized by a critical phosphate or pyrophosphate moiety. As used herein, Vγ2Vδ2 T cells refer to thymus-derived cells in the immune system which mediate cellular immune reactions and regulate immune response, and characterized by the presence of specific γδ T cell antigen receptors expressed on their cell surface. These specific γδ T cell antigen receptors (TCR) are composed of rearranged (TCR) γ and (TCR) δ chains which incorporate variable domains encoded by the germline variable (V) domain gene segments Vγ2 (Vγ9 is alternate nomenclature) and Vδ2, respectively. These cells may include certain T cell clones, T cell lines, T cell hybridomas, as well as naturally occurring populations of T cells arising or present in a mammal.

As discussed above, depending upon the intra- and extra-cellular environment, activation of the Vγ2Vδ2 T cells can initiate a number of cellular changes including, but not limited to, proliferation of Vγ2Vδ2 T cells, stimulation or inhibition (e.g., blocking) of cytokine production by the Vγ2Vδ2 T cells, stimulation or inhibition of cytotoxic activity by Vγ2Vδ2 T cells, and stimulation of the Vγ2Vδ2 T cells to undergo apoptosis and die. Thus, the invention also embraces methods and compositions for these particular aspects of activating Vγ2Vδ2 T cells. Each of these activities has been well documented in, for example, α/β T cells, and can be assessed using standard procedures (e.g., ELISA assays to measure cytokine release, apoptosis assays). For example, following activation by antigen, CD4 T lymphocytes produce one of two distinctive cytokine profiles which has led to their classification as T helper 1 (T_H1) and T helper 2 (T_H2) cells. The different cytokines produced by these cells lead to differences in immune function, i.e., upregulation or downregulation of the immune response. According to one particular aspect of the invention, a method for stimulating activation of Vγ2Vδ2 T cells is provided. The method for stimulating activation involves contacting the cells with a Vγ2Vδ2 T cell activation stimulating amount of an alkylamine agent, in vivo or ex vivo, to stimulate activation of the Vγ2Vδ2 T cells. As used herein, "contacting the cells" with the alkylamine agent means placing the cells in sufficient proximity to the alkylamine agent for a time and under conditions sufficient for the alkylamine to stimulate the activation of the Vγ2Vδ2 T cells. Activation is assessed using routine procedures. Contacting may be performed in vivo or ex vivo.

According to another aspect of the invention, a method for stimulating the proliferation of Vγ2Vδ2 T cells is provided. The method involves contacting the cells with a Vγ2Vδ2 T cell proliferation stimulating amount of an alkylamine agent, in vivo or ex vivo, to stimulate proliferation of the Vγ2Vδ2 T cells. As used herein, a "Vγ2Vδ2 T cell proliferation stimulating amount" refers to the amount effective to induce the proliferation of the Vγ2Vδ2 T cells. Such proliferation can be detected by using standard procedures to measure the number of cells before and after treatment with the alkylamine agents. A statistically significant increase in the number of cells following treatment with the alkylamine agent is a Vγ2Vδ2 T cell proliferation stimulating amount. Preferably, for ex vivo applications, this amount is from about 0.01 mM to about 100 mM, more preferably from about 0.1 mM to about 20 mM, and most preferably from about 1 mM to about 10 mM. For in vivo applications, the preferred amounts are from about 5 mg/kg to about 100 mg/kg, more preferably from about 10 mg/kg to about 50 mg/kg, and most preferably from about 20 mg/kg to about 30 mg/kg.

Contacting the Vγ2Vδ2 T cells with the alkylamine agents of the invention can be effected in vivo or ex vivo. Thus, contacting refers to exposing the Vγ2Vδ2 T cells to the alkylamine agent under conditions and for a sufficient period of time to permit the alkylamine agent to stimulate proliferation of the Vγ2Vδ2 T cells. Contacting may be effected by administering to a subject in need of such treatment a Vγ2Vδ2 T cell proliferation inducing amount of the alkylamine agent. Alternatively, biological fluid (preferably containing Vγ2Vδ2 T cells) can be removed from the subject, treated with the alkylamine agents of the invention, and reinfused into the subject in accordance with standard practice. Such biological fluids include, but are not limited to sera, cerebrospinal fluid, and synovial fluid. The preferred fluid is blood.

In one aspect, the invention is directed to a method for treating a subject diagnosed as having a condition that is associated with an antigen that is recognized by Vγ2Vδ2 T cells. By stimulating the proliferation of Vγ2Vδ2 T cells, it is believed that an enhanced immune response is generated against the condition. The method of treatment involves administering to the subject an isolated alkylamine agent in an amount and in a manner effective to induce proliferation of Vγ2Vδ2 T cells and/or to activate the γδ T cell receptor. The particular mode of administration will depend upon the nature of the condition and the location of the Vγ2Vδ2 T cells that are being targeted by the alkylamine agents. Exemplary conditions that are associated with an antigen that is recognized by Vγ2Vδ2 T cells are known to those of ordinary skill in the art and include, but are not limited to, the following diseases: infectious diseases such as tuberculosis, salmonellosis, brucellosis, ehlichiosis, tularemia, malaria, leishmaniasis, mononucleosis, and in HIV. Additional conditions in which increased numbers of Vγ2Vδ2 T cells may be desirable include autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and sarcoidosis.

The alkylamine agents of the invention are particularly useful for treating gingivitis that is associated with an antigen that is recognized by Vγ2Vδ2 T cells. Most studies examining the reactivity of Vγ2Vδ2 T cells to defined antigens have used peripheral blood mononuclear cells (PBMC) as a source of γδ T cells and phosphate antigens isolated from mycobacteria as stimulants. Although not intending the invention to be limited to a particular theory or mechanism, we believe that γδ T cells found in other anatomic compartments such as the gingiva, in which 30% of isolated leukocytes are TCR γδ+, are more likely to encounter a distinct set of pathogens, γδ T cells represent up to 60% of T cells in the female reproductive tissues such as the cervix and decidua. For example, there have been recent reports that γδ T cells are activated in the gingiva of patients with chronic gingivitis, which is caused by periodontopathic mouth anaerobic bacteria such as Bacteroides forsythus and Porphyromonas gingivalis. Such bacteria are also opportunistic pathogens in the female reproductive tract in diseases such as pelvic inflammatory disease, chorioamnionitis, postpartum endometritis, preterm labor, and postoperative infections. Our experimental results indicate that human γδ T cells recognize alkylamines which are major products (produced in millimolar concentrations) of certain bacteria including the Bacteroides species. Accordingly, we believe that alkylamines can be used to treat gingivitis that is associated with an infection that is associated with an antigen that is recognized by the gingival γδ T cell, as well as infections of the female reproductive tract. For embodiments which target the gingival tissue, the preferred compositions of the invention are formulated for localized delivery to the tissue (e.g., chewing gum, floss that is impregnated or otherwise coated with alkylamine agents of the invention, gingival tissue implants). Other variations on the formulation will depend upon the nature of the target tissue source of γδ T cells to which the therapy is directed.

The alkylamine agents of the invention are administered in effective amounts. An effective amount is a dosage of the alkylamine agent sufficient to provide a medically desirable result. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. For example, in connection with a diagnosed bacterial or viral infection, an effective amount is that amount which slows or inhibits the extent of the bacterial or viral infection as measured in accordance with standard practice for determining bacterial or viral loads in a subject (intracorporeally or extracorporeally) or in cell culture. Likewise, an effective amount for treating gingivitis is that amount which slows, inhibits, or halts the progression of gingivitis. Thus, it will be understood that the alkylamines of the invention can be used to treat the above-noted conditions prophylactically in subjects at risk of developing the foregoing conditions. As used in the claims, "inhibit" embraces all of the foregoing. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.

A subject, as used herein, refers to any mammal (preferably, a primate and, more preferably, a human) that may be susceptible to a condition associated with an infection or an autoimmune condition in which an antigen that is recognized by Vγ2Vδ2 T cells is implicated, provided that the mammal is otherwise free of symptoms calling for alkylamine agent treatment. The preferred subjects are free of symptoms calling for treatment with an alkylamine agent for any reason.

An alkylamine agent, as used herein, refers to an alkylamine of formula I or III and an alkylamine precursor of formula II (discussed below). Alkylamine agents are capable of inducing stimulation of Vγ2Vδ2 T cells and/or activating a γδ T cell receptor, in vivo or ex vivo. Accordingly, the alkylamine agents of the invention are capable of reducing or preventing the proliferation of conditions that are associated with antigens that are recognized by Vγ2Vδ2 T cells, such as the above-describedinfectious diseases and autoimmune conditions.

As used herein an "alkylamine" refers to a compound of formula I or III. The preferred alkylamines are compounds of formula I: R-NH2 (formula I), wherein R is a straight- or branched chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive. Exemplary alkylamines include: monoalkylamines such as methyl, ethyl, n-propyl, n-butyl, isopropyl and alkenylamines such as allyl, crotyl, dimethylallyl, isopentenyl, geranyl, farnesyl, geranylgeranyl, 3-methyl-2-pentenyl, and 3-methyl-2-hexenyl. See also U.S. 5,639,653, issued to Bloom et al., (e.g., columns 4-7) for examples of alkylgroups and substituted alkyl groups (e.g., hydroxymethyl -, β-hydroxyethyl-, δ-hydroxy propyl-, hydroxy isopropyl-) that contain six carbon atoms or less and that can be used in accordance with the instant invention. The Bloom compounds differ from those disclosed herein in that the former compounds have an absolute requirement for a phosphate or pyrophosphate moiety whereas the compounds of the instant invention have a positively charged amine moiety in place of a negatively charged phosphate group. The preferred alkylamines are ethyl-, n-propyl-, -iso-propyl-, n-butyl, iso-butyl-, sec-butyl-, and iso- amylamines.

As used herein, an "alkylamine precursor" refers to a compound of formula II, O I

Rl-C-NH-R (formula II),

wherein R is a straight- or branched chain alkyl or alkenyl group containing from one to six carbon atoms, and wherein Rl is a straight- or branched chain alkyl or alkenyl group containing from one to twenty carbon atoms, inclusive. Thus, R may contain 1, 2, 3, 4, 5, or 6 carbon atoms and Rl may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Preferably, Rl contains from one to ten carbon atoms, inclusive. The R and Rl groups are, independently, straight-chained or branched-chain and may be saturated or unsaturated. R and Rl, optionally and independently, contain substituted functional groups including, e.g., hydroxy, amino, carboxy and methyl groups. The preferred alkylamine precursors are theanine, N⁵- isopropylglutamine, N4-ethyl asparagine, and N⁵-^ec- butylglutamine.

Although a primary amine is shown in formula I, it is to be understood that the invention embraces alkylamines that are secondary or tertiary amines, i.e., compounds of formula III: R-NR2R3 (formula III) wherein R2 is a straight- or branched-chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive; wherein R3 is hydrogen or a straight- or branched-chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive; and wherein R2 and R3 may be the same or different from one another and from R. The invention also embraces alkylamine precursors that can be cleaved to form the secondary or tertiary alkyl amines of formula III.

As used herein in reference to Rl, an "alkyl or alkenyl group" refers to an alkyl or alkenyl chain containing from one to twenty carbon atoms, inclusive. Exemplary alkyl groups include propyl, butyl, and amyl. Exemplary dicarboxylic acids include the amino acids and, in particular, glutamic acid or aspartic acid. The preferred alkylamine precursors are selected from the group consisting of theanine, N⁵- z^'sø-propylglutamine, N4-ethyl asparagine, and N⁵-sec- butylglutamine.

The term "isolated", as used herein, refers to a compound which is substantially free of contaminating substances which render the compound unsuitable for therapeutic applications. When used therapeutically, the isolated alkylamine agents of the invention are administered in therapeutically effective amounts. In general, a therapeutically effective amount means that amount necessary to delay the onset of, inhibit the progression of, or halt altogether the particular condition being treated. Generally, a therapeutically effective amount will vary with the subject's age, condition, and sex, as well as the nature and extent of the disease in the subject, all of which can be determined by one of ordinary skill in the art. The dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg//kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days.

The therapeutically effective amount of the isolated alkylamine agent is that amount effective to induce stimulation of Vγ2Vδ2 T cells and/or activate the γδ T cell receptor as determined by, for example, standard tests known in the art. It is believed that by increasing the number of Vγ2Vδ2 T cells, the alkylamine agents of the invention enhance the immune response against diseases which are associated with agents that are recognized by the Vγ2Vδ2 T cells.

Optionally, the isolated alkylamine agents are administered to the subject in combination with other agents for treating the disease, e.g., an antibiotic for treating a bacterial infection, an antiviral agent for treating a viral infection. As used herein, antibiotic and antiviral agents are terms of art that would be understood by one of skill in the art to refer to a wide spectrum of therapeutic agents which have these functional activities. See, e.g., Harrisons, Principles of Internal Medicine (McGraw Hill, Inc., New York) for a more detailed description of these therapeutic agents.

The above-described drug therapies are well known to those of ordinary skill in the art and are administered by modes know to those of skill in the art. The drug therapies are administered in amounts which are effective to achieve the physiological goals (to prevent or reduce the physiological consequences of the infection or autoimmune condition), in combination with the isolated alkylamine agents of the invention. Thus, it is contemplated that the drug therapies may be administered in amounts which are not capable of preventing or reducing the physiological consequences of the condition when the drug therapies are administered alone but which are capable of preventing or reducing the physiological consequences of the condition when administered in combination with the isolated alkylamine agents of the invention.

The isolated alkylamine agents may be administered alone or in combination with the above-described drug therapies as part of a pharmaceutical composition. Such a pharmaceutical composition may include the isolated alkylamine agent in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount of the isolated alkylamine agent in a unit of weight or volume suitable for administration to a patient. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human or other animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. Pharmaceutically acceptable further means a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the alkylamine agent, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

As noted above, variety of administration routes are available for delivering the alkylamine agents of the invention to the subject. The particular mode of delivery that is selected will depend, of course, upon the particular drug selected, the severity of the condition being treated, and the dosage required for therapeutic efficacy. The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, interdermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intranasal, inhalation, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. There could, however, be preferred in emergency situations. Oral administration is preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule.

The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the alkylamine agents into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the alkylamine agents into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the alkylamine agents. Preferred formulations for oral administration include: implants that are constructed and arranged for implantation into gingival tissue, chewing gum containing the alkylamine agents, floss that is impregnated with alkylamine agents, and toothpaste containing the alkylamine agents. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.

Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the alkylamine agents described above, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include the above-described polymeric systems, as well as polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the alkylamine agent is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,832,253, and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

Administration of the alkylamine agents may be effected using any means known to those of skill in the art, including oral, rectoral, topical, intravenous, subcutaneous, intramuscular, or intraperitoneal routes of delivery. The alkylamine agents may be formulated to achieve sustained release of the agent in vivo. Thus, for example, in one embodiment, the preferred vehicle is a biocompatible micro particle or implant that is suitable for implantation into the subject. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US/03307 (Publication No. WO 95/24929, entitled "Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no. 213,668, filed March 15, 1994). PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix. In accordance with the instant invention, the alkylamine agents are encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307. The polymeric matrix preferably is in the form of a micro particle such as a micro sphere (wherein the alkylamine agent(s) are dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the alkylamine agent(s) are stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the alkylamine agents include films, coatings (e.g., coatings onto floss), gels, and implants. The alkylamines, alone or formulated into a sustained release composition, can be used as a food or hygienic product additive. For example, in one embodiment, the alkylamines, alone or formulated as a sustained release composition, can be added to toothpaste to help control gingivitis. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted. The size of the polymeric matrix devise further is selected according to the method of delivery which is to be used, e.g., injection or implantation into a tissue or area of inflammation (e.g., a gingival tissue, the synovial fluid within an arthritic joint), administration of a suspension by aerosol into the nasal and/or pulmonary areas. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the devise is administered to a surface in vivo. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time. Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the alkylamine agents of the invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.

In general, the alkylamine agents of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylme hacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof. Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly (hexylmethacry late), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). Thus, the invention provides a composition of the above-described alkylamine agents for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo.

Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions, e.g., chronic gingivitis. Long-term release, are used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.

The isolated alkylamine agents may be administered alone or in combination with the above-described drug therapies by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intra-cavity, subcutaneous, or transdermal. When using the isolated alkylamine agents of the invention, direct administration to the affected site is preferred.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

In general, the alkylamine agents can be administered to the subject (any mammalian recipient) using the same modes of administration that currently are used for the administration of low molecular weight organic molecules.

Another aspect of the invention includes a screening assay method for determining whether a putative therapeutic agent modulates (upregulates or downregulates) alkylamine- induced stimulation of Vγ2Vδ2 T cell proliferation and/or alkylamine-mediated activation of the γδ T cell receptor, i.e., the method is useful for identifying alkylamine agonists that can be used in place of an alkylamine agent of the invention for stimulating Vγ2Vδ2 T cell proliferation and/or activation of the γδ T cell receptor. According to one embodiment of the invention, the method involves contacting a putative alkylamine agonist (e.g., contained in a combinatorial library) with Vγ2Vδ2 T cells under conditions to stimulate Vγ2Vδ2 T cell proliferation if an alkylamine agonist was present and determining whether the putative alkylamine agonist stimulates Vγ2Vδ2 T cell proliferation. Alternatively, antagonists of alkylamine activity can be identified by stimulating Vγ2Vδ2 T cells using an alkylamine agent of the invention in the presence and absence of a putative alkylamine agent antagonist and determining whether the putative alkylamine antagonists reduces the extent of Vγ2Vδ2 T cell proliferation. In another embodiment, the screening tests are employed which measure activation of the γδ T cell receptor (instead of Vγ2Vδ2 T cell proliferation) to identify alkylamine agent agonists and/or alkylamine agent antagonists.

The invention will be more fully understood by reference to the following examples. These examples, however, are merely intended to illustrate the embodiments of the invention and are not to be construed to limit the scope of the invention. It is also to be understood that the referenced figures are illustrative only and are not essential to the enablement of the claimed invention.

Examples Example 1. Human γδ T Cells Recognize Alkylamines A study in our laboratory of naturally occurring compounds potentially capable of stimulating or suppressing γδ T cell cytokine secretion showed that ethylamine was capable of inducing IL-2 release from γδ T cells. Ethylamine, a byproduct of L-theanine (Figure la) hydrolysis found in the urine of tea drinkers ²², differs from the known synthetic γδ T cell antigen ethyl pyrophosphate (EPP) ²³ in that the amine group of ethylamine replaces the pyrophosphate group of EPP (Figure lb). To further explore the reactivity of ethylamine with Vγ2Vδ2 T cells, we measured the IL-2 release of a polyclonal γδ T cell line coexpressing γδ variable region T cell receptor (TCR) chains (Figure 2a), four T cell clones coexpressing Vγ2Vδ2 (Figure 2b), five αβ T cell clones (Figure 2c), one VγlVδl T cell transfectant (Figure 2d, open circles) and one Vγ2Vδ2 T cell transfectant (Figure 2d, closed circles) in response to ethylamine. The Vγ2Vδ2 T cell line, the Vγ2Vδ2 transfectant and all four Vγ2Vδ2 T cell clones, but neither the VγlVδl transfectant nor the αβ T cell clones, responded to ethylamine in a dose dependent manner. The Vγ2Vδ2 T cell line (Figure 2a) and the Vγ2Vδ2 T cell clones (Figure 2b) respectively showed on average a 4-fold and a 5 -fold greater response to ethylamine as compared to media. The Vγ2Vδ2 transfectant (Figure 2d) responded to ethylamine in a manner comparable to the Vγ2Vδ2 T cell line (Figure 2a). In contrast, there was no significant difference in the amount of IL-2 released in response to ethylamine by the 5 αβ T cell clones when compared to media (Figure 2c), or by the VγlVδl TCR transfectant (Figure 2d). These data were unexpected since only phosphate containing antigens have been shown to stimulate Vγ2Vδ2 T cells ^23"27. As expected, EPP stimulated IL-2 release from the Vγ2Vδ2 T cell line and all four Vγ2Vδ2 T cell clones. Alkaline phosphatase treatment of EPP prior to culture with the PBMC abrogated its γδ T cell reactivity, whereas this treatment had no effect of the reactivity of ethylamine (data not shown).

Tea beverage typically has a 2-10 mM concentration of L-theanine, an amino acid unique to all varieties of tea (Camellia sinesis) ^28"30. To determine if tea beverage could cause expansion of γδ T cells in vitro, we mixed unfractionated PBMC isolated from healthy donors with green or black tea purchased commercially from a local food store and brewed according to instructions. Neither green nor black teas expanded the number of γδ T cells (data not shown). However, green and black teas that were acid hydrolyzed by reflux in 5 N HCI to liberate ethylamine from L-theanine ²² caused a three- to five-fold expansion of γδ T cells (Figure 3a). Purified L-theanine failed to cause γδ T cell expansion, but acid hydrolyzed L-theanine caused a fifteen-fold expansion of γδ T cells from PBMC (Figure 3b). L-theanine hydrolysis in vivo most likely occurs by acid hydrolysis in the gut and by the enzymatic action of tissue amidases in the liver ²². Our results here suggest that antigen presenting cells (APC) in the peripheral blood cannot hydrolyze L-theanine, since only acid hydrolyzed tea or purified L-theanine preparations stimulated γδ T cells. To verify that ethylamine was released by acid hydrolysis from the L-theanine in tea or from purified L-theanine, GC-mass spectrometry analysis was performed on acid hydrolyzed or unhydrolyzed tea extract or L-theanine. Ethylamine was not detected in unhydrolyzed samples. In contrast, 7.2 mM ethylamine was detected in the biologically active acid hydrolyzed tea sample that was used to stimulate γδ cells. GC-mass spectrometry analysis of a purified acid hydrolyzed 1 mM L-theanine sample revealed the presence of a roughly equimolar amount (1.1 mM) of ethylamine. Taken together, these results show that ethylamine resulting from hydrolysis of L-theanine found in tea is capable of expanding γδ T cells.

To examine the reactivity of human γδ T cells with other naturally occurring alkylamines, peripheral blood mononuclear cells (PBMC) from healthy donors were cultured into 400 mM concentrations of either EPP or various alkylamines. After 7 to 13 days of culture, we performed cell counts and flow cytometric analysis with TCR specific monoclonal antibodies. The alkylamines ethyl-, «-propyl-, «-butyl-, wø-propyl-, iso-buXyl-, sec-butyl-, tert-butyl, and wo-amylamine expanded γδ T cells 2- to 15-fold compared to media alone or to tetanus toxoid, which did not expand γδ T cells (Fig 4). Methyl- and rc-amylamines as well as the polyamines, putrescine and spermidine, failed to expand γδ T cells from PBMC of several donors (data not shown), αβ T cells were not expanded, and flow cytometry using Vγ and Vδ-specific monoclonal antibodies revealed that all the expanded γδ T cells expressed Vγ2 and Vδ2 gene segments. Such Vγ2Vδ2 TCR classes have no homologues in nonprimates. The alkylamines here shown to be capable of expanding γδ T cells are found in apples ³¹, wine ³², tea (in precursor form as L-theanine) ²², as secreted products of bacteria ^{33 35}, human urine ^36"38, breast milk, amniotic fluid ³⁹, and in vaginal secretions from healthy women.⁴⁰ Thus, alkylamine antigens may be derived from either plant foodstuffs or from bacteria and are routinely found in human body fluids. Antigenic alkylamines reportedly are found in vivo in humans in concentrations of up to 10 mM, well above the 400 μM concentrations that elicited the γδ T cell expansions from PBMC in vitro (Figure 4). γδ T cell expansions in man have been strikingly correlated with certain microbial infections although such reactivity has been linked to alkyl phosphate antigens ^{27, 4}'. Bacteroides fragϊlis and Clostridium perfringens also produce and secrete millimolar concentrations of alkylamines and polyamines such as methylamine, dimethylamine, ft-propylamine, pyrrolidine, piperidine, «-butylamine, and putrescine ³⁴. Proteus, Salmonella, Shigella and Escherichia coli produce amines such as cadaverine, b-phenylethylamine, putrescine, wo-amylamine, 2-methylbutylamine, and z^'so-butylamine ³⁵. Listeria monocytogenes produces tt-butylamine and putrescine during fermentation ³³. Escherichia coli, Salmonella species, Listeria monocytogenes and Yersinia enterocolitica are known to cause expansion of Vγ2Vδ2 T cells in vitro ¹²'⁴²-⁴³ and patients with Salmonellosis and Listeriosis have increased numbers of peripheral blood Vγ2Vδ2 T cells ^{12, 15}. Moreover, abnormally high numbers of activated Vγ2Vδ2 T cells are found in patients with periodontal diseases such as gingivitis ⁴⁴, which is associated with Bacteroides or Porphyromonas species ⁴⁵. The parasite Trichinella pseudospiralis, which causes regressive and regenerative changes in muscle tissue produces large amounts of π-butylamine. Infusion of «-butylamine into mouse muscle induces microscopic pathologic changes identical to those in the muscle tissue of Trichinella pseudospiralis infected mice. Inflammatory changes are not seen, and this may be because mice lack γδ T cells that are able to recognize alkylamine antigens⁴⁶. Thus, alkylamine antigens produced in large quantities by pathogenic organisms are capable of expanding Vγ2Vδ2 T cells in vitro, and they may have in vivo effects such as tissue destruction and expansion of Vγ2Vδ2 T cells during certain microbial infections. To identify potential microbial sources of alkylamine antigens, fresh supematants from bacterial broth cultures and supernatant extracts enriched for alkylamines were prepared from Proteus morganii, an important cause of urinary tract infections and urosepsis and from the anaerobic periodontopathic bacteria, Porphyromonas intermedius or Porphyromonas gingivalis, (formerly genus Bacteroides). These supematants were mock-treated or treated with alkaline phosphatase to destroy any alkyl phosphate activity that might be present and then added to cultures of PBMC from healthy donors. Cultures were maintained for 12 days and subsequently analyzed by cell counting and flow cytometry (Figure 5). Crude bacterial supernatant (circles) and alkylamine-enriched extract (triangles) from Proteus morganii induced in a dose-dependent manner a 3 -fold and a 7-fold increase, respectively, in Vγ2Vδ2 T cell numbers, whereas the bacteriological media alone (squares) had no effect (Figure 5a). Quantitative headspace GC-mass spectrometry analysis revealed that the crude bacterial supernatant contained 3.4 mM z^'sø-butylamine and 3.9 mM z^'sø-amylamine, the alkylamine- enriched extract of the bacterial supernatant contained 4.1 mM wø-butylamine and 8.4 mM iso- amylamine, and the uninoculated culture media had no detectable alkylamines (data not shown). Whereas alkaline phosphatase treatment totally abrogated the ability of the alkyl phosphate EPP to expand γδ T cells (data not shown), alkaline phosphatase treated or mock treated crude bacterial supematants from Porphyromonas intermedius and Porphyromonas gingivalis both caused up to a 2-fold expansion of γδ T cells (Figure 5b). This result is consistent with the idea that alkylamines found in bacterial supematants expanded γδ T cells.

To determine whether reactivity to alkylamines was TCR-dependent, we tested TCR transfectants for their ability to release IL-2 in response to bacterial supematants (Figure 6a) and a panel of alkylamines (Figure 6b). DBS43 was made by cotransfecting cDNA encoding the Vγ2 and Vδ2 TCR chains from the EPP reactive clone DGSF.13 into a TCR deficient mutant of Jurkat T cells. For comparison, another transfectant, 27/3.62 containing cDNA from the T cell clone F7 encoding a VγlVδl chain pair which lacks reactivity to EPP ⁴⁷ was also used. An extract of Proteus supernatant enriched for alkylamines (triangles), a solution of pure iso- butylamine (squares), and a solution of monoethylphosphate (circles) (i.e., as an example of a phosphate antigen) were either mock-treated (closed symbols) or alkaline phosphatase treated (open symbols) (Figure 6a). Alkaline phosphatase treatment of monoethylphosphate reduced by 7-fold its ability to induce IL-2 release from the Vγ2Vδ2 TCR transfectant (Figure 6a), but not the VγlVδl TCR transfectant (data not shown), thus totally abrogating antigenic activity. In contrast, this treatment failed to significantly reduce the antigenic activity of the Proteus extract or pure iso-butylamine, emphasizing that alkylamines, and not phosphate antigens, were responsible for this activity (Figure 6a). The Vγ2Vδ2 TCR transfectant DBS43 also released significant levels of IL-2 in response to a series of other alkylamines tested (Figure 6b) whereas the VγlVδl TCR transfectant did not (data not shown). These data show also that z^'sø-butyl- and sec-butylamines were the most potent antigens on a molar basis for this particular transfected TCR, π-propyl-, wø-propyl-, π-butyl-, and wo-amylamines had intermediate potency, and ethylamine was the least potent antigen. Antigen titrations using a panel of Vγ2Vδ2 T cell clones will reveal whether the rank order of potency seen for these antigens' reactivity with the DBS43 transfectant holds for all Vγ2Vδ2 T cell clones, or if there exist T cell clones specific for only one alkylamine. These results together with the expansion of only certain γδ T cells from PBMC, indicate that reactivity to alkylamines is critically dependent on the expression of a Vγ2Vδ2 TCR.

To determine the in vivo effect of alkylamines antigens on T cells, we administered intravenously 50 mg/kg /so-butylamine to each of 2 rhesus monkeys 3 times in a one week period, without co-administration of IL-2. At t₀ (i.e., before any infusions), blood γδ T cells could be expanded up to 30-fold in vitro in response to z^'sø-butylamine and IL-2. However, blood γδ T cells harvested after one week of injections were unable to respond in vitro to iso- butylamine and IL-2. These results show that in vivo infusions of w -butylamine can influence the in vitro reactivity of monkey γδ T cells. Infusion of pure antigens without cytokines such as IL-2 can lead to T cell unresponsiveness (i.e., anergy). To test the hypothesis that infusion of IL-2 and wo-butylamine together can cause in vivo expansion of blood γδ T cells, monkeys are intravenously administered w -butylamine with IL-2 for 3 times during a one-week period, with measurements of T cell expansion in vitro prior to and following the administration schedule. It is expected that co-administration of wø-butylamine and IL-2 will lead to in vivo expansion of blood γδ T cells rather than anergy.

These results show that the same Vγ2Vδ2 TCR can recognize a positively charged molecule (e.g., ethylamine) as well as the previously characterized negatively charged molecules such as EPP and isopentenyl pyrophosphate (IPP) ²³-²⁷-⁴¹. This suggests that the TCR might bind these two different molecules in different locations on the TCR. This is easily possible, given the small sizes of these antigens. Alternatively, the TCR may recognize the alkyl chain of either alkyl phosphates or alkyl amines at one TCR site. Distinct TCR residues adjacent to such a site may be able to provide stabilization of the TCR-antigen complex by accommodating either positive or negative charges. The functional parts of the alkyl phosphate, EPP, are the hydrophobic alkyl chain and the negatively charged phosphate moiety. Data from the present report show that substitution of the phosphate by an amine is permitted. This suggests that the nature of the charge on the molecule is less important than the presence of the alkyl group. Previous data showed that alkyl groups of 1-4 carbons are necessary for straight chain alkyl phosphate reactivity and that substitutions with phenyl rings are not permitted ²³. This suggests that the TCR specificity may be directed more at the hydrophobic alkyl chain than at the charged part of the EPP or ethylamine molecule. The TCR dependent reactivity of either negatively charged alkyl phosphates or positively charged alkylamine antigens must therefore be accounted for in any molecular model of Vγ2Vδ2 TCR interaction with antigen.

These results also show that alkylamines recognized by γδ T cells are characterized by a straight or branched alkyl chain of two to five carbons with a single primary amine group as the only substituent (Figure 7). Conversely, alkylamines with one carbon (e.g., methylamine) or more than five carbons, or any substituent in addition to the primary amino group were not antigenic (Figure 7). These structural constraints on alkylamine antigens are reminiscent of those of alkyl phosphates and prenyl pyrophosphates. Alkylamines recognized by γδ T cells and having antigenic activity can be identified using in vitro screening assays as described herein. In this way, a few compounds lacking such activity have been identified. For instance, the five carbon iso-amyl phosphate is not recognized, while iso-amylamine is recognized. As another example, methylamine is not antigenic, while methylphosphate is antigenic. In summary, straight or branched chain alkyl phosphates of one to four or five carbons and primary alkylamines of two to five carbons were antigenic for Vγ2Vδ2 T cells (Figure 7).

To assess the importance of APC in the T cell-mediated response to alkylamines, we compared the reactivities of several alkylamines and EPP in the presence or absence of glutaraldehyde fixed SH-5YSY neuroblastoma cells as APC in IL-2 release assays. Neither the potency nor the magnitude of the response to ethylamine (Figure 8, upper panel) or several other antigenic alkylamines (data not shown) was affected by the addition of SH-5YSY APC. As expected, EPP also showed a significant reactivity in the absence of APC although reactivity was increased both in magnitude (about 4-fold) and in potency (about 100-fold) by the addition of APC (Figure 8, lower panel). The use of another APC, EBV-transformed B lymphoblastoid cells, increased EPP potency by 10-fold ²³ but again had no effect on the response to ethylamine (data not shown). This suggests that the TCR-dependent recognition of ethylamine and EPP each have different requirements for antigen presentation or costimulation. These data show that APC have no effect on TCR dependent recognition and argue for direct recognition of alkylamine antigens, similar to that seen in immunoglobulin (Ig)-hapten interactions. Several lines of evidence suggest that some γδ T cells recognize antigen in a way that is more like that of an immunoglobulin than like that of an αβ TCR. In the mouse, γδ T cells directly recognize the MHC class II molecule I-E^k and the nonclassical MHC molecules T10 and T22, without peptide dependence. Site directed mutagenesis of the I-E^k MHC molecule showed that γδ T cells recognized MHC residues outside the peptide binding groove ⁴⁸- ⁴⁹. Moreover, γδ T cells recognize native HSV glycoprotein I even when bound to plastic without professional APC ⁵⁰- ⁵¹. Analysis of CDR3 length distributions of TCRδ chains indicates that they are more similar to those of IgH than to TCRα and TCRβ chains ⁵², and finally, atomic structure analysis of human Vδ3 indicates that this TCR models more closely on Ig V domains than on TCRα or β V domains^{54 55}. Crystallographic studies have shown that immunoglobulins recognize small phosphate-containing molecules such as phosphorylcholine in a way that is highly dependent on both the heavy and light chain CDR3 regions ⁵³. Thus, the Vγ2Vδ2 TCR-alkylamine complex may be very similar to an Ig-hapten complex. However, preliminary evidence in our laboratory suggests that cell to cell contact may be necessary for IL-2 release by γδ T cells in response to alkylamines. Detailed knowledge of the antigenic structures recognized by αβ T cells has deepened our understanding of their role in immunity. One key to understanding the role of γδ T cells in immunity is to identify the antigens they recognize. The alkylamines identified herein as antigens point to recognition of bacteria in host defense, and to the potential in plant foods such as tea, apples, and wine for enhancing immunity, and also for a potential role for γδ T cells in autoimmunity and enhancing immune tolerance to allergens. Further study will be needed to determine the precise nature of interaction between the Vγ2Vδ2 TCR and their antigens. However, this study clearly shows that alkylamines are a large class of newly identified Vγ2Vδ2 T cell antigens with chemical and biological properties distinct from phosphate antigens. Found in plant foods, human body fluids, and bacteria, alkylamines are likely to play a major role in Vγ2Vδ2 T cell activity in vivo.

Vγ2Vδ2 T cells may be considered part of the adaptive immune system in that they have a memory phenotype⁵⁸, they express junctionally diverse TCR⁵⁹ that require gene rearrangement for their cell surface expression⁶⁰, and their ability to undergo either anergy or expansion depending on the availability of costimulation^{61, 62}. On the other hand, Vγ2Vδ2 T cells also may be considered part of the innate immune response since their frequently paired TCR variable region genes Vγ2 and Vδ2 reflect limited germ line diversity. This V gene pairing enables each Vγ2Vδ2 TCR to immediately recognize families of unprocessed antigens with conserved molecular patterns such as the alkylamines and alkyl phosphates. This TCR- dependent recognition of the conserved one- to five-carbon straight and branched-chain alkyl groups found in antigenic alkylamines and alkyl phosphates that are products of multiple pathogens is reminiscent of the CD14-mediated pattern recognition of pathogen-associated molecular patterns such as the repeating sugar residues found in various bacterial lipopolysaccharides⁶³- ⁶⁴. This pattern of recognition by the Vγ2Vδ2 TCR allows the expansion of memory γδ T cells to large numbers in normal adults (mean 4.5% of CD3⁺ T cells) and to 2- to 10-fold higher levels (8-60% of CD3⁺ T cells) during a host of microbial infections. These large numbers of memory T cells capable of responding to alkylamine antigens produced by microbes thus may bridge the gap between innate and adaptive immune responses.

Example 2. Alkylamine Precursor Reactions

The alkylamine agents of the invention are alkylamines of formula I or III or alkylamine precursors of formula II, as described above. Precursors of alkylamines of the type in formula I may be found in nature as naturally occurring compounds or may be chemically synthesized. Examples of naturally occurring precursors include the N-substituted primary amides N⁵- ethylglutamine (L-theanine) in Camellia sinesis, N⁴-ethylasparagine in Tulipa gesneriana, and N⁵-w -propylglutamine in Lunaria annua.

Figure 9a illustrates an exemplary synthetic reaction for forming the alkylamine precursors of the invention. The synthesis reaction is based upon that described by Y. Sakato, et al., in Nippon Nugei Kagadu Kaishi 23:262-271 (1949). (See, also, Morrison & Boyd, 3rd ed, Section 18 for exemplary reactions for forming and hydrolyzing an amide compound.) Exemplary Rl groups in the acid structure include but are not limited to ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl and sec-butyl. Figure 10b illustrates an exemplary reaction in which an acid (e.g., L-glutamic acid) is allowed to react with an amine (e.g., ethylamine) to form an ester (e.g., L-glutamic acid γ-ethyl ester) which is derivatized to form a derivatized ester. Thereafter, the derivatized ester is allowed to form the alkylamine precursor (amide). Figure 10 illustrates the hydrolysis of an alkylamine precursor of formula II. Hydrolysis can be effected in the presence either of acid or base, or by enzymatic cleavage of the amide bond (e.g., by an amidase) to yield an alkylamine of formula I or III and a mild carboxylic acid. For example, hydrolysis of N⁵-ethylglutamine (L-theanine) yields ethylamine plus glutamic 5 acid. Naturally occurring alkylamine precursors include N⁵-ethylglutarnine (L-theanine), as found in Camellia sinesis i.e., tea; N⁵-wø-propylglutamine, as found in Lunaria annua i.e., an ornamental plant; and N5 ethylasparagine, as found in Tulipe gesneriana i.e., tulip, Echallium elaterium, i.e., squirting cucumber and Bryonia dioica.

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All references, patents and patent publications that are recited in this application are incorporated in their entirety herein by reference.

It should be understood that the preceding is merely a detailed description of certain preferred embodiments. It therefore should be apparent to those skilled in the art that various 30 modifications and equivalents can be made without departing from the spirit and scope of the invention. It is intended to encompass all such modifications within the scope of the appended claims.

Claims

1. A method for stimulating the proliferation of V╬│2V╬┤2 T cells comprising: contacting the cells with a V╬│2V╬┤2 T cell proliferation stimulating amount of an alkylamine agent selected from the group consisting of an alkylamine of formula I: R-NH2 (formula I), an alkylamine of formula III:

R-NR2R3 (formula III) and an alkylamine precursor of formula II, O I

Rl-C-NH-R (formula II),

wherein R is a straight- or branched chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive, wherein Rl is a straight- or branched chain alkyl or alkenyl group containing from one to twenty carbon atoms, inclusive, wherein R2 is a straight- or branched-chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive, that may be the same or different from R, and wherein R3 is hydrogen or a straight- or branched-chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive, that may be the same or different from R or R2.

2. The method of claim 1, wherein stimulating the proliferation comprises increasing the number of V╬│2V╬┤2 T cells by at least two-fold.

3. The method of claim 1 , wherein stimulating the proliferation comprises increasing the number of V╬│2V╬┤2 T cells by at least ten-fold.

4. The method of claim 1 , wherein contacting the cells with the alkylamine agent is performed ex vivo.

5. The method of claim 1, wherein contacting the cells with the alkylamine agent is performed in vivo.

6. The method of claim 4, wherein the V╬│2V╬┤2 T cell proliferation stimulating amount of the alkylamine agent is from about 0.01 mM to about 100 mM.

7. The method of claim 4, wherein the V╬│2V╬┤2 T cell proliferation stimulating amount of 5 the alkylamine agent is from about 0.1 mM to about 0.1 mM to about 20 mM.

8. The method of claim 4, wherein the V╬│2V╬┤2 T cell proliferation stimulating amount of the alkylamine agent is from about 1 mM to about 10 mM.

10 9. The method of claim 5, wherein the V╬│2V╬┤2 T cell proliferation stimulating amount of the alkylamine agent is from about 5 mg/kg to about 100 mg/kg.

10. The method of claim 5, wherein the V╬│2V╬┤2 T cell proliferation stimulating amount of the alkylamine agent is from about 10 mg/kg to about 50 mg/kg.

15

11. The method of claim 4, wherein the V╬│2V╬┤2 T cell proliferation stimulating amount of the alkylamine agent is from about 20 mg/kg to about 30 mg/kg.

12. The method of claim 1 , wherein the alkylamine agent is an alkylamine.

20

13. The method of claim 1 , wherein the alkylamine agent is an alkylamine precursor.

14. The method of claim 1 , wherein R is a straight-chained alkyl group.

25 15. The method of claim 1 , wherein R is a branched-chained alkyl group.

16. The method of claim 14, wherein R is saturated.

17. The method of claim 15, wherein R is unsaturated. 30

18. The method of claim 1 , wherein the alkylamine is selected from the group consisting of methyl-, ethyl-, n-propyl-, -iso-propyl-, n-butyl, iso-butyl-, sec-butyl-, iso-amyl-, - J O - hydroxymethyl -, ╬▓-hydroxyethyl-, ╬┤-hydroxy propyl-, hydroxy isopropyl-, allyl, crotyl, dimethylallyl, isopentenyl, geranyl, farnesyl, geranylgeranyl, 3-methyl-2-pentenyl, and 3- methyl-2-hexenyl-amine.

5 19. The method of claim 1, wherein R is a straight-chained alkyl group.

20. The method of claim 1 , wherein R is a branched-chained alkyl group.

10

21. The method of claim 19, wherein R is saturated.

22. The method of claim 20, wherein R is unsaturated.

15

23. The method of claim 1, wherein Rl is a straight- or branched chain alkyl or alkenyl group containing from one to ten carbon atoms.

20 24. The method of claim 1, wherein the Rl alkyl group or the Rl alkenyl group is selected from the group consisting of propyl, butyl, and amyl.

25. The method of claim 1, wherein Rl is a straight- or branched chain alkyl or alkenyl group of a naturally-occurring dicarboxylic acid.

25

26. The method of claim 1, wherein the Rl alkyl group or the Rl alkenyl group is selected from the group consisting of propyl, butyl, and amyl.

27. The method of claim 1 , wherein the monocarboxylic acid and the dicarboxylic acid are 30 selected from the group of naturally-occurring amino acids.

28. The method of claim 1, wherein the dicarboxylic acid is glutamatic acid or aspartic acid.

29. The method of claim 1 , wherein the alkylamine precursor is selected from the group

35 consisting of theanine, N⁵- isopropylglutamine, N4-ethyl asparagine, and N⁵-secbutylglutamine.

30. A method for activating a ╬│╬┤ T cell receptor comprising: contacting the receptor with an alkylamine under conditions to permit alkylamine- mediated activation of the receptor, wherein the alkylamine is a compound of formula I:

R-NH2 (formula I), 5 or a compound of formula III:

R-NR2R3 (formula III) wherein R is a straight- or branched chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive, wherein R2 is a straight- or branched-chain alkyl or alkenyl group containing from one 10 to six carbon atoms, inclusive, that may be the same or different from R, and wherein R3 is a hydrogen or a straight- or branched-chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive, that may be the same or different from R or R2.

15 31. The method of claim 30, wherein contacting the receptor with the alkylamine agent is performed ex vivo.

32. The method of claim 30, wherein contacting the receptor with the alkylamine agent is performed in vivo.

20

33. The method of claim 30, wherein R is a straight-chained alkyl group.

34. The method of claim 30, wherein R is a branched-chained alkyl group.

25 35. The method of claim 33, wherein R is saturated.

36. The method of claim 34, wherein R is unsaturated.

37. The method of claim 30, wherein the alkylamine is selected from the group consisting of 30 ethyl-, n-propyl-, -iso-prolyl-, n-butyl, iso-butyl-, sec-butyl-, and iso-amylamines.

38. A composition comprising : an alkylamine of formula I

R-NH2 (formula I), or a compound of formula III:

R-NR2R3 (formula III) 5 wherein R is a straight- or branched chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive, wherein R2 is a straight- or branched-chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive, that may be the same or different from R, wherein R3 is a hydrogen or a straight- or branched-chain alkyl or alkenyl group 10 containing from one to six carbon atoms, inclusive, that may be the same or different from R or R2; and . a pharmaceutically acceptable carrier.

39. The composition of claim 38, wherein at least one of R and R2 is a straight-chained 15 alkyl group.

40. The composition of claim 38, wherein at least one of R and R2 is a branched-chained alkyl group.

20 41. The composition of claim 38, wherein at least one of R and R2 is saturated.

42. The composition of claim 38, wherein at least one of R and R2 is unsaturated.

43. The composition of claim 38, wherein the alkylamine is selected from the group

25 consisting of ethyl-, n-propyl-, -iso-prolyl-, n-butyl, iso-butyl-, sec-butyl-, and iso-amylamines.

44. The composition of claim 38, wherein the composition is formulated as an oral formulation.

30 45. The composition of claim 38, wherein the composition is formulated as an oral formulation for administration to a gingival tissue.

46. The composition of claim 38, wherein the composition is formulated as a food additive.

47. The composition of claim 38, wherein the composition is formulated for delivery to the female reproductive tract

5

48. The composition of claim 38, wherein the composition is formulated for vaginal delivery.

49. A compound of formula II : 10 O

Rl-C-NH-R (formula II),

wherein R is a straight- or branched chain alkyl group containing from one to six carbon 15 atoms, and wherein Rl is a straight- or branched chain alkyl group containing from one to twenty carbon atoms.

50. The compound of claim 49, wherein R is a straight-chained alkyl group. 20

51. The compound of claim 49, wherein R is a branched-chained alkyl group.

52. The compound of claim 50, wherein R is saturated.

25 53. The compound of claim 51 , wherein R is unsaturated.

54. The compound of claim 49, wherein Rl is a straight- or branched chain alkyl or alkenyl group containing from one to ten carbon atoms.

30 55. The compound of claim 49, wherein the Rl alkyl group or the Rl alkenyl group is selected from the group consisting of propyl, butyl, and amyl.

56. The compound of claim 49, wherein Rl is a straight- or branched chain alkyl or alkenyl group of a naturally-occurring dicarboxylic acid.

57. The compound of claim 49, wherein the alkylamine precursor is selected from the group consisting of theanine, N⁵- isopropylglutamine, N4-ethyl asparagine, and N⁵-secbutylglutamine. 5

58. The compound of claim 49, wherein the monocarboxylic acid and the dicarboxylic acid are selected from the group of amino acids.

59. The compound of claim 49, wherein the dicarboxylic acid is glutamatic acid or aspartic acid.

10

60. A composition comprising: any of the compounds of claims 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59, and a pharmaceutically acceptable carrier.

15 61. The composition of claim 60, wherein the composition is formulated as an oral formulation.

62. The composition of claim 60, wherein the composition is formulated as an oral formulation for administration to a gingival tissue.

20

63. The composition of claim 60, wherein the composition is formulated as a food additive.

64. The composition of claim 60, wherein the composition is formulated as a food additive for delivery to the female reproductive track.

25

65. The compound of claim 49, wherein the compound is not theanine.

66. The compound of claim 49, wherein the compound is isolated from contaminants that render it unsuitable for therapeutic applications.

30

67. A method for stimulating the activation of V╬│2V╬┤2 T cells comprising: contacting the cells with a V╬│2V╬┤2 T cell activation stimulating amount of an alkylamine agent selected from the group consisting of an alkylamine of formula I:

R-NH2 (formula I), an alkylamine of formula III: R-NR2R3 (formula III) 5 and an alkylamine precursor of formula II, O

Rl-C-NH-R (formula II),

10 wherein R is a straight- or branched chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive, wherein Rl is a straight- or branched chain alkyl or alkenyl group containing from one to twenty carbon atoms, inclusive, wherein R2 is a straight- or branched-chain alkyl or alkenyl group containing from one 15 to six carbon atoms, inclusive, that may be the same or different from R, and wherein R3 is hydrogen or a straight- or branched-chain alkyl or alkenyl group containing from one to six carbon atoms, inclusive, that may be the same or different from R or R2.

20 68. The method of claim 67, wherein stimulating the activation comprises stimulating cytokine production by V╬│2V╬┤2 T cells.

69. The method of claim 68, wherein the cytokines are selected from the group consisting of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), tumor necrosis factor-beta (TNF-beta,

25 Lymphotoxin), interleukin-4 (IL-4), IL-5, IL-10, and TNF-╬▒.

70. The method of claim 67, wherein stimulating the activation comprises stimulating proliferation of V╬│2V╬┤2 T cells.

30 71. The method of claim 67, wherein stimulating the activation comprises stimulating cytotoxic activity by V╬│2V╬┤2 T cells.

72. The method of claim 67, wherein stimulating the activation comprises stimulating blocking the activity of V╬│2V╬┤2 T cells.

73. The method of claim 67, wherein stimulating the activation comprises stimulating the V╬│2V╬┤2 T cells to apoptose and die.

74. The method of claim 67, wherein contacting the cells with the alkylamine agent is 5 performed ex vivo.

75. The method of claim 67, wherein contacting the cells with the alkylamine agent is performed in vivo.

10 76. The method of claim 74, wherein the V╬│2V╬┤2 T cell activation stimulating amount of the alkylamine agent is from about 0.01 mM to about 100 mM.

77. The method of claim 74, wherein the V╬│2V╬┤2 T cell activation stimulating amount of the alkylamine agent is from about 0.1 mM to about 0.1 mM to about 20 mM.

15

78. The method of claim 74, wherein the V╬│2V╬┤2 T cell activation stimulating amount of the alkylamine agent is from about 1 mM to about 10 mM.

79. The method of claim 75, wherein the V╬│2V╬┤2 T cell activation stimulating amount of 20 the alkylamine agent is from about 5 mg/kg to about 100 mg/kg.

80. The method of claim 75, wherein the V╬│2V╬┤2 T cell activation stimulating amount of the alkylamine agent is from about 10 mg/kg to about 50 mg/kg.

25 81. The method of claim 75, wherein the V╬│2V╬┤2 T cell proliferation stimulating amount of the alkylamine agent is from about 20 mg/kg to about 30 mg/kg.

82. The method of claim 67, wherein the alkylamine agent is an alkylamine.

30 83. The method of claim 67, wherein the alkylamine agent is an alkylamine precursor.

84. The method of claim 67, wherein R is a straight-chained alkyl group.

85. The method of claim 67, wherein R is a branched-chained alkyl group.

86. The method of claim 84, wherein R is saturated.

5 87. The method of claim 85, wherein R is unsaturated.

88. The method of claim 67, wherein the alkylamine is selected from the group consisting of methyl-, ethyl-, n-propyl-, -iso-propyl-, n-butyl, iso-butyl-, sec-butyl-, iso-amyl-, hydroxymethyl -, ╬▓-hydroxyethyl-, ╬┤-hydroxy propyl-, hydroxy isopropyl-, allyl, crotyl,

10 dimethylallyl, isopentenyl, geranyl, farnesyl, geranylgeranyl, 3-methyl-2-pentenyl, and 3- methyl-2-hexenyl-amine.

89. The method of claim 67, wherein R is a straight-chained alkyl group.

15 90. The method of claim 67, wherein R is a branched-chained alkyl group.

91. The method of claim 89, wherein R is saturated.

92. The method of claim 90, wherein R is unsaturated.

20

93. The method of claim 67, wherein Rl is a straight- or branched chain alkyl or alkenyl group containing from one to ten carbon atoms.

94. The method of claim 67, wherein the Rl alkyl group or the Rl alkenyl group is selected 25 from the group consisting of propyl, butyl, and amyl.

95. The method of claim 67, wherein Rl is a straight- or branched chain alkyl or alkenyl group of a naturally-occurring dicarboxylic acid.

30 96. The method of claim 67, wherein the Rl alkyl group or the Rl alkenyl group is selected from the group consisting of propyl, butyl, and amyl.

97. The method of claim 67, wherein the monocarboxylic acid and the dicarboxylic acid are selected from the group of naturally-occurring amino acids.

98. The method of claim 67, wherein the dicarboxylic acid is glutamatic acid or aspartic acid.

99. The method of claim 67, wherein the alkylamine precursor is selected from the group consisting of theanine, N⁵- isopropylglutamine, N4-ethyl asparagine, and N⁵-secbutylglutamine.