JP2002519007A - Alkylamines and their precursors as specific modulators of human gamma-delta T cell function - Google Patents

Abstract

  (57) [Summary] Methods and compositions for inducing proliferation of Vγ2Vδ2 T cells are provided. The composition contains an alkylamine agent such as an alkylamine and an alkylamine precursor. This agent is also useful for the γδ T cell receptor.

Description

DETAILED DESCRIPTION OF THE INVENTION

(Government Assistance) The work described herein was partially funded by the National Institutes of Health with grant number A.
Assisted by I01330-3 and CA / AI47724-11. The government may have certain rights in these inventions.

FIELD OF THE INVENTION [0002] The present invention relates to methods and compositions for stimulating a γδ T cell-mediated immune response against tumors and infectious diseases such as bacterial and viral infections. Also,
Such γδ stimulation can also be used to reduce autoimmunity or enhance immune tolerance to allergens. These methods involve administering an alkylamine or an alkylamine precursor to a subject or cells isolated from the subject to obtain V
stimulating activation, proliferation, cytokine release, cytotoxic activity or apoptosis of γ2Vδ2 T cells.

BACKGROUND OF THE INVENTION T cells are thymus-derived cells that mediate a cellular immune response in the immune system. T
Lymphocytes (T cells) include two subgroups: αβ T cells and γδ T cells. αβ T cells have αβ receptors that recognize antigenic peptides that bind to major histocompatibility complex (MHC) I or II molecules;
It accounts for 98%. γδ T cells have γδ T receptors; they make up 3-5% of T cells.

[0004] γδ T cells play an important role in defense against bacterial and viral infections and in autoimmunity. γδ T cells are present in humans with infectious diseases (eg, tuberculosis, salmonellosis, brucellosis, ehlichiosis, hare, malaria, leishmaniasis, mononucleosis), and in HIV (early stage). Multiply. These cells grow in the synovium of patients with chronic rheumatoid arthritis, CSF and CNS plaques with multiple sclerosis, and lungs of patients with sarcoidosis. The predominant subset of γδ T cells in human peripheral blood (referred to as Vγ2Vδ2 T cells), in contrast to αβ T cells that recognize peptide antigens associated with MHC molecules, is characterized by professional antigen presenting cells (AP
Recognizes unprocessed non-peptide phosphate antigen in the absence of C). In contrast to the abundant information available on the nature of the interaction between αβ T cell receptors and MHC-binding peptide antigens, information on the nature of the interaction between γδ T cell receptors and their ligands is available. rare.

[0005] Antigens responsible for the proliferation of γδ T cells have been most extensively studied for mycobacteria, where they were identified as prenyl pyrophosphates.
Such a natural antigen is iso-pentenyl pyrophosphate secreted by mycobacteria. TUBags 1 and 2 are another group of closely related antigen molecules isolated from the cytoplasm of mycobacteria. These are also
Pyrophosphate-containing molecules whose complete structure has not been determined.

[0006] Other less powerful natural antigens include 2,3-diphosphoglycerate, glycerol-3-phosphate, ribose-1-phosphate, and xylose-1-.
Phosphates. In addition to these natural antigens, there are several alkyl and alkenyl phosphate analogs and alkyl and alkenyl pyrophosphate analogs, which have been synthesized and reported to be recognized by Vγ2Vδ2 T cells. (See, for example, US Pat. No. 5,639,653, issued to Bloom et al. For a complete description of these phosphate or pyrophosphate containing compounds.) Γδ T cells protect against a variety of bacterial and viral diseases. And is involved in the regulation of autoimmunity and immune tolerance, thus selectively stimulating T cells of this subpopulation to enhance the immune response to these diseases and conditions, and providing the basis for a γδ T cell-mediated immune response. There is a need to better understand the molecular processes that make up. In view of the above, improved drug therapies to replace or augment existing methods for stimulating Vγ2Vδ2 T cell-mediated immune responses have been developed, preferably for bacterial or viral infections and their earliest stages. There is also a need to develop new drugs that reduce the autoimmune status of and increase immune tolerance.

SUMMARY OF THE INVENTION [0007] The present invention relates to alkylamines (which include sepsis, chorioamnionitis, preterm labor, enteritis,
Discovery that it is a major product of certain bacteria associated with gingivitis, and also found in plant foods and human body fluids) causes Vγ2Vδ2 T cell proliferation in a T cell receptor (TCR) -specific manner. based on. Alkylamine antigens are the first phosphate-free antigens described for Vγ2Vδ2 T cells and are therefore a different chemical class of ligand for Vγ2Vδ2 T cells. All antigens described to date for Vγ2Vδ2 T cells are characterized by a critical phosphate or pyrophosphate moiety, and the finding that alkylamines stimulate γδ T cell proliferation is surprising and unexpected. . Thus, contrary to expectation, the examples show that the same Vγ2Vδ2 T cell receptor that recognizes a previously characterized negatively charged alkyl phosphate compound,
Provides evidence that it can be similarly activated by positively charged alkylamine molecules. Based on these findings, Applicants describe herein a function newly found for alkylamines, namely the ability to stimulate the activation and / or proliferation of Vγ2Vδ2 T cells. Accordingly, the present invention relates to compositions of alkylamines and novel alkylamine precursors, and methods based on the discovery of this newly discovered function.

[0008] The methods of the invention comprise administering an alkylamine agent to a subject or cells isolated from the subject to stimulate activation and / or proliferation of Vγ2Vδ2 T cells. Activation of Vγ2Vδ2 T cells is a process mediated by the γδ T cell receptor. Generally, activation of the T cell receptor results from binding of the receptor to each of these antigens. Without intending to limit the invention to any particular theory and mechanism, the evidence provided by the examples indicates that activation of the γδ T cell receptor is also initiated by the binding of the alkylamines of the invention to the γδ T cell receptor. Suggest that you get.

[0009] Depending on the intracellular and extracellular environment, activation of Vγ2Vδ2 T cells can be numerous.
Cell changes can be initiated, including the expansion of Vγ2Vδ2T cells, Vγ2Vδ2T
Stimulation or inhibition of cytokine production by cells, cells by Vγ2Vδ2 T cells
Stimulation or inhibition of toxic activity and V to induce apoptosis and death
Stimulation of γ2Vδ2 T cells includes, but is not limited to. Therefore,
Ming also describes a method for activation of Vγ2Vδ2 T cells in these particular aspects.
Methods and compositions. Each of these activities, for example, is associated with α / β T cells.
Well-documented and standard procedures (eg, measuring cytokine release)
ELISA assays, apoptosis assays).
You. For example, following activation by an antigen, CD4 T lymphocytes become a type 1 helper.
T (T_H1) Cells and type 2 helper T (T_H2) causes cell-like classification
Produces one of two different cytokine profiles. By these cells
The different cytokines produced produce differences in immune function. T_HOne cell
Mainly interferon-γ (IFN-γ) as well as interleukins
-2 (IL-2) and tumor necrosis factor-β (TNF-β, lymphotoxin)
It excretes and induces cellular immunity. T_H2 cells are mainly interleukins
Secretes not only IL-4 (IL-4), but also IL-5 and IL-10, and
Antibodies mediated by plasma cells while down-regulating sexual immunity
Plays a major role in eliciting a response. The cytokine TNF-α is T_H1 and T_H
Two immune responses can be produced in both_HIn one response
High, this cytokine has a cytolytic effect that contributes to the effectiveness of cellular immunity.
And is well known. T_H1 and T_HFirst studies on two lymphocyte subsets
Most were performed using mouse helper T cell clones, but today
CD4 T cells secrete a similar cytokine profile, and these _H 1 and T_HIt has been recognized that they can be classified into two subsets (Powellie
, F. Et al., 1993, Immunol. Today 14: 270; Romag
nani, S .; Et al., 1992, Int. Arch. Allergy Immun
ol. 4: 279; Romagnani, S .; Et al., 1991, Immunolo.
. Today 8: 256). Intends to limit the invention to any particular theory or mechanism
However, given the findings disclosed herein, the alkylamines of the present invention
Is Vγ2Vδ2T in a manner similar to that described in the literature for α / β T cells.
It is believed that cytokine production by cells can be stimulated. Accordingly, the present invention
The ability of killamine to induce Vγ2Vδ2 T cell activation is well established
Determined using routine experimentation and standard procedures for measuring cell properties
obtain.

[0010] According to one aspect of the present invention, there is provided a method for stimulating activation of Vγ2Vδ2 T cells. Methods of stimulating activation include contacting these cells with an alkylamine agent in an amount that stimulates Vγ2Vδ2 T cell activation in vivo or ex vivo to stimulate activation of Vγ2Vδ2 T cells. As used herein, “contacting a cell with an alkylamine agent” means that the alkylamine is Vγ
Meaning that the cells are placed close enough to the alkylamine agent for a time and under conditions sufficient to stimulate the activation of 2Vδ2T cells. Activation is assessed using conventional procedures. Contacting can be performed in vivo or ex vivo.

[0011] According to another aspect of the present invention, there is provided a method for stimulating the proliferation of Vγ2Vδ2 T cells. This method involves contacting these cells with an alkylamine agent in an amount that stimulates Vγ2Vδ2 T cell proliferation in vivo or ex vivo to produce Vγ2Vδ2 cells.
Stimulating T cell proliferation. A growth stimulating amount of an alkylamine agent can be the same as an active stimulating amount of the agent; such an amount can be such that the amount is proliferated (eg, by counting cells) or activated (eg, cytokine production by the cells). To determine whether it elicits) by performing conventional tests.

As used herein, “contacting a cell with an alkylamine agent” refers to
Alkylamine stimulates the proliferation of Vγ2Vδ2 T cells and / or
This means placing the cells sufficiently close to the alkylamine agent for a time and under conditions sufficient to activate the T cell receptor. Contacting can be performed in vivo or ex vivo.

As used herein, “stimulating the proliferation of Vγ2Vδ2 T cells” refers to a detectable, statistically significant amount of a phenotype in a subject or cell that is contacted with an alkylamine agent. It means increasing the number of Vγ2Vδ2 T cells by an amount that leads to a change. By stimulating the proliferation of Vγ2Vδ2 T cells, an enhanced immune response is likely to be generated against microorganisms and tumor cells that express the antigen recognized by Vγ2Vδ2 T cells. In an autoimmune condition in which Vγ2Vδ2 T cells can limit tissue damage, thereby effectively treating the disease or condition, increased proliferation of such Vγ2Vδ2 T cells is desirable. For ease of discussion, such diseases or conditions are referred to collectively as "disease" or "condition" throughout this document. Exemplary diseases that can be treated (preventable, inhibit progression, or alleviate symptoms) that can be treated by stimulating proliferation of Vγ2Vδ2 T cells include infectious diseases (eg, bacterial or viral infections, eg, Bacteria causing gingivitis, Escherichia coli infection, Listeria infection, tuberculosis, salmonellosis, Plasmodium infection, bacteriodes infection, Porphyromonas (p
or phyromonas infection, Klebsiella infection, Erzinia infection, Clostridium infection, Brucellosis, Ehrlichiosis, Tularemia, Malaria, Leishmaniasis, mononucleosis, Epstein-Barr virus infection (EBV), HIV infection, and herpes simplex infection) ). 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, virus Hepatitis, and Crohn's disease. While not intending to limit the invention to any particular mechanism of action, Vγ2Vδ2 T cells
Autoreactive α / β T cells known to cause disease by responding to self antigens or allergens can act by down-regulation. Thus, alkylamine recognition by Vγ2Vδ2 T cells can lead to greater down regulation of α / β T cells and less tissue damage or allergic reactions.

[0014] In in vivo applications, the alkylamine agent administered to the subject is an isolated alkylamine agent ("alkylamine of formula I or III") in an amount effective to stimulate the growth of Vγ2Vδ2 T cells in vivo. Or "alkylamine precursor of formula II"). As used herein, a subject refers to a mammal, preferably a primate, more preferably a human. It is noted that preferred subjects treated according to the above method have no other symptoms requiring alkylamine treatment, either by administration of the alkylamine or by administration of the alkylamine precursor. . Preferably, the alkylamine agent is administered to the subject in combination with other methods (eg, antibiotic therapy) to treat the condition, such as an infectious condition. Thus, the compositions and methods of the present invention improve the efficacy of existing therapies to replace existing drug therapies and to treat conditions characterized by an inappropriate number or function of Vγ2Vδ2 T cells. Useful for. Generally, such conditions are identified as infectious diseases as identified herein or by assessing the number of Vγ2Vδ2 T cells in peripheral blood mononuclear cells (PBMCs) using standard procedures. Or associated with an autoimmune condition. For example, γδ T cells are
Increased in certain autoimmune diseases (eg, in the synovium of patients with chronic rheumatoid arthritis, CSF and CNS plaques with multiple sclerosis, and lungs of patients with sarcoidosis).

[0015] In certain preferred embodiments, the alkylamine agent is delivered directly to the site of the infection. For example, treating bacterial or virus-mediated gingivitis may be a drug that is suitable for oral delivery of an alkylamine agent and retains it proximal to gingival tissue (eg,
Chewing gum, floss, gingival tissue implant). In this way, the composition can be targeted within the gum to a particular site, where it stimulates Vγ2Vδ2 T cell proliferation. According to yet another preferred embodiment, the alkylamine agent is delivered directly to the female reproductive tract to treat the progression of the opportunistic infection. For example, the alkylamine agents of the present invention can be used, for example, for vaginal delivery.
delivery). Optimally, the alkylamine agent is
It is delivered in combination with an anti-exogen agent such as an antibiotic or anti-viral agent to treat the infectious agent.

[0016] In accordance with yet another aspect of the present invention, there is provided a method for activating a γδ T cell receptor. The method involves contacting the receptor with an alkylamine of Formula I as defined below under conditions permitting alkylamine-mediated activation of the receptor. As used herein, γδ receptor is a term in the art that recognizes antigenic molecules and describes the T cell receptor present on γδ T cells. Thus, the methods of the invention may be used ex vivo (eg, for γδT expressed in isolated cells).
Cell receptor) or in vivo.

[0017] An alkylamine agent useful for practicing the claimed invention is an alkylamine or an alkylamine precursor. Preferably, the alkylamine agent is in an isolated form (ie, substantially free of contaminants which prevent pharmaceutical use).
An alkylamine or alkylamine precursor of the invention is used to treat an infectious disease, allergy, or autoimmune condition that can be treated by increasing the proliferation of Vγ2Vδ2 T cells, or to activate the γδ T cell receptor. No precedent to use has been proposed.

As used herein, “alkylamine” refers to a compound of Formula I or Formula III. Preferred alkylamines are compounds of formula I: R-NH2 (formula I), wherein R is a straight or branched chain alkyl or alkenyl containing 1 to 6 carbon atoms inclusive. Group. Preferred alkylamines are ethyl-, n
-Propyl, iso-propyl-, n-butyl, iso-butyl-, sec-butyl-, and iso-amylamine.

R optionally includes a substituted functional group (eg, hydroxy, amino, and carboxy). A primary amine is shown in Formula I, but a secondary amine of Formula III
Tertiary amines and alkylamine precursors of Formula II, which upon hydrolysis form primary and secondary amines, are also included within the scope of the invention.

There are a number of other amine molecules that are also found as components of natural bacterial products or vegetable foods and human bodily fluids, which are slightly structurally related to the alkylamine antigens described above. The major of these is putrescine (1,4-butanediamine), which is n-butylamine with an additional amine group on the fourth carbon. Putrescine is essential for the growth and differentiation of mammalian cells. Putrescine is a precursor of GABA and a major neuroinhibitor of the vertebrate CNS. Putrescine causes gastrointestinal mucosal cell proliferation in vitro and in vivo and causes certain bacterial species (Listeria, Clostridia, Bacteroides,
Escherichia, Salmonella, Shigella, and P
roteus). This is Vγ2Vδ because putrescine has four carbon chains and only the addition of an amine group differs from the antigen molecule n-butylamine.
There is a high possibility that it will be recognized by 2T cells. The higher polyamine spermidine is the addition product of the n-propylamine moiety to putrescine derived from the decarboxylation of S-adenosylmethionine. It is important in cell growth and is found in bacteria. Therefore, spermidine is also tested as a potential antigen against Vγ2Vδ2 T cells.

In accordance with yet another aspect of the present invention, there is provided a novel alkylamine precursor. As used herein, an "alkylamine precursor" is a compound of formula II

[0022]

Embedded image Wherein R is a linear or branched alkyl or alkenyl group containing 1 to 6 carbon atoms (inclusive), wherein R 1 is 1 to 20 carbon atoms Straight or branched chain alkyl or alkenyl group (including both ends). Thus, R may include 1, 2, 3, 4, 5, or 6 carbon atoms and R
1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
It may contain 15, 16, 17, 18, 19, or 20 carbon atoms. Preferably, R1 contains 1 to 10 carbon atoms (inclusive). The R and R1 groups can independently be linear or branched and saturated or unsaturated. R and R1 optionally and independently include substituted functional groups, including, for example, hydroxy, amino, and carboxy. Preferred alkylamine precursor, theanine, N ⁵ - isopropyl glutamine, N ⁴ - ethyl asparagine, and N ⁵ -s
ec-butylglutamine. Upon cleavage (eg, in vivo), the precursor of Formula II forms a mild acid (R1COO ⁽⁻⁾ ) and an alkylamine of Formula I or III.

As used for R 1, an “alkyl or alkenyl group” is 1-2
Represents an alkyl or alkenyl chain containing 0 carbon atoms (inclusive). Preferably, the alkyl or alkenyl group is present on a natural mono- or dicarboxylic acid. Exemplary monocarboxylic acids include those where the alkyl group is propyl, butyl, and amyl. Exemplary dicarboxylic acids include amino acids, especially glutamic or aspartic acid. Preferred alkylamine precursor, theanine, N ^{5-iso-propyl-glutamine,} is selected from the group consisting of N4- ethyl asparagine, and N ^{5-sec-butyl-glutamine.}

According to another aspect of the present invention, there is provided a novel composition for use according to the method of the present invention. The novel pharmaceutical composition comprises an alkylamine of the formula I or III and / or
Alternatively, an alkylamine precursor of Formula II is included 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 composition is formulated as an oral formulation, more preferably, for administration to gingival tissue (eg, chewing gum, toothpaste additive, gingival tissue implant). Alternatively, the novel compositions of the present invention may be used to reduce the likelihood of infection of food transmission and / or to increase immune resistance to infectious diseases or tumors by stimulating the proliferation of Vγ2Vδ2 T cells. It can be formulated as a food additive or vitamin-type dietary supplement. In addition, the novel compositions of the present invention can be formulated for delivery to the female reproductive tract, for example, to treat the progression of opportunistic infections. For example, an alkylamine agent of the invention can be formulated for vaginal delivery (eg, jelly, tampon, etc.).

According to yet another aspect of the present invention, there is provided a method for making a medicament.
The method comprises the steps of disposing 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, intramuscularly, intranasally or as an inhalant, the nature of the pharmaceutically acceptable carrier being appropriately varied according to the mode of administration. Is done.

In accordance with yet another aspect of the present invention, there is provided a method for making a food additive or vitamin-type dietary supplement. The method comprises the steps of disposing an alkylamine of formula I or III and / or an alkylamine precursor of formula II in a pharmaceutically acceptable carrier, and the composition as a food additive or vitamin-type dietary supplement. Prescribing. The alkylamine of Formula I or III and the alkylamine precursor of Formula II are as defined herein.

[0027] The present invention is also directed to determining the role that Vγ2Vδ2 T cell proliferation plays in the fight against infectious diseases and to determine the role these molecules play in activating the γδ T cell receptor. In a model system, the use of an alkylamine of the formula I or III and / or an alkylamine precursor of the formula II is contemplated. Accordingly, the present invention also relates to novel screening assays (eg, by testing combinatorial libraries) to identify molecules that interfere with the functional activity of the alkylamine agents of the present invention.

[0028] These and other aspects of the invention are described in further 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 otherwise defined.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the discovery that alkylamines cause the proliferation of Vγ2Vδ2 T cells in a T cell receptor (TCR) -specific manner. The alkylamine antigen is the first phosphate-free antigen described for Vγ2Vδ2 T cells,
Thus, it represents a distinct species of ligand for Vγ2Vδ2 T cells.
The finding that alkylamines stimulate γδ T cell proliferation is surprising and unexpected since all antigens previously described for Vγ2Vδ2 T cells are characterized by important phosphate or pyrophosphate moieties. Things.

As used herein, Vγ2Vδ2 T cells refer to thymus-derived cells in the immune system that mediate the cellular immune response and regulate the immune response and are expressed on the surface of these cells Γδ T cell antigen receptor. These specific γδ T cell antigen receptors (TCRs) are composed of rearranged (TCR) γ and (TCR) δ chains, which comprise the germline modified (V) domain gene segment Vγ2 (Vγ9 And the modified domains encoded by each of Vδ2. These cells can include specific T cell clones, T cell lines, T cell hybridomas, and the natural population of T cells that arise from or are present in mammals.

As noted above, depending on the intracellular or extracellular environment, activation of Vγ2Vδ2T cells can initiate a number of cellular changes, including but not limited to: Vγ2Vδ2T cell proliferation, Stimulation or inhibition (eg, blockade) of cytokine production by Vγ2Vδ2T cells, stimulation or inhibition of cytotoxic activity by Vγ2Vδ2T cells, and stimulation of Vγ2Vδ2T cells that undergo apoptosis and killing. Accordingly, the present invention also encompasses methods and compositions relating to these particular aspects of activating Vγ2Vδ2 T cells. Each of these activities is, for example, α / βT
In cells, they have been well documented and can be evaluated using standard procedures (eg, ELISA assays to measure cytokine release, apoptosis assays). For example, the following activation by the antigen, CD4 T lymphocytes, results in their classification as T helper 1 (T _H 1) and T helper 2 (T _H 2) cells, one of two different cytokine profiles. To produce
The different cytokines produced by these cells have resulted in differences in immune function (ie, up-regulation or down-regulation of the immune response).

According to one particular aspect of the present invention, there is provided a method for stimulating the activation of Vγ2Vδ2 T cells. The method for stimulating activation involves contacting the cell with an amount of an alkylamine agent that stimulates Vγ2Vδ2 T cell activation in vivo or ex vivo to stimulate activation of the Vγ2Vδ2 T cell. As used herein, the step of "contacting a cell with an alkylamine agent" refers to an alkylamine for a period of time under conditions sufficient for the alkylamine to stimulate the activation of Vγ2Vδ2 T cells. It refers to the step of placing cells sufficiently proximal to the agent. Activation is assessed using conventional procedures. The contacting step can be performed in vivo or ex vivo.

According to another aspect of the present invention, there is provided a method for stimulating the proliferation of Vγ2Vδ2 T cells. The method includes contacting the cells with a Vγ2Vδ2 T cell growth stimulating amount of an alkylamine agent in vivo or ex vivo to stimulate the growth of Vγ2Vδ2 T cells. As used herein, “Vγ2Vδ2T
The “cell proliferation stimulating amount” refers to an amount effective to induce the proliferation of Vγ2Vδ2 T cells. Such proliferation can be detected using standard procedures to measure cell numbers before and after treatment with the alkylamine agent. A statistically significant increase in cell number after treatment with an alkylamine agent is the amount of growth stimulation of Vγ2Vδ2 T cells. Preferably, for ex vivo applications, this amount will be 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, 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.

The step of contacting the Vγ2Vδ2 T cells with the alkylamine agent of the present invention can be performed in vivo or ex vivo. Thus, the contacting step refers to exposing the Vγ2Vδ2 T cells to the alkylamine agent for a sufficient time and under conditions that allow the alkylamine agent to stimulate the growth of Vγ2Vδ2 T cells. The contacting can be performed by administering a Vγ2Vδ2 T cell proliferation inducing amount of the alkylamine agent to a subject in need of such treatment. Alternatively, a biological fluid, preferably containing Vγ2Vδ2 T cells, can be removed from a subject, treated with an alkylamine of the invention, and re-infused into the subject by standard methods. Such biological fluids include, but are not limited to, serum, cerebrospinal fluid, and synovial fluid. The preferred fluid is blood.

[0035] In one aspect the present invention relates to a method for treating a subject diagnosed as having a condition associated with an antigen recognized by Vγ2Vδ2 T cells. V
It is thought that by stimulating the proliferation of γ2Vδ2 T cells, an increased immune response is generated for these conditions. The method of treatment comprises 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 activate γδ T cell receptors. The particular mode of administration will be the treated Vγ2V targeted by the alkylamine agent.
Depends on the nature of the state and location of the δ2T cells. Exemplary conditions associated with antigens recognized by Vγ2Vδ2 T cells are known to those of skill in the art, and include, but are not limited to, the following diseases: tuberculosis, salmonellosis, brucellosis, Infectious diseases such as ehlichiosis, tularemia, malaria, leishmaniasis, mononucleosis and HIV. Vγ2
Additional conditions in which increased numbers of Vδ2 T cells may be desirable include rheumatoid arthritis,
Autoimmune diseases such as multiple sclerosis and sarcoidosis can be mentioned.

[0036] The alkylamine agents of the present invention are particularly useful for treating gingivitis associated with an antigen recognized by Vγ2Vδ2 T cells. Vγ2Vδ2 against defined antigen
Most studies testing T cell reactivity use peripheral blood mononuclear cells (PBMC) as the source of γδ T cells and phosphate antigens isolated from mycobacteria as stimulants. While not intending to limit the invention to any particular theory or mechanism, we believe that γδ T cells found in other anatomical compartments such as gingiva (
Here, 30% of the isolated leukocytes are TCRγδ +), which is more likely to encounter a different set of pathogens. γδ T cells represent up to 60% of T cells in female reproductive tissues (eg, cervix and decidua). For example, recent reports indicate that γδ T cells are activated in the gingiva of patients suffering from chronic gingivitis, which is associated with Bacteroides forsythus and Porph.
anaerobic bacteria in the mouth of periodontal disease such as
iodotopatic mousanaerobic vector
ia). Such bacteria are also opportunistic pathogens of diseases such as pelvic inflammatory disease, chorioamnionitis, postpartum endometritis, preterm labor, and postoperative infection in the female reproductive tract. The experimental results show that human γδ T cells
Major bacterial main products, including cteroides species (produced in millimolar concentrations)
It is shown that the alkylamine is recognized. Thus, it is believed that alkylamines can be used to treat gingivitis for infections associated with antigens recognized by gingival γδ T cells, as well as infections of the female reproductive tract. For embodiments targeting gingival tissue, preferred compositions of the invention are formulated for topical delivery to the tissue (eg, chewing gum, floss, which may be impregnated or otherwise impregnated with an alkylamine agent of the invention). Gingival tissue implant). Other variations of the formulation depend on the nature of the target tissue source of γδ T cells to which the treatment is directed.

[0037] The alkylamine agent of the present invention is administered in an effective amount. An effective amount is a dose of the alkylamine agent that is sufficient to provide a medically desired 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 current treatment, if any, the particular route of administration. , As well as any factors known and the professional opinion of the physician. For example, in the context of a diagnosed bacterial or viral infection, an effective amount can refer to a subject (in vivo,
Or the amount that slows or inhibits the extent of bacterial or viral infection measured according to standard practices to determine bacterial or viral load in cell culture. Similarly, an amount effective to treat gingivitis refers to an amount that slows, inhibits, or halts the progression of gingivitis. Thus, it is understood that the alkylamines of the present invention can be used to prophylactically treat the above symptoms in subjects at risk of developing the above symptoms. As used in the claims, "inhibition" includes all of the above. It is generally preferred that the largest dose be used (ie, the safest dose according to sonic medical judgment).

As used herein, a subject is any mammal that may be susceptible to a condition associated with an infection or an autoimmune condition, where an antigen recognized by Vγ2Vδ2 T cells is involved. Refers to an animal (preferably a primate, more preferably a human), except that the mammal is free of conditions requiring alkylamine treatment. Preferred subjects do not include a condition that requires treatment with an alkylamine for some reason.

An alkylamine agent as used herein refers to an alkylamine of Formula I or III and an alkylamine precursor of Formula II (described below).
. The alkylamine agent is capable of inducing stimulation of Vγ2Vδ2 T cells and / or activating γδ T cell receptors in vivo or ex vivo. Thus, the alkylamine agents of the present invention can reduce or prevent proliferative conditions (eg, the infectious diseases and autoimmune conditions described above) associated with antibodies recognized by Vγ2Vδ2 T cells.

As used herein, “alkylamine” refers to a compound of formula I or III
Of the compound. Preferred alkylamines are compounds of the following Formula I: R-NH2 (Formula I), wherein R is a straight or branched chain containing 1 to 6 (including 1 and 6) carbon atoms. A chain alkyl or alkenyl group. Exemplary alkylamines include: monoalkylamines (eg, methyl, ethyl, n-propyl, n-
Butyl, isopropyl) and alkenylamines (eg, allyl, crotyl,
Dimethylallyl, isopentenyl, geranyl, farnesyl, geranylgeranyl, 3-methyl-2-pentenyl, and 3-methyl-2-hexenyl). Alkyl and substituted alkyl groups containing up to 6 carbon atoms and that can be used in accordance with the present invention (eg, hydroxymethyl-, β-hydroxyethyl-, δ-
For examples of hydroxypropyl-, hydroxyisopropyl-), see Blood
See also U.S. Patent No. 5,639,653 issued to M et al. (e.g., columns 4-7). The compounds of Bloom are different from the compounds disclosed herein (where the former compounds require an absolute phosphate or pyrophosphate moiety, while the compounds of the present invention are negatively charged. With a positively charged amine moiety instead of a phosphate group). Preferred alkylamines are ethyl-, n-propyl-, -is
o-propyl-, n-butyl, iso-butyl-, sec-butyl-, and i
So-amylamine.

As used herein, an “alkylamine precursor” has the following formula II
Refers to the compound of:

[0042]

Embedded image Wherein R is a linear or branched alkyl or alkenyl group containing 1 to 6 carbon atoms, and R1 is 1 to 20 (including 1 and 20) carbon atoms And a linear or branched alkyl or alkenyl group. Thus, R can include 1, 2, 3, 4, 5, or 6 carbon atoms, and R1 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15
, 16, 17, 18, 19, or 20 carbon atoms. Preferably,
R1 contains 1 to 10 (including 1 and 10) carbon atoms. The R and R1 groups are independently linear or branched and may be saturated or unsaturated. R and R1 are optionally and independently, for example,
Includes substituted functional groups including hydroxy, amino, carboxy and methyl groups. Preferred alkylamine precursor, theanine, N ⁵ - isopropyl glutamine, N4
- ethyl asparagine, and N ^{5-sec-butyl-glutamine.}

While primary amines are shown in Formula I, it is understood that the present invention encompasses secondary or tertiary amines, ie, alkylamines that are compounds of Formula III: R-NR2R3 (Formula III Wherein R2 is a linear or branched alkyl or alkenyl group containing 1 to 6 (including 1 and 6) carbon atoms; wherein R3 is hydrogen or 1 to 6
A linear or branched alkyl or alkenyl group containing 6 (including 1 and 6) carbon atoms; and wherein R2 and R3 may be the same or different from each other , And may be the same as or different from R. The invention also includes an alkylamine precursor, wherein the precursor comprises
It can be cleaved to form a secondary or tertiary alkylamine of Formula III.

With reference to R 1 and as used herein, “alkyl or alkenyl group” refers to an alkyl or alkenyl chain containing from 1 to 20 (including 1 and 20) carbon atoms. Exemplary alkyl groups include propyl, butyl, and amyl. Exemplary dicarboxylic acids include amino acids, especially glutamic or aspartic acid. Preferred alkylamine precursor is selected from the group consisting of theanine, N ⁵ - iso - propyl glutamine, N4-ethyl asparagine, and N ^{5-sec-butyl-glutamine.}

As used herein, the term “isolated” refers to a compound that is substantially free of contaminants, which renders the compound unsuitable for therapeutic applications. When used therapeutically, the isolated alkylamines of the present invention are administered in a therapeutically effective amount. Generally, a therapeutically effective amount is the amount required to delay the onset of a particular condition being treated, inhibit the progress of the particular condition being treated, or completely stop the particular condition being treated. Means In general, a therapeutically effective amount will vary with the age, condition, and sex of the subject, and the nature and extent of the disease in the subject, all of which can be determined by one of skill in the art. The dose can be adjusted by the individual physician or veterinarian, especially if there are any complications. A therapeutically effective amount is typically in one or more dose administrations per day for one or more days.
0.01 mg / kg to about 1000 mg / kg, preferably about 0.1 mg / kg to
About 200 mg / kg, and most preferably from about 0.2 mg / kg to about 20 mg / kg.
Varies with kg.

A therapeutically effective amount of an isolated alkylamine agent will induce stimulation of Vγ2Vδ2 T cells and / or inhibit γδ T cell receptors, as determined, for example, by standard tests known in the art. An effective amount to activate. Vγ2
By increasing the number of Vδ2 T cells, the alkylamine agents of the present invention
It is believed to increase the immune response to diseases associated with drugs that are recognized by γ2Vδ2 T cells.

[0047] Optionally, the isolated alkylamine agent can be combined with other agents for treating disease, such as antibiotics for treating bacterial infections, antiviral agents for treating viral infections. The combination is administered to the subject. As used herein, antibiotics and antivirals are technical terms understood by those skilled in the art to refer to a wide range of therapeutic agents having their functional activity. For example, for a more detailed description of these therapeutic agents, see Harrisons, Princip.
les of Internal Medicine (McGraw Hill
, Inc. , New York).

[0048] The above-mentioned drug therapies are well known to those skilled in the art and are performed according to embodiments known to those skilled in the art. Pharmacotherapy is in combination with the isolated alkylamine of the present invention,
It is performed in an amount effective to achieve its physiological purpose (preventing or reducing the physiological consequences of an infection or autoimmune condition). Therefore, the physiologic consequences of the condition cannot be prevented or reduced when the pharmacotherapy is administered alone, but the physiologic consequences of the condition are prevented when administered in combination with the isolated alkylamine agent of the present invention. It is contemplated that drug therapy may be administered in an amount that can be reduced or.

An isolated alkylamine agent can be administered alone or as part of a pharmaceutical composition in combination with the above drug therapy. Such pharmaceutical compositions may include an isolated alkylamine agent in combination with any standard physiologically and / or pharmaceutically acceptable carriers known in the art. These compositions should be sterile and should contain a pharmaceutically effective amount of the isolated alkylamine in a suitable weight or volume for administration to the patient. The term "pharmaceutically acceptable carrier" as used herein refers to one or more compatible solid or liquid fillers, diluents or capsules suitable for administration to a human or other animal. Chemical substance. The term "carrier" refers to an organic or inorganic component, natural or synthetic, with which the active component is combined to facilitate application. The components of the pharmaceutical composition can also be mixed simultaneously with the molecules of the present invention in such a way that there is no interaction with one another that would substantially impair the desired pharmaceutical efficacy. Pharmaceutically acceptable further means non-toxic substances that are compatible with the biological system (eg, cells, cell cultures, tissues or organisms). 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 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 can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. 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 conveniently 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 (eg, oleic acid)
It can be used for preparing injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration are described in Remington's Pharmaceuticals
l Sciences, Mack Publishing Co. , Easto
n, PA. Can be seen in

As noted above, various routes of administration are applicable to the delivery of the alkylamine agents of the present invention to a subject. The particular mode of delivery chosen will, of course, depend on the particular drug chosen, the severity of the condition being treated, and the dosage required for therapeutic efficacy. The methods of the present invention are generally practiced using any mode of administration that is medically acceptable, meaning any mode that produces an effective level of an active compound without causing clinically unacceptable adverse effects. Can be done. Such modes of administration include oral, rectal, topical, nasal, interdermal, parenteral routes. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intranasal, inhalation or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term treatment and prevention. However, it may be preferred in emergency situations. Oral administration is preferred for prophylactic treatment because it is convenient for the patient and the dosage regimen.

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 agent into association with a carrier which constitutes one or more accessory ingredients.
Generally, the composition will uniformly and closely associate the alkylamine agent with the liquid carrier, the finely divided solid carrier, or both, and then, if desired,
It is prepared by molding the product.

Compositions suitable for oral administration may be presented as separate units, for example, capsules, tablets, troches, each of which contains a predetermined amount of an alkylamine. Preferred formulations for oral administration include: an implant configured and implanted for gingival tissue, a chewing gum containing an alkylamine, a floss impregnated with an alkylamine, and an alkylamine. Toothpaste. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrups, elixirs or emulsions.

Other delivery systems may include time release, delayed release or sustained release delivery systems. Such a system may avoid repeated administration of the alkylamines described above, which may be more convenient for subjects and physicians. Many types of release delivery systems
It is applicable and known to those skilled in the art. They include the above-described polymer systems and polymer-based systems such as poly (lactide-glycolide), copolyoxalate, polycaprolactone, polyesteramide, polyorthoester, polyhydroxybutyric acid and polyanhydride. Microcapsules of the above polymers containing drugs are described, for example, in US Pat. No. 5,075,109. Delivery systems also include the following non-polymeric systems: cholesterol, sterols such as cholesterol esters, and lipids including fatty acids or neutral fats such as mono-, di- and tri-glycerides; hydrogel release systems; silastic (syl)
peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants;
Particular examples include, but are not limited to: (a) US Pat.
The alkylamine agent is included in a matrix form such as those described in US Pat. Nos. 2,775, 4,667,014, 4,748,034 and 5,239,660. An erosion system, and (b) US Pat. No. 3,832,253.
No. 3,854,480. Diffusion systems in which the active ingredient penetrates at a controlled rate from polymers such as those described in US Pat. In addition, pump-based hardware delivery systems may be used, some of which are compatible with implantation.

[0055] Administration of the alkylamine agent can be performed using any means known to those of skill in the art, including oral, rectal, topical, intravenous, subcutaneous, intramuscular or intraperitoneal routes. Alkylamine agents can be formulated to achieve sustained release of the agent in vivo. Thus, for example, in one embodiment, a preferred vehicle is a biocompatible microparticle or implant, which is suitable for implantation into a subject. An exemplary bioerodible implant useful according to this method is PCT International Application No. PCT / US / 03307 (publication number WO95).
/ 24929, Title "Polymeric Gene Delivery S
ystem ", claiming priority in U.S. Patent Application No. 213,668, filed March 15, 1994). PCT / US / 0307 describes a biocompatible, preferably biodegradable, polymer matrix. According to the present invention, the alkylamine agent is biocompatible, preferably PCT / US / 033.
07 is encapsulated or dispersed within a biodegradable polymer matrix. The polymer matrix is preferably a microsphere (where the alkylamine (s) are dispersed throughout the solid polymer matrix),
Or in the form of microparticles such as microcapsules, where the alkylamine agent (s) are stored in the core of the polymer shell. Other forms of polymer matrices for containing the alkylamine agent include films, coatings (eg, coating on floss), gels and implants. Alkylamines (alone or formulated into sustained release compositions) can be used as food or hygiene product additives. For example, in one embodiment,
Alkylamines (alone or formulated as a sustained release composition) can be added to toothpastes to aid in gingivitis control.

[0056] The size and composition of the polymer matrix device are selected to produce advantageous release kinetics in the tissue into which the matrix device is implanted. The size of the polymer matrix device will further depend on the method of delivery to be used (eg,
It is selected according to infusion or implantation into the inflamed tissue or area (eg, gingival tissue, synovial fluid in the arthritic joint), administration of the aerosol suspension to the nasal and / or pulmonary areas). The polymer matrix composition can be selected to have both advantageous degradation rates, and can also be selected to be formed from a material that is bioadhesive, such that the device is administered to a surface in vivo. This further increases the effectiveness of the transfer. The matrix composition may also be selected not to degrade, but rather to release by diffusion over an extended period of time.

[0057] Both non-biodegradable and biodegradable polymer matrices can be used to deliver the alkylamine agents of the invention to a subject. Biodegradable matrices are preferred. Such a polymer may be a natural polymer or a synthetic polymer. Synthetic polymers are preferred. The polymer is selected based on the time period over which release is desired, generally on the order of hours to one year or more. Typically, release over a period ranging from a few hours to 3 to 12 months is most desirable. The polymer is optionally in the form of a hydrogel that can absorb up to about 90% of its weight in water and is optionally crosslinked with polyvalent ions or other polymers.

In general, the alkylamine agents of the present invention are delivered using a bioerodible implant by diffusion, or more preferably, by degradation of the polymer matrix. Exemplary synthetic polymers that can be used to form a biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohol, Polyvinyl ether, polyvinyl ester, polyvinyl halide, polyvinyl pyrrolidone, polyglycolide, polysiloxane, polyurethane and their copolymers, alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose,
Acrylic and methacrylic ester polymers, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate Sodium salt, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), 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 alcohol), polyvinyl acetate,
Polyvinyl chloride, polystyrene and polyvinyl pyrrolidone.

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 (eg, lactic acid and glycolic acid polymers, polyanhydrides, poly (ortho) esters, polyurethanes, poly (butyric acid (b
utic acid)), poly (valeric acid) and poly (lactide-co-caprolactone)), and natural polymers such as alginate and other polysaccharides (including dextran and cellulose), collagen, chemical derivatives thereof (substituted). Body, adducts of chemical groups (eg, alkyl, alkylene), hydroxides, oxides and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamins And hydrophobic proteins, copolymers, and mixtures thereof). Generally, these substances degrade either by enzymatic hydrolysis or by exposure to water in vivo by surface or internal erosion.

[0061] Bioadhesive polymers of particular interest include those described in H.E. S. Sawhney, C.A. P. P
atak and J.M. A. Hubbell (Macromolecules, 19
93, 26, 581-587, the teachings of which are incorporated herein by reference), including: polyhyaluronic acid, casein, gelatin, glutin. , Polyanhydride, polyacrylic acid, alginate, chitosan, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), 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 compositions of the above-described alkylamine agents for use as a medicament, and methods for sustained release of the medicament in vivo and methods of preparing the medicament.

The use of a long-term sustained release implant may be particularly suitable for the treatment of chronic conditions (eg, chronic gingivitis). Prolonged release, as used herein, means that the implant is configured and arranged to deliver a therapeutic level of the active ingredient for at least 30 days, preferably 60 days. Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above.

[0063] The isolated alkylamine agent can be administered alone or in combination with the above drug therapy by any conventional route, including by injection or by gradual infusion over time. Administration can be, for example, oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous or transdermal. When using the isolated alkylamine agent of the present 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 (eg, olive oil), and injectable organic esters (eg, ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer
Dextrose, dextrose and sodium chloride, lactated Ringer oil or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte supplements (eg, those based on Ringer's dextrose), and the like. Preservatives and other additives may also be presented as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases and the like.

In general, the alkylamine agent can be administered to the subject (any mammalian receptor) using the same mode of administration currently used for the administration of low molecular weight organic molecules.

Another aspect of the invention is whether the contemplated therapeutic agent modulates (up-regulates or down-regulates) alkylamine-induced stimulation of Vγ2Vδ2 T cell proliferation and / or alkylamine-mediated activation of γδ T cell receptors. Screening assays to determine That is, the method is useful for identifying alkylamine agonists that can be used to stimulate Vγ2Vδ2 T cell proliferation and / or activate γδ T cell receptors instead of the alkylamine agents of the present invention. According to one embodiment of the present invention, the method comprises the steps of providing a putative alkylamine agonist (eg, contained in a combinatorial library) under conditions that stimulate Vγ2Vδ2 T cell proliferation when the alkylamine agonist is present.
With Vγ2Vδ2 T cells, and determining whether the putative alkylamine agonist stimulates Vγ2Vδ2 T cell proliferation. Alternatively, an antagonist of alkylamine activity stimulates Vγ2Vδ2 T cells using an alkylamine agent of the invention in the presence and absence of a putative alkylamine agent antagonist, and the putative alkylamine antagonist stimulates Vγ2Vδ2 T cells. It can be identified by determining whether to reduce the degree of proliferation. In another embodiment, a screening test that measures the activity of the γδ T cell receptor (as an alternative to Vγ2Vδ2 T cell proliferation) is employed to identify alkylamine agent agonists and / or alkylamine agent antagonists.

The present invention will be more fully understood by reference to the following examples. However, these examples are only intended to illustrate embodiments of the present invention and are not intended to limit the scope of the present invention. The drawings referred to are exemplary only,
It is also understood that it is not essential to the enablement of the claimed invention.

EXAMPLES Example 1. Human γδ T Cells Recognize Alkylamine Studies in our laboratory indicate that natural compounds can potentially stimulate or suppress γδ T cell cytokine secretion. Showed that ethylamine could induce IL-2 release from γδ T cells. Ethylamine, L-theanine (Fig. 1
by-products of the hydrolysis of a) is found in the urine of those who drink tea ^22, known synthetic γδ
It differs from the T cell antigen ethyl pyrophosphate (EPP) ^{23 in} that the amine group of ethylamine replaces the pyrophosphate group of EPP (FIG. 1b). To further investigate the reaction of ethylamine with Vγ2Vδ2T cells, we developed a polyclonal γδT cell line (FIG. 2a), Vγ2Vδ2T, which co-expresses the γδ variable region T cell receptor (TCR) chain in response to ethylamine. Co-expressed 4
T cell clones (FIG. 2b), 5 αβ T cell clones (FIG. 2c), one Vγ1Vδ1 T cell transfectant (FIG. 2d, open circle), and one V
IL-2 release of γ2Vδ2 T cell transfectants (FIG. 2d, closed circle) was measured. The Vγ2Vδ2T cell line, the Vγ2Vδ2T transfectant and all four Vγ2Vδ2T cell clones, but not the Vγ1Vδ1 transfectant and αβT cell clone, respond to ethylamine in a dose-dependent manner. The Vγ2Vδ2T cell line (FIG. 2a) and the Vγ2Vδ2T cell clone (FIG. 2b) showed an average of 4-fold and 5-fold greater response to ethylamine, respectively, compared to the medium. Vγ2Vδ2 transfectants (FIG. 2d) respond to ethylamine in a manner comparable to the Vγ2Vδ2 T cell line (FIG. 2a). In contrast, when compared to the medium, the 5αβ T cell clone (FIG. 2c)
Alternatively, there was no significant difference in the amount of IL-2 released in response to ethylamine by Vγ1Vδ1 TCR transfectants (FIG. 2d).

These data were unexpected because they showed that only phosphate-containing antigens stimulate Vγ2Vδ2 T cells ^23-27 . As expected, EPP is V
IL from γ2Vδ2 T cell line and all four Vγ2Vδ2 T cell clones
-2 stimulated release. Alkaline phosphatase treatment of EPP before culturing in PBMC abrogated its γδ T cell reactivity, while this treatment had no effect on ethylamine reactivity (data not shown).

[0070] Tea beverages are typically L-theanine (all types of tea) at a concentration of 2-10 mM.
Has a unique amino acid (Camellia sinesis)^{28-3 0} . Determine if tea beverage can cause in vitro expansion of γδ cells
In order to achieve this, we have used unfractionated PBM isolated from healthy donors.
C and green or black tea purchased commercially from a local food store and placed according to the instructions
And were mixed. Neither green tea nor black tea increased the number of γδ cells (data shown
Not). However, acid hydrolysis is carried out by refluxing in 5N HCl, and L-te
Green tea and black tea with ethylamine released from anine^{twenty two}Is three times larger than that of γδ T cells.
Caused a 5-fold growth (FIG. 3a). Purified L-theanine is
Although it did not cause expression, acid hydrolyzed L-theanine was found to be
Caused a 15-fold growth of the vesicles (FIG. 3b). L-theanine hydrolysis in vivo
The solution is probably due to acid hydrolysis in the gut and to the enzyme of tissue amidase in the liver.
Caused by elementary action^{twenty two}. Here, our results show that acid-hydrolyzed tea
Or antigen presentation of peripheral blood since only L-theanine preparation stimulated γδ T cells
It proposes that cells (APC) cannot hydrolyze L-theanine. Ethyl a
Min is acid-hydrolyzed from L-theanine in tea or from purified L-theanine.
GC-mass spectrometry analysis was performed to prove that it was released by the solution.
Made with hydrolyzed or non-hydrolyzed tea extract or L-theanine. D
No tilamine was detected in the unhydrolyzed sample. In contrast, 7.2 mM
Is the biologically active acid used to stimulate γδ cells
Detected in hydrolyzed tea samples. Purified acid hydrolyzed
GC-mass spectrometry analysis of a 1 mM L-theanine sample showed approximately equivalent (1.
1 mM) ethylamine. In summary, these results are:
The ethylamine resulting from the hydrolysis of L-theanine found in tea is
Indicates that the vesicles can grow.

To test the reactivity of human γδ T cells with other natural alkylamines,
Peripheral blood mononuclear cells (PBMC) from a healthy donor were
Was cultured in any of a variety of alkylamines. 7-13 days after culture
The authors used a TCR-specific monoclonal antibody to analyze flow cytometry.
Analysis and cell count were performed. Alkylamine (ethyl-, n-propyl-
, N-butyl, iso-propyl, iso-butyl, sec-butyl, tert
-Butyl, and iso-amylamine) in medium alone or in tetanus toxoid
(Which did not proliferate γδ T cells)
Proliferated (FIG. 4). Methyl- and n-amylamine and polyamines,
Resin and spermidine are responsible for γδ T cells from some donor PBMCs.
Did not proliferate (data not shown). αβ T cells are not expanded, Vγ and
Cytometry using monoclonal antibodies specific for V and Vδ
It has been shown that cultured γδ T cells express Vγ2 and Vδ2 gene segments.
I did it. Such Vγ2Vδ2 TCR class has homologs in non-primates.
I do not have. Alkylamines shown here to be able to proliferate γδ T cells are
Ngo³¹,wine³², Tea (particularly in the form of L-theanine)^{twenty two}, Bacterial secretion products ^33-35 , Human urine^36-38, Breast milk, amniotic fluid³⁹In vaginal discharge, and from healthy women⁴⁰Look at
Was issued. Thus, alkylamine antigens are either present on plant foodstuffs or bacteria.
And are commonly found in human body fluids. Antigenic alkylamine (in
Induced γδ T cell proliferation from PBMC in vitro) at concentrations up to 10 mM (
At concentrations well above 400 μM) in vivo in humans.
(FIG. 4).

Such reactivity has been linked to alkyl phosphate antigens, but
Γδ T cell proliferation was significantly associated with certain microbial infections.^27,41
. Bacteroides fragilis and Clostridium
perfringens also provides millimolar concentrations of alkylamines and polyamines.
Mines (eg, methylamine, dimethylamine, n-propylamine, pyrrolidi
, Piperidine, n-butylamine, and putrescine)³⁴ . Proteus, Salmonella, Shigella and Esche
richia coli is cadaverine, b-phenylethylamine, putresci
Iso-amylamine, 2-methylbutylamine, and iso-butylamine
Produce amines like min³⁵. Listeria monocytogen
s produces n-butylamine and putrescine during fermentation³³. Esche
richia coli, Salmonella species, Listeria mon
ocytogenes and Yersinia enterocolitica
Is known to cause the proliferation of Vγ2Vδ2 T cells in vitro.^{12 , 42,43} , Patients with salmonellosis and listeriosis have peripheral blood Vγ2V
The number of δ2 T cells is increasing^12,15. In addition, differences in activated Vγ2Vδ2 T cells
Always high numbers are found in patients with periodontal disease such as gingivitis⁴⁴This
It is associated with Bacteroides or Porphyromonas species
Is⁴⁵. Parasite Trichinella pseudospiralis (muscle
Causing degenerative and regenerative changes in meat tissue)
Produces amines. Injection of n-butylamine into mouse muscle was performed by Trichin
microscopy in muscle tissue of mice infected with Pseudospiralis
Induces the same changes as pathological changes. No inflammatory changes were seen, indicating that mice
May be due to lack of γδ T cells that can recognize alkylamine antigens⁴⁶. Obedience
Thus, alkylamine antigens produced in large quantities by pathological organisms
It allows the growth of Vγ2Vδ2 T cells in vitro, and they
During infection, it has in vivo effects such as Vγ2Vδ2 T cell proliferation and tissue destruction.
I can do it.

To identify potential microbial sources of alkylamine antigens, fresh supernatants from bacterial cultures and supernatant extracts enriched for alkylamine were purified from urinary tract infections and urinary tracts. From Proteus morganii, an important cause of sepsis, and the anaerobic periodontopathic bacteria, Por
phyromonas intermedius or Porphyromon
as gingivalis (formerly Bacteroides). These supernatants are treated with alkaline phosphatase or mock treated (moc) to destroy any alkyl phosphate activity that may be present.
k-treat) and then added to a culture of PBMC from a healthy donor.
Cultures were maintained for 12 days and subsequently analyzed by cell count and flow cytometry (FIG. 5). Alkylamine-enriched extracts (Triangles) and crude bacterial supernatants (Circles) from Proteus morganii have Vγ
A 7-fold and 3-fold increase, respectively, in 2Vδ2 T cell counts was induced in a dose-dependent manner, whereas the bacteriological medium alone (squares) had no effect (FIG. 5a). Quantitative headspace GC-mass spectrometry analysis showed that the crude bacterial supernatant contained 3.4 mM iso-butylamine and 3.9 mM iso-amylamine, and the alkylamine-enriched extract of bacterial supernatant was 4.1 mM. iso-
Uninoculated media containing butylamine and 8.4 mM iso-amylamine revealed no detectable alkylamine (data not shown). Alkaline phosphate treatment totally abrogated the ability of the alkyl phosphate EPP to proliferate γδ T cells (data not shown), but the Porphyromonas interterm
Crude alkaline phosphatase-treated or mock-treated bacterial supernatants from both edius and Porphyromonas gingivalis caused up to 2-fold growth of γδT cells (FIGS. 5b and 5c). This result is consistent with the idea that alkylamines found in bacterial supernatants proliferate γδ T cells.

To determine whether the reactivity to alkylamines was TCR-dependent, we determined in response to bacterial supernatant (FIG. 6a) and panel alkylamines (FIG. 6b). TCR for their ability to release IL-2
The transfectants were tested. DBS43 is used for Vγ2 and Vδ2 TCR
The cDNA encoding the strand was designated as EPP reactive clone DGSF. 13 was made by co-transfecting a TCR-deficient mutant from Jurkat T cells. For comparison, another transfectants containing cDNA from T cell clones F7 encoding Vγ1Vδ1 chain pair lacking reactive (chain pair) for EPP, it is used Kamata 27 / 3.62 ^47. Extracts of Proteus supernatant concentrated against alkylamine (triangles), a solution of pure iso-butylamine (squares), and a solution of monoethyl phosphate (circles) (ie, an example of a phosphate antigen) were mock treated. (Open symbols) or alkaline phosphatase treatment (open symbols) (FIG. 6a). Alkaline phosphatase treatment of monoethyl phosphate reduced its ability to induce IL-2 release from Vγ2Vδ2 TCR transfectants by 7-fold (FIG. 6a), while Vγ1Vδ1 TCR transfectants did not (data not shown). (Not shown), thus totally abrogating the antigenic activity. In contrast, this treatment did not significantly reduce the antigenic activity of Proteus extract or pure iso-butylamine, highlighting that alkylamine, but not phosphate antigen, was responsible for these activities (FIG. 6a). Vγ2Vδ
The 2 TCR transfectant DBS43 also released significant IL-2 levels in response to a series of other alkylamines tested (FIG. 6b), while Vγ
The 1Vδ1 TCR transfectant was not (data not shown). These data also show that iso-butyl- and sec-butylamine are the most potent antigens on a molar basis for this particular transfected TCR, n-propyl-, iso-propyl-, n- Butyl- and is
It shows that o-amylamine has a medium strength and ethylamine was the lowest strength antigen. Antigen titration using Vγ2Vδ2 T cell clones in the panel indicates that the order of intensity seen for the reactivity of these antigens with the DBS43 transfectant is retained for all Vγ2Vδ2 T cell clones, or 1 Determine whether there are T cell clones specific for only one alkylamine. These results, together with the proliferation of only specific γδ T cells from PBMC, indicate that the reactivity to alkylamines is Vγ2Vδ2 TCR
Shows that it is critically dependent on the growth of E. coli.

To determine the in vivo effects of alkylamine antigens on T cells, we administered 50 mg / kg iso-butylamine intravenously without co-administration of IL-2 to each of two rhesus monkeys. Was administered three times a week. t ₀ (that is, before any infusion), blood γδT cells obtained grown to 30 times in vitro in response to iso- butylamine and IL-2. However, blood γδ T cells harvested one week after injection failed to respond to iso-butylamine and IL-2 in vitro. These results indicated that in vivo injection of iso-butylamine could affect in vitro reactivity of monkey γδ T cells. Injection of pure antigen without cytokines such as IL-2 can lead to T cell unresponsiveness (ie, anergy). To test the hypothesis that co-injection of IL-2 and iso-butylamine causes in vivo proliferation of blood γδ T cells, monkeys were dosed with iso-butylamine with IL-2 three times a week with IL-2. Before and according to its schedule, with intravenous T cell proliferation measurements. It is expected that co-administration of iso-butylamine and IL-2 will lead to in vivo proliferation of blood γδ T cells rather than anergy.

These results indicate that the same Vγ2Vδ2 TCR has a positively charged molecule (eg,
Ethylamine) as well as previously characterized negatively charged molecules (eg, E
It can recognize PP and isopentenyl pyrophosphate (IPP) ^23,27,41 .
This indicates that the TCR can bind these two molecules at different positions on the TCR. This suggests that the TCR may bind to these two different molecules at different locations on the TCR. This is easily possible given the small size of these antigens. Alternatively, the TCR recognizes an alkyl chain of either an alkyl phosphate or an alkylamine at the TCR site. Different TCR residues flanking such sites may provide for stabilization of the TCR-antigen complex by adapting to either a positive or negative charge. The functional part of the alkyl phosphate, EPP, is a hydrophobic alkyl chain and a negatively charged phosphate moiety.
The data from the report of the present invention shows that displacement of the phosphate by the amine is acceptable. This suggests that the nature of the charge on the molecule is less important than the presence of the alkyl group. ²³ previous data, 1-4 alkyl groups of carbon is required for the reactive linear alkyl phosphate, which showed that substitution of the phenyl ring is not permitted. This indicates that TCR specificity can be more directed at the hydrophobic alkyl chains than at the charged portion of the ethylamine molecule or EPP. The TCR-dependent reactivity of either the negatively charged alkylphosphate or the positively charged alkylamine antigen must therefore be accounted for in any molecular model of Vγ2Vδ2 TCR interaction with the antigen.

These results also indicate that the alkylamine recognized by the γδ T cells is a linear or branched alkyl chain of 2 to 5 carbons having a single primary amine group as the only substituent. (Figure 7). Conversely, an alkylamine having one carbon (eg, methylamine) or an alkylamine having more than 5 carbons, or an alkylamine having any substituents in addition to the primary amine group is not antigenic (see FIG. 7). The structural constraints on these alkylamine antigens are reminiscent of those of alkyl phosphate and prenyl pyrophosphate. Alkylamines recognized by γδ T cells and having antigenic activity can be identified using the in vitro screening assays described herein. In this way, a few compounds lacking such activity were identified. For example, 5-carbon iso-amyl phosphate is not recognized, while
iso-Amylamine is recognized. As another example, methylamine is not antigenic, while methyl phosphate is antigenic. In summary, linear or branched alkyl phosphates of 1-4 or 5 carbons and primary alkyl amines of 2-5 carbons were antigenic to Vγ2Vδ2 T cells (FIG. 7).

Assessing the Importance of APC in T Cell-Mediated Responses to Alkylamines
To this end, the inventors have determined that some alkylamines and the reactivity of
Glutaraldehyde-immobilized SH-5YSY as APC in -2 release assay
Comparisons were made in the presence or absence of neuroblastoma cells. Ethylamine (Figure 8a and
8c) or some other antigenic alkylamine (data not shown)
Neither the potency nor the magnitude of the resulting response was affected by the addition of SH-5YSY APC.
won. As expected, EPP also showed significant reactivity in the absence of APC.
However, the addition of APC increased the reactivity (about 4 times) and potency (about 100 times).
Fold) (FIGS. 8b and 8d). Another APC, EBV-trait
Use of transformed B lymphoblastoid cells increased EPP potency by 10-fold^{twenty three}But again, d
It had no effect on the response to tilamine (data not shown). this is
, Ethylamine and TPP-dependent recognition of EPP,
We propose to have different requirements for the stimuli. These data are
No effect on TCR-dependent recognition, indicating that immunoglobulin (Ig) -hap
Discuss the direct recognition of alkylamines that is the same as that found in ten interactions
. Several lines of evidence suggest that some γδ T cells may not recognize αβ TCRs.
Suggest that antigens be recognized in a more similar way to immunoglobulin recognition.
You. In mice, γδ T cells are transformed into MHC class II molecules IE^kAnd non-old
The typical MHC molecules T10 and T22 are recognized directly without peptide dependence. I
-E^kSite-directed mutagenesis of MHC molecules requires that γδT
Cells showed recognition of MHC residues^48,49. Furthermore, γδ T cells are specialized
Even when bonded to plastic without the APC of the
Recognizes HSV glycoprotein I^50,51. Analysis of the CDR3 length distribution of the TCR chain
They are more similar to IgH than to the TCRα and TCRβ chains. ⁵² Finally, the atomic structure analysis of human Vδ3 showed that this TCR model
Indicates closer to IgV domain than βV domain^54,55. Crystallographic
Studies have shown that immunoglobulins are small phosphate-containing molecules (eg, phosphoryl
Phosphorus) in a manner that is highly dependent on both the heavy and light chain CDR3 regions. ⁵³ . Therefore, the Vγ2Vδ2 TCR-alkylamine complex is replaced with the Ig-hapten complex.
Can be very similar to coalescence. However, preliminary evidence in our laboratory is
Cell-cell contact in response to alkylamine in response to IL-2 by γδ T cells.
It proposes that it may be necessary for release.

Detailed knowledge of the antigenic structures recognized by αβ T cells has enhanced our understanding of their role in immunity. One key to understanding the role of γδ cells in immunity is to identify the antigens they recognize. Alkylamines identified herein as antigens can be used as host defenses.
Potential in plant foods such as tea, apples, and wine, for enhancing bacterial recognition and immunity in plants, and also for the potential of γδ T cells in autoimmunity and enhanced immune resistance to allergens Suitable for Further studies are needed to determine the exact nature of the interaction between Vγ2Vδ2 TCRs and their antigens. However, this study clearly shows that alkylamines are a large class of newly identified Vγ2Vδ2 T cell antigens with different chemical and biological properties than phosphate antigens.
When found in plant foods, human body fluids, and bacteria, alkylamines appear to play a major role in Vγ2Vδ2 T cell activity in vivo.

Vγ2Vδ2 T cells have a memory phenotype (memory phenotype).
point with pe)⁵⁸, They are their cell surface expression⁶⁰Gene rearrangement for
Junctionally diverse TCR requiring ⁵⁹ Anergy or gain depending on the point of expression of
Their ability to receive any of the breeding^61,62For adaptive (adaptive)
) Can be considered part of the immune system. On the other hand, Vγ2Vδ2 T cells also
Closely paired TCR variable region genes Vγ2 and Vδ2 are restricted germ cells
Reflecting lineage diversity, it can be considered as part of the innate immune response. This V heredity
Due to child pairing, each of the Vγ2Vδ2 TCRs has not been processed
A family of antigens that are conserved, such as alkylamines and alkyl phosphates
Can be readily recognized by the molecular pattern of sex. Antigen archi, a product of multiple pathogens
Of the conservative one to five carbons found in amines and alkyl phosphates
This TCR-dependent recognition of chain or branched chain alkyl groups has been demonstrated in various bacterial lipopolysaccharides.
CD-14-mediated patterning of pathogen-associated molecular patterns such as recurring sugar residues found
Reminds turn recognition^63,64. This pattern of recognition by Vγ2Vδ2 TCR
By the number of memory γδ T cells in normal adults (4.5% C average).
D3⁺T cells) and between 2 and 10 fold higher levels (8
~ 60% CD3⁺T cells). Therefore, by microorganisms
These large numbers of memory T cells capable of responding to alkylamine antigens produced are
It can bridge the gap between the immune response and the adaptive immune response.

Example 2 Alkylamine Precursor Reaction The alkylamine agent of the present invention is an alkylamine of Formula I or III or an alkylamine precursor of Formula II, as described above. Precursors of alkylamines of the type of formula I can be found in nature as naturally occurring compounds or can be chemically synthesized. Examples of naturally occurring precursor is N- substituted primary amides, N ⁵ - ethyl glutamate (L- theanine) (Camellia Sines
is), N ⁴ -ethyl asparagine (Tulipa gesneri)
included in ana), and N ⁵ - iso - propyl glutamine (Lunaria
annua).

FIG. 9 a illustrates an exemplary synthetic reaction to form an alkylamine precursor of the present invention. This synthesis reaction is described in Y. Description by Sakato et al. (Nippon Nu
gei Kagadu Kaishi 23: 262-271 (1949) (Mo
Rrison and Boyd, 3rd ed., Sec. 18, see also exemplary reactions for the formation and hydrolysis of amide compounds)). Exemplary R1 groups in the acid structure include, but are not limited to, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl and sec-butyl. FIG. 9b shows that an acid (eg, L-glutamic acid) can be converted to an amine (eg, L-glutamic acid).
2 shows an exemplary reaction to form an ester (e.g., L-glutamic acid [gamma] -ethyl ester) with ethylamine. Here, the ester is derived to form a derived ester. Thus, the derived ester is capable of forming an alkylamine precursor (amide).

FIG. 10 shows the hydrolysis of the alkylamine precursor of Formula II. Hydrolysis can be effected by the presence of either an acid or a base, or by enzymatic cleavage of an amide bond (eg, by an amidase) to give an alkylamine of formula I or III and a moderate carboxylic acid. For example, N ⁵ -ethylglutamine (
Hydrolysis of L-theanine) gives ethylamine and glutamic acid. Alkylamine precursor occurring naturally, N ⁵ - ethyl glutamate (L- theanine) (C
amellia sinesis, i.e. is found in tea); N ⁵ - iso - propyl glutamine (Lunaria annua, i.e. is found in ornamental plants); and N5 ethyl asparagine (Tulipe gesneria
na, that is, tulip, Echalium elate
rium, which is found in the cucumbers and Bryonia dioica).

[0084]

(Equation 1) All references, patents and patent publications listed in this application are incorporated by reference herein in their entirety.

It should be understood that the preceding description is only a detailed description of certain preferred embodiments. Thus, it should be apparent to one skilled in the art that various modifications and equivalents may be achieved without departing from the spirit and scope of the invention. All such modifications are intended to be included within the scope of the appended claims.

[Brief description of the drawings]

FIG. 1a shows the conversion of L-theanine into ethylamine and glutamic acid (glutam).
ate).

FIG. 1b is a structural diagram of ethyl pyrophosphate and iso-pentenyl pyrophosphate.

FIG. 2 is a series of line and bar graphs showing the effect of ethylamine on the induction of IL-2 release from Vγ2Vδ2 and Vγ1Vδ1 cells. Ethylamine caused release of IL-2 in the polyclonal Vγ2Vδ2T cell line (a), Vγ2Vδ2T cell clone (b), however, αβT
Cell clone (c) or Vγ1Vδ1 T cell transfectants (d, open circles) did not cause this. Vγ2Vδ2 T cell clone 12G12, D
G. FIG. SPF6 and CP1.15 were derived from healthy donor PBMC by stimulation of Mycobacterium extract, whereas DGSF. 13 was derived from synovial fluid of patients with rheumatoid arthritis. αβ T cell clones were isolated from PBMCs of healthy donors by limiting dilution and expanded with PHA. The Vγ2Vδ2 T cell line was derived from a healthy donor PBMC using magnetic beads.
Generated by positive selection of anti-TCRδ1 ⁺ cells from These T cells are expanded in PHA and 1 nM IL-2, then undergo a negative selection procedure using monoclonal antibodies OKT4 and BMA-031 treatment, followed by packing magnetic beads with goat anti-mouse Ig. The final cells were> 99% Vγ2Vδ2 TCR ⁺ by flow cytometry analysis.
Vγ2Vδ2 T cell transfectants were obtained from TCR-J. RT3-T3.5 cells were isolated from DG.T3 cells obtained by stimulating synovial fluid mononuclear cells with Mycobacterium extract. It was made by transfecting with SF13, a cDNA made from Vγ2Vδ2 TCR + phosphate antigen reactive γδ T cell clone. Vγ1Vδ1 T cell transfectants were obtained from TCR-J. RT3-T3
. 5 cells were obtained by transfecting F7, a cDNA generated from the Vγ1Vδ1 TCR + γδT cell clone. Stimulation of T cell clones and transfectants were performed in 96-well flat bottom plate with 1 × 10 ⁵ cells of responder cells per well in 0.2 ml ^47. In one experiment, 5 × 10 ⁴ mitomycin-treated or glutaraldehyde-fixed B-lymphoblastoid cells (LCL) or SH-5YSY neuroblastoma cells per well were used as feeders or antigen presenting cells (APCs). However, these APCs are not required to obtain IL-2 release from transfected Jurkat cells. Starting from 100 mM (a, d) or 30 mM
Logarithmic dilutions of ethylamine in fixed volumes (b, c), or as positive controls, calcium ionophore, ionomycin (at 1 mg / 1 ml) with 10 nM
⁵⁶ was added as a costimulator in the presence of g / ml PMA. Twenty-four hours later, supernatants were harvested and tested at a final dilution of 1/8 for their ability to stimulate the growth of an IL-2-dependent HT-2 cell line. The proliferation assay is 96
Using 5 × 10 ³ HT-2 cells per flat bottom well of a well plate,
Performed in triplicate. Eighteen hours later, the cells were applied with ³ H thymidine (1 mCi / well), harvested 24 hours later, and the beta plate system (Bet
The count was performed by liquid scintillation on an apply system. The standard deviation of triplicate measurements was less than 10% of the mean.

FIG. 3 shows Vγ2Vδ2T of acid-hydrolyzed tea extract or L-theanine.
4 is a bar graph showing the effect on cells. Hydrolyzed green and black tea (a) and hydrolyzed L-theanine (b) caused the proliferation of γδ T cells from PBMC. A concentrated green tea extract was made by mixing 15 g of green tea leaves with 500 ml of boiling water and soaking for 10 minutes. This mixture is
Centrifuge at 00 × g for 20 minutes and filter to remove residual large particles, yielding 400 ml of concentrated tea extract. This was lyophilized to give 4 g of solid material. 2 grams of this extract, 2 g of Nestea ice tea (1
00% tea; Nestle, Lausanne, Switzerland),
And 500 mg of L-theanine (Sigma) in 5N HCl for 2 hours,
Individually refluxed and brought to pH 7.4 with NaOH. These substances are then
γδ T cells were tested for their ability to expand from PBMC. The hydrolyzed tea extract was used at a dilution of 1:30, and the hydrolyzed L-theanine was used at 10 mM.

FIG. 4 is a bar graph showing the effect of alkylamines on Vγ2Vδ2 T cell proliferation. PBMC were mixed with either alkylamine or EPP at a concentration of 400 mM, and after 7 days, γδ T cells were quantified by flow cytometry. The total number of cells in the culture remained constant over 7 days.
Data is expressed as a percentage of CD3 ⁺ cells containing the γδ TCR.
Significant proliferation of γδ T cells occurred using alkylamine concentrations as low as 50 mM. Total γδ T cells had Vγ when assessed using V chain specific antibodies.
2Vδ2. The monoclonal antibodies for ascites induction against the T cell antigen used were as follows: control antibody (P3), pan γδ TCR (anti-T
CRδ1), Vδ1 / Vδ1 (dTCS1), Vδ2 (BB3), Vγ2 (7A
5), and CD3 (OKT3). The specificity of these antibodies is outlined ^57. FITC-conjugated (Fab ') ₂ goat anti-mouse IgG was purchased from Tago (Burlin
game, CA). Ethyl pyrophosphate (EPP) was synthesized as described ^23. Alkylamines are available from Sigma Chemical Compa
ny (St. Louis, MO).

FIG. 5a is a line graph showing the effect of Proteus morganii bacterial supernatants and their extracts on Vγ2Vδ2PBMCT cell proliferation.
The supernatants and their extracts caused the proliferation of Vγ2Vδ2 T cells from PBMC. Supernatant from a Proteus morganii broth culture (
(Circles), extracts from this supernatant enriched for amines (triangles), and unseeded bacterial culture medium (squares) were mixed with PBMC. On day 3, 0.3 nM I
L-2 was added to all cultures. On day 12, cells were analyzed by flow cytometry using an anti-γδ TCR monoclonal antibody and the number of γδ T cells was counted. The total number of cells in the culture remained constant over a period of 12 days. Data are expressed as percentage of CD3 + cells with γδ TCR. Greater than 99% γδ T cells were Vγ2Vδ2 + T cells when evaluated using V-chain specific antibodies. Ploteus morganii,
Strain 235 (National Collection of Types Cu
ltures, London) were grown in LB broth at 37 ° C. overnight. Culture supernatants were obtained by centrifugation at 2000 g for 10 minutes. To concentrate for alkylamines, 10 ml of the 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 is
Extract with 7 ml of diethyl ether and extract the aqueous extract with 10 N NaO
PH was adjusted to 12 with H. The fraction was then extracted three times with 7 ml of chloroform, followed by extraction of the organic phase with 3 ml of 5N HCl. The aqueous phase was then dried in an oven at 95 ° C., reconstituted with 1 ml of H ₂ O, brought to pH 7.4, and passed through a 0.45 micron filter.

FIG. 5b is a bar graph showing the effect of alkaline phosphatase treatment on the ability of bacterial supernatants to proliferate γδ T cells. Porphyromonas g
ingivalis and Porphyromonas intermediu
Treatment of the supernatants of bacterial broth cultures with alkaline phosphatase had no effect on their ability to proliferate γδ T cells. Bacterial supernatant or E
PP was treated with alkaline phosphatase ²³ and PB at a 1: 6 dilution.
They were cultured with MC and tested for their ability to expand γδ T cells. As a control, alkaline phosphatase treatment was performed on PBMC in γδ T cells
PP-mediated proliferation reduced CD3 + cells from 9.6 to 6.1% in Experiment 1.

FIG. 5c is a bar graph showing the effect of alkaline phosphatase treatment on the ability of bacterial supernatants to grow γδ T cells. Porphyromonas g
ingivalis and Porphyromonas intermediu
Treatment of the supernatants of bacterial broth cultures with alkaline phosphatase had no effect on their ability to proliferate γδ T cells. Bacterial supernatant or E
PP was treated with alkaline phosphatase ²³ and PB at a 1: 6 dilution.
They were cultured with MC and tested for their ability to expand γδ T cells. As a control, alkaline phosphatase treatment was performed on PBMC in γδ T cells
PP-mediated proliferation was reduced in Experiment 2 from 26.9 to 6.0%.

FIG. 6a is a line graph showing the ability of bacterial supernatants to induce IL-2 release from T cells. Alkaline phosphatase treatment of extracts of Proteus morganii broth culture supernatants had no effect on their antigenic capacity. Monoethyl phosphate (circle), iso-butylamine (square) or P
extract of Roteus supernatant (triangles) was treated with alkaline phosphatase (open symbols) or mock (closed symbols) and Vγ2Vδ
Used as an antigen to stimulate IL-2 release from 2TCR transfectant DBS43. A semi-log dilution solution of the alkylamine antigen stock solution was added to 10 n
Phorbol myristate acetate M were added to 10 ⁵ responders TCR transfectants present as a co-stimulant. Twenty-four hours later, supernatants were collected and tested at a final dilution of 1/8 for their ability to stimulate the growth of an IL-2-dependent HT-2 cell line. Alkaline phosphatase treatment was performed as follows: undiluted bacterial supernatant or bacterial supernatant extracted to concentrate for amines (as described above) or 10 m
M solution of purified alkylamine (eg, 10 mM iso-butylamine or 10 mM monoethyl phosphate) in 500 μl of 5 units of shrimp alkaline phosphatase (Sigma) at 37 ° C. for 2 hours at 37 ° C. Treated or treated and used as antigen.

FIG. 6b is a line graph showing the ability of purified alkylamine to induce IL-2 release from Vγ2Vδ2 TCR transfectant, DBS43. DBS
43 requires almost 100-fold more alkylamine antigen concentration for detectable IL-2 response compared to expansion of γδ T cells from PBMC. Vγ1V
There was no detectable IL-2 release from the δ1 TCR transfectant (data not shown).

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

FIG. 8 is a series of bar graphs showing the effect of APC on ethylamine recognition by Vγ2Vδ2 T cells. APC has no effect on TCR-dependent recognition of ethylamine by Vγ2Vδ2 TCR transfectants. SH-5YS
Y neuroblastoma cells (FIGS. 8c and 8d), or EBV transformed lymphoblastoid cells (not shown) were treated with mitomycin C and Vγ as APC.
Added to 2Vδ2 TCR transfectants and the response to ethylamine was measured by IL-2 release and IL-L seen in the absence of APC
Two responses (FIGS. 8a and 8b) were compared. APC enhanced the response to ethyl pyrophosphate by approximately 100-fold (FIGS. 8b and 8d), while there was no detectable effect on the response of APC to ethylamine (FIGS. 8a and 8c).
.

FIG. 9a illustrates an exemplary synthetic reaction to form an alkylamine precursor of the present invention.

FIG. 9b illustrates an exemplary synthetic reaction for forming an alkylamine precursor of the present invention.

FIG. 10 shows the hydrolysis of an alkylamine precursor of Formula II.

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventors Brenner and Michael B. United States Massachusetts 02159, Newton, The Ledges Road 27 F-term (Reference) 4B063 QA01 QA06 QA18 QQ08 QQ79 QR41 QR49 QS33 4B065 AA90X AC14 BB40 BC12 BD32 BD33 CA24 CA41 CA44 4C206 AA01 AA02 AA03 GA03 GA67 ZC41 4H006 AA03 AB81 AC29

Claims (99)

[Claims] 1. A method for stimulating the proliferation of Vγ2Vδ2 T cells, comprising:
The method comprises: contacting the cells with a Vγ2Vδ2T cell growth stimulating amount of an alkylamine agent, wherein the alkylamine agent is an alkylamine of Formula I: R-NH2 (Formula I) Alkylamines of Formula III: R-NR2R3 (Formula III) and alkylamine precursors of Formula II below: Wherein R is a straight or branched alkyl or alkenyl group containing 1 to 6 carbon atoms, wherein R 1 is a straight or branched chain Is an alkyl or alkenyl group containing 1 to 20 carbon atoms, wherein R2 is a linear or branched alkyl or alkenyl group containing 1 to 6 carbon atoms, wherein R2 is , R and may be the same or different, and R3 is hydrogen or a linear or branched alkyl or alkenyl group containing 1 to 6 carbon atoms, R3 may be the same or different from R or R2. 2. The method of claim 1, wherein stimulating proliferation comprises increasing the number of Vγ2Vδ2 T cells by at least two fold. 3. The method of claim 1, wherein the step of stimulating proliferation comprises increasing the number of Vγ2Vδ2 T cells by at least 10-fold. 4. The method of claim 1, wherein the step of 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 said Vγ2Vδ2 T cell proliferation stimulating amount of said alkylamine agent is from about 0.01 mM to about 100 mM. 7. The method of claim 4, wherein said Vγ2Vδ2 T cell proliferation stimulating amount of said alkylamine agent is from about 0.1 mM to about 20 mM. 8. The method of claim 4, wherein said Vγ2Vδ2 T cell proliferation stimulating amount of said alkylamine agent is from about 1 mM to about 10 mM. 9. The method of claim 5, wherein said Vγ2Vδ2 T cell proliferation stimulating amount of said alkylamine agent is from about 5 mg / kg to about 100 mg / kg. 10. The method of claim 5, wherein said Vγ2Vδ2 T cell proliferation stimulating amount of said alkylamine agent is from about 10 mg / kg to about 50 mg / kg. 11. The method of claim 4, wherein said Vγ2Vδ2 T cell proliferation stimulating amount of said alkylamine agent is from about 20 mg / kg to about 30 mg / kg. 12. The method of claim 1, wherein said alkylamine agent is an alkylamine.
The method described in. 13. The method of claim 1, wherein said alkylamine agent is an alkylamine precursor. 14. The method according to claim 1, wherein R is a linear alkyl group. 15. The method of claim 1, wherein R is a branched alkyl group. 16. The method of claim 14, wherein R is saturated. 17. The method according to claim 15, wherein R is unsaturated. 18. The method according to claim 18, wherein the alkylamine is methyl-, ethyl-, n-propyl-, -iso-propyl-, n-butyl-, iso-butyl-, sec-butyl-.
, Iso-amyl-, hydroxymethyl-, β-hydroxyethyl-, δ-hydroxypropyl-, hydroxyisopropyl-, allyl, crotyl, dimethylallyl, isopentenyl, geranyl, farnesyl, geranylgeranyl, 3-methyl-2-pentenyl, The method of claim 1, wherein the method is selected from the group consisting of and 3-methyl-2-hexenyl-amine. 19. The method according to claim 1, wherein R is a straight chain alkyl group. 20. The method of claim 1, wherein R is a branched alkyl group. 21. The method of claim 19, wherein R is saturated. 22. The method of claim 20, wherein R is unsaturated. 23. The method of claim 1, wherein R1 is a linear or branched alkyl or alkenyl group containing from 1 to 10 carbon atoms. 24. The method of claim 1, wherein said R1 alkyl or R1 alkenyl group is selected from the group consisting of propyl, butyl, and amyl. 25. The method of claim 1, wherein R1 is a linear or branched alkyl or alkenyl group of a natural dicarboxylic acid. 26. The method of claim 1, wherein said R1 alkyl or R1 alkenyl group is selected from the group consisting of propyl, butyl, and amyl. 27. The method of claim 1, wherein said monocarboxylic acid and said dicarboxylic acid are selected from the group of natural amino acids. 28. The method of claim 1, wherein said dicarboxylic acid is glutamic acid or aspartic acid. 29. The alkylamine precursor, theanine, N ⁵ - isopropyl glutamine, is selected from the group consisting of N4- ethyl asparagine, and N ⁵ -sec-butyl glutamine method of claim 1. 30. A method for activating a γδ T cell receptor, comprising:
The method comprises: contacting the receptor with an alkylamine under conditions that allow for the alkylamine-mediated activity of the receptor, wherein the alkylamine is a compound of Formula I: R-NH2 (Formula I), or a compound of the following Formula III: R-NR2R3 (Formula III) wherein R is a straight or branched alkyl containing 1 to 6 carbon atoms. Or an alkenyl group, wherein R2 is a linear or branched alkyl or alkenyl group containing 1 to 6 carbon atoms, and R2 may be the same as or different from R; And wherein R3 is hydrogen or a linear or branched alkyl or alkenyl group containing from 1 to 6 carbon atoms, wherein R3 is the same as R or R2; May be different , Method. 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. 33. The method according to claim 30, wherein R is a linear alkyl group. 34. The method of claim 30, wherein R is a branched alkyl group. 35. The method of claim 33, wherein R is saturated. 36. The method of claim 34, wherein R is unsaturated. 37. The alkylamine is ethyl-, n-propyl, -iso-propyl-, n-butyl, iso-butyl-, sec-butyl-, and iso-
31. The method of claim 30, wherein the method is selected from the group consisting of amylamine. 38. A composition comprising: an alkylamine of the following formula I: R-NH2 (Formula I), or a compound of the following Formula III: R-NR2R3 (Formula III) and a pharmaceutically acceptable carrier. Wherein R is a linear or branched alkyl or alkenyl group containing 1 to 6 carbon atoms, wherein R2 is a linear or branched, An alkyl or alkenyl group containing 6 carbon atoms, wherein R2 may be the same as or different from R, wherein R3 is hydrogen or a linear or branched, A composition, wherein the composition is an alkyl or alkenyl group containing 6 carbon atoms, wherein R3 is the same as or different from R or R2. 39. The composition of claim 38, wherein at least one of R and R2 is a straight chain alkyl group. 40. The composition of claim 38, wherein at least one of R and R2 is a branched alkyl group. 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 alkylamine is selected from the group consisting of ethyl-, n-propyl-, -iso-propyl-, n-butyl, iso-butyl-, sec-butyl-, and iso-amylamine. 39. The composition of claim 38. 44. The composition of claim 38, wherein said composition is formulated as an oral formulation. 45. The composition of claim 38, wherein said composition is formulated as an oral formulation for administration to gingival tissue. 46. The composition of claim 38, wherein said composition is formulated as a food additive. 47. The composition of claim 38, wherein said composition is formulated for delivery to the female reproductive tract. 48. The composition of claim 38, wherein said composition is formulated for vaginal delivery. 49. A compound of the following formula II: Wherein R is a linear or branched alkyl group containing 1 to 6 carbon atoms, and wherein R1 is a linear or branched 1 to 20 carbon atom A compound which is an alkyl group containing an atom. 50. The compound according to claim 49, wherein R is a straight chain alkyl group. 51. The compound according to claim 49, wherein R is a branched alkyl group. 52. The compound of para 50, wherein R is saturated. 53. The compound according to claim 51, wherein R is unsaturated. 54. The compound according to claim 49, wherein R1 is a linear or branched alkyl or alkenyl group containing from 1 to 10 carbon atoms. 55. The compound of claim 49, wherein said R1 alkyl group or said R1 alkenyl group is selected from the group consisting of propyl, butyl, and amyl. 56. The compound according to claim 49, wherein R1 is a linear or branched alkyl or alkenyl group of a natural dicarboxylic acid. 57. The alkylamine precursor, theanine, N ⁵ - isopropyl glutamine, N4-ethyl asparagine, and N ⁵ is selected from the group consisting -sec-butyl glutamine The compound of claim 49. 58. The compound of claim 49, wherein said monocarboxylic acid and said dicarboxylic acid are selected from the group of amino acids. 59. The compound of claim 49, wherein said dicarboxylic acid is glutamic acid or aspartic acid. 60. Any of the compounds of claims 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59, and comprising a pharmaceutically acceptable carrier. ,Composition. 61. The composition of claim 60, wherein said composition is formulated as an oral formulation. 62. The composition of claim 60, wherein said composition is formulated as an oral formulation for administration to gingival tissue. 63. The composition of claim 60, wherein said composition is formulated as a food additive. 64. The composition of claim 60, wherein said composition is formulated as a food additive for delivery to the female reproductive tract. 65. The compound of claim 49, wherein said compound is not theanine. 66. The compound of claim 49, wherein said compound is isolated from contaminants making said compound unsuitable for therapeutic use. 67. A method for stimulating activation of Vγ2Vδ2 T cells, comprising: contacting the cells with a Vγ2Vδ2T cell activating stimulating amount of an alkylamine agent. The agent is an alkylamine of formula I below: R-NH2 (formula I), an alkylamine of formula III below: R-NR2R3 (formula III) and an alkylamine precursor of formula II below: Wherein R is a straight or branched alkyl or alkenyl group containing 1 to 6 carbon atoms, wherein R 1 is a straight or branched chain Is an alkyl or alkenyl group containing 1 to 20 carbon atoms, wherein R2 is a linear or branched alkyl or alkenyl group containing 1 to 6 carbon atoms, wherein R2 is , R and may be the same or different, and R3 is hydrogen or a linear or branched alkyl or alkenyl group containing 1 to 6 carbon atoms, R3 may be the same or different from R or R2. 68. The method of claim 67, wherein stimulating activation comprises stimulating cytokine production by Vγ2Vδ2 T cells. 69. The cytokine, wherein the cytokine is interferon γ (IFN-γ)
, Interleukin 2 (IL-2), tumor necrosis factor β (TNF-β, lymphotoxin), interleukin 4 (IL-4), IL-5, IL-10, and TN
70. The method of claim 68, wherein the method is selected from the group consisting of F- [alpha]. 70. The method of claim 67, wherein stimulating activation comprises stimulating proliferation of Vγ2Vδ2 T cells. 71. The method of claim 67, wherein stimulating activation comprises stimulating cytotoxic activity by Vγ2Vδ2 T cells. 72. The method of claim 67, wherein stimulating activation comprises stimulating blocking of the activity of Vγ2Vδ2 T cells. 73. The method of claim 67, wherein stimulating activation comprises stimulating the Vγ2Vδ2 T cells to apoptosis and killing. 74. The method of claim 67, wherein contacting said cells with said alkylamine agent is performed ex vivo. 75. The method of claim 67, wherein contacting said cells with said alkylamine agent is performed in vivo. 76. The method of claim 74, wherein said Vγ2Vδ2 T cell activation stimulating amount of said alkylamine agent is from about 0.01 mM to about 100 mM. 77. The method of claim 74, wherein said Vγ2Vδ2 T cell activation stimulating amount of said alkylamine agent is from about 0.1 mM to about 20 mM. 78. The method of claim 74, wherein said Vγ2Vδ2 T cell activation stimulating amount of said alkylamine agent is from about 1 mM to about 10 mM. 79. The method of claim 75, wherein said Vγ2Vδ2 T cell activation stimulating amount of said alkylamine agent is from about 5 mg / kg to about 100 mg / kg. 80. The method of claim 75, wherein said Vγ2Vδ2 T cell activation stimulating amount of said alkylamine agent is from about 10 mg / kg to about 50 mg / kg. 81. The method of claim 75, wherein said Vγ2Vδ2 T cell proliferation stimulating amount of said alkylamine agent is from about 20 mg / kg to about 30 mg / kg. 82. The method of claim 6, wherein the alkylamine agent is an alkylamine.
7. The method according to 7. 83. The method of claim 67, wherein said alkylamine agent is an alkylamine precursor. 84. The method of claim 67, wherein R is a straight chain alkyl group. 85. The method of claim 67, wherein R is a branched alkyl group. 86. The method of claim 84, wherein R is saturated. 87. The method of claim 85, wherein R is unsaturated. 88. The alkylamine is methyl-, ethyl-, n-propyl-, -iso-propyl-, n-butyl-, iso-butyl-, sec-butyl-
, Iso-amyl-, hydroxymethyl-, β-hydroxyethyl-, δ-hydroxypropyl-, hydroxyisopropyl-, allyl, crotyl, dimethylallyl, isopentenyl, geranyl, farnesyl, geranylgeranyl, 3-methyl-2-pentenyl, 68. The method of claim 67, wherein the method is selected from the group consisting of and 3-methyl-2-hexenyl-amine. 89. The method according to claim 67, wherein R is a straight chain alkyl group. 90. The method of claim 67, wherein R is a branched alkyl group. 91. The method of claim 89, wherein R is saturated. 92. The method of claim 90, wherein R is unsaturated. 93. The method of claim 67, wherein R1 is a linear or branched alkyl or alkenyl group containing from 1 to 10 carbon atoms. 94. The method of claim 67, wherein said R1 alkyl or R1 alkenyl group is selected from the group consisting of propyl, butyl, and amyl. 95. The method of claim 67, wherein R1 is a linear or branched alkyl or alkenyl group of a natural dicarboxylic acid. 96. The method of claim 67, wherein said R1 alkyl or R1 alkenyl group is selected from the group consisting of propyl, butyl, and amyl. 97. The method of claim 67, wherein said monocarboxylic acid and said dicarboxylic acid are selected from the group of natural amino acids. 98. The method of claim 67, wherein said dicarboxylic acid is glutamic acid or aspartic acid. 99. The alkylamine precursor, theanine, N ⁵ - isopropyl glutamine, N4-ethyl asparagine, and N ⁵ is selected from the group consisting -sec-butyl glutamine The method of claim 67.