WO1995020673A1 - Organo-phosphorus compounds as activators of gamma delta t cells - Google Patents
Organo-phosphorus compounds as activators of gamma delta t cells Download PDFInfo
- Publication number
- WO1995020673A1 WO1995020673A1 PCT/FR1995/000092 FR9500092W WO9520673A1 WO 1995020673 A1 WO1995020673 A1 WO 1995020673A1 FR 9500092 W FR9500092 W FR 9500092W WO 9520673 A1 WO9520673 A1 WO 9520673A1
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- WIPO (PCT)
- Prior art keywords
- lymphocytes
- compound according
- compound
- thymidine
- phosphoric
- Prior art date
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Classifications
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- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
- A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
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- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/28—Phosphorus compounds with one or more P—C bonds
- C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
- C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
- C07F9/3886—Acids containing the structure -C(=X)-P(=X)(XH)2 or NC-P(=X)(XH)2, (X = O, S, Se)
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
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- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
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- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
- C12Y306/01009—Nucleotide diphosphatase (3.6.1.9), i.e. nucleotide-pyrophosphatase
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Definitions
- the present invention relates to non-peptide water-soluble organophosphorus compounds that stimulate human lymphocytes.
- the invention also relates to a process for preparing and / or isolating and / or characterizing such organophosphorus compounds, a pharmaceutical composition, in particular a vaccinating composition comprising at least one such organophosphorus compound, various uses of these compounds for obtaining pharmaceutical compositions , a pharmaceutical composition inhibiting the activity of these compounds on y5 lymphocytes and the uses of this composition for obtaining medicaments.
- human Ty lymphocytes with TCR receptors comprising the variable regions Vy and V (CD3 + lymphocytes, TCR ⁇ -, CD4-, CD8-) play a role in the immune system of humans and animals.
- TCR receptors comprising the variable regions Vy and V (CD3 + lymphocytes, TCR ⁇ -, CD4-, CD8-)
- Vy and V CD3 + lymphocytes, TCR ⁇ -, CD4-, CD8-
- their presence has been demonstrated in the context of various diseases such as bacterial, in particular mycobacterial (in particular tuberculosis and leprosy) or streptococcal infections, tumor or leukemic affections, certain parasites (for example Plasmodi um) , AIDS and autoimmune diseases (multiple sclerosis, lupus, ).
- T lymphocytes carrying variable regions Vyg V 2 TCRs represent the majority (of the order of 80%) of y 5 lymphocytes.
- Ty5 lymphocytes respond to an antigen formed by a protein such as HSP60. Other authors have, however, contradicted this thesis. However, it has not yet been possible to isolate an antigen from Ty ⁇ lymphocytes. Ty5 lymphocytes have also been shown to recognize and lyse cancer cells and infected cells, including ycobacteria, and accumulate at certain infectious sites. However, the above-mentioned conditions in which Tyg lymphocytes are involved are for the most part considered to date to be particularly severe. Consequently, since the discovery of the y 5 lymphocytes, it is sought to highlight the antigen (s) capable of activating them and to explain the reason for their presence in large numbers in the peripheral blood of the adult.
- the Ty5 lymphocytes act as pathogenic agents which it would be desirable to be able to inhibit. This is particularly the case in the context of autoimmune diseases, such as multiple sclerosis.
- the invention therefore aims, in general, to propose means for controlling the proliferation and / or cytotoxic activity of y5 lymphocytes, in particular human Tyg ⁇ 2 lymphocytes, whether it is to stimulate them in order to promote their immune role. , or to inhibit them when they are involved in a pathology, in particular autoimmune.
- the invention aims to propose isolated compounds which activate y5 lymphocytes, in particular Ty952 lymphocytes, their preparation process, their use in a pharmaceutical composition, in particular a vaccinating composition, and their various therapeutic uses for the preventive or curative treatment of the man or animal.
- the invention aims in particular to provide a method and a composition for stimulating the immune response mediated by Ty lymphocytes. And the invention aims to propose a use of this process or of this composition in the preventive (vaccine) or curative (im unomodulator) treatment: infectious diseases with prokaryotic or eukaryotic germs -in particular mycobacterial, streptococcal or plasmodium-; and / or tumor or leukemic pathologies; and / or immunodeficiency pathologies such as AIDS.
- the invention also aims to propose a method for isolating and / or characterizing compounds activating Ty ⁇ lymphocytes, in particular human Ty952 lymphocytes.
- the invention further aims to provide a composition intended for inactivating Ty5 lymphocytes, and more particularly a therapeutic composition inactivating the cytotoxicity of Ty5 lymphocytes involved in autoimmune pathologies such as multiple sclerosis.
- the invention relates to a water-soluble organophosphorus compound of a non-peptide nature, activator of human Tyg lymphocytes with TCR receptors comprising the variable regions Vyg and V52, this compound comprising at least one acidolabile ester bond of phosphoric acid.
- the activating character of this compound with respect to the y5 lymphocytes disappearing when it is placed in the presence of an enzyme mixture comprising at least one phosphoric phosphohydrolase monoester and at least one phosphoric phosphohydrolase diester.
- the enzymatic mixture can comprise a pyrophosphohydrolase as phosphoric diester phosphohydrolase.
- a compound according to the invention has an anionic character measured by HPAEC chromatography with conductometric detection in chemical suppression mode which is higher than that of phosphonoformic acid and lower than that of thymidine 5 'triphosphate.
- the invention relates to a compound characterized in that, after treatment with a phosphoric diester phosphohydrolase enzyme, it has an anionic character measured by HPAEC chromatography with conductimetric detection in chemical suppression mode which is superior to that of phosphonoformic acid and lower than that of thymidine 5 'triphosphate.
- the invention comprises the phosphodiester X-Phosphate-R compounds, the degradation of which by the enzyme diesterase releases the minimum active principle X-Phosphate.
- the invention relates to a compound which has a hydrophobic character measured by reverse phase HPLC chromatography of type C18 eluted in ion pair with ammonium acetate which is lower than that of thymidine 5 'monophosphate. More particularly, a compound according to the invention is characterized in that its activation on Ty5 lymphocytes is inhibited in the presence of monoclonal antibodies specific for human TCRs comprising the variable regions Vyg or V52-
- a compound according to the invention comprises at least one substituent X of molecular mass less than 500, which is distinct from a nucleic acid, from an oligosaccharide, from a fatty acid (that is to say a saturated carboxylic acid comprising at least four carbons), a protide, an alkaloid and a steroid, and which is linked in the compound to a phosphoryl group by an acidolabile covalent bond capable of being cleaved in the presence of a monoester phosphatase .
- X-Phosphate sequence is sufficient to confer an antigenic power to the compound according to the invention with regard to Tyg lymphocytes.
- the organophosphorus compounds according to the invention comprises at least one nucleotide group.
- the invention relates to a compound corresponding to the formula X-Phosphate — R where R is a hydrogen or an inorganic or organic substituent, in particular a nucleoside group, and X is a substituent as mentioned above.
- the invention relates to a compound corresponding to the formula:
- R is hydrogen or an inorganic or organic substituent, n is a non-zero integer.
- the invention also relates to a compound consisting of a hydrolysis product obtained from a compound according to the invention which comprises thymidine linked to a phosphoryl group by the fifth carbon of its 2-deoxyribose radical, this hydrolysis possibly in particular be obtained by the enzymatic action of a nucleotide pyrophosphatase and / or a phosphoric diester phosphohydrolase.
- the invention relates to a compound consisting of an acidolabile phosphoric monoester of molecular mass less than 500 distinct from thymidine 5 'monophosphate, thymidine 5' diphosphate, thymidine 5 'diphosphate glucose and thymidine 5' triphosphate .
- the invention relates to a compound consisting of an acid-labile phosphoric diester with a molecular mass of less than 1000, distinct from thymidine 5 'monophosphate, thymidine 5' diphosphate, thymidine 5 'diphosphate glucose and thymidine 5 'triphosphate.
- a compound according to the invention constitutes an antigen isolated from human Tyg lymphocytes, in particular T lymphocytes with TCR receptors comprising the variable regions Vyg and V 2, and can be used as such.
- an antigen according to the invention is not peptide. It also does not consist of a protein such as hsp60 or hsp65, nor of a peptide derived therefrom. This is why the traditional methods used in the prior art to isolate these antigens from an antigenic solution, and which assume that the antigens are of a protein nature, have failed.
- the invention also relates to a pharmaceutical composition, and more particularly to a composition vaccinating comprising at least one organophosphorus compound according to the invention.
- the invention relates to the use of at least one organophosphorus compound according to the invention for the preventive or curative treatment of bacterial infectious diseases, in particular mycobacterials such as leprosy or tuberculosis, and / or tumor or leukemic diseases and / or parasitic diseases and / or immunodeficiency diseases such as AIDS and / or parasitic diseases, in particular malaria, humans or animals.
- the invention thus relates to the use of at least one organophosphorus compound according to the invention for obtaining a pharmaceutical composition intended for the preventive or curative treatment of these diseases, in particular by general route.
- the pharmaceutical composition is formulated in a galenical form allowing its administration by intravascular or intramuscular route, that is to say directly into the blood in contact with the Ty5 lymphocytes which must be activated.
- the invention also relates to a method for characterizing an antigen of human T y lymphocytes, in particular a compound according to the invention, characterized in that:
- the invention also relates to a process for preparing and / or isolating and / or characterizing at least one compound according to the invention, characterized in that an aqueous solution activating human Ty lymphocytes is subjected to at least one step of separation by chromatography preparative in different fractions, and in that the activity of each fraction is tested on human Tyg lymphocytes.
- the method according to the invention for isolating at least a compound according to the invention is characterized by the following stages:
- a strain of bacteria in particular mycobacteria, is cultivated, a lipid extraction is carried out, and the aqueous phase is separated from the crude lipid extract.
- Mycobacteri um tuberculosis strain H37Rv or H37Ra is cultivated.
- a strain of mycobacteria whose growth is rapid is cultivated.
- the separation step by chromatography comprises an anionic separation by anion exchange chromatography with a saline solution from which the saline eluate which activates the Tyg lymphocytes is recovered.
- the step of separation by chromatography comprises at least one preparative separation of the different active principles of the eluate by HPLC chromatography on CI 8 reverse phase eluted in ion pair and / or by HPAEC anion exchange chromatography with conductimetric detection operating in chemical suppression mode, each separation step being followed by a measurement of the activity of the various fractions obtained on the yg lymphocytes.
- an organophosphorus compound according to the invention or subject an aqueous solution of this compound to an enzymatic treatment by incorporating it into a mixture comprising at least one phosphoric phosphohydrolase monoester and / or at least one phosphoric phosphohydrolase diester, and measures the activity of this mixture on Tyg lymphocytes. If the aqueous starting solution comprises a compound according to the invention, there is a disappearance of its activity on T ⁇ ⁇ lymphocytes.
- the aqueous starting solution is subjected to at least one chromatographic analysis of its various active ingredients by HPLC chromatography on reverse phase C18 eluted in ion pair and / or by HPAEC anion exchange chromatography with conductimetric detection operating in chemical suppression mode, each separation step being followed by a measurement of the activity of the different fractions obtained on Tyg lymphocytes.
- the invention also relates to organophosphorus compounds which can be isolated and / or characterized according to the method according to the invention.
- the invention also relates to a therapeutic composition intended to inhibit the proliferation and / or cytotoxicity of Tygg2 lymphocytes, characterized in that it contains a pharmaceutically acceptable proportion of at least one principle having an enzymatic phosphatase activity which is capable of cleave at least one compound activating human Ty5 lymphocytes with TCR receptors comprising the variable regions Vyg and V52.
- the composition comprises a phosphoric phosphohydrolase monoester.
- the composition comprises at least one nucleotide pyrophosphatase and / or at least one phosphoric diester phosphohydrolase.
- the composition comprises at least one enzyme alkaline phosphatase.
- this composition is formulated in a galenic form allowing its administration directly in contact with the medium incorporating the pathogenic autoimmune Ty5 lymphocytes or in contact with a medium capable of transporting the composition in contact with the pathogenic autoimmune Ty lymphocytes .
- composition according to the invention is advantageously formulated in a galenical form allowing its administration by injection into the circulating blood for example, by intravascular route, in particular intravenous, or intramuscular, or intradermal, or other.
- the invention also relates to the use of a composition according to the invention for obtaining a medicament inhibiting the cytotoxicity of Tyg52 lymphocytes implicated in at least one autoimmune pathology, in particular of multiple sclerosis.
- T 962 lymphocytes are involved in the neurological degradations of multiple sclerosis, and the composition according to the invention aims to inhibit the cytotoxic activity in situ of Tyg lymphocytes, that is to say, their destructive activity. cells of the central nervous system.
- FIG. 1 is a gel permeation chromatogram of a mycobacterial extract incorporating the organophosphorus compounds according to the invention, with an indication of the estimated molecular weights,
- FIG. 2 is an HPLC C18 chromatogram in an ion pair obtained by a chromatographic separation step of a process for the preparation of the organophosphorus compounds according to the invention
- FIG. 3 is a dot graph illustrating the amplification of the Tyg lymphocytes in culture with an organophosphorus compound according to the invention
- - Figures 4a and 4b illustrate the two-dimensional etric cytofluori profile of y9 2 lymphocytes in culture respectively without and with an organophosphorus compound according to the invention
- - Figure 5 is a titration curve of the lymphoproliferative effect of a compound organophosphorus according to the invention, for a Ty952- lymphocyte clone
- FIG. 6 is a nuclear magnetic resonance spectrum H of an organophosphorus compound according to the invention.
- FIG. 7 is a mass spectrum of an organophosphorus compound according to the invention.
- FIG. 8 illustrates three superimposed chromatograms HPLC C18 er pair of ions respectively of inactive control phosphorylated molecules; an organophosphorus compound according to the invention treated with an alkaline phosphohydrolase monoester; and a nucleotide pyrophosphatase,
- FIG. 9 is a histogram illustrating the cytotoxicity induced by an organophosphorus compound according to the invention, on Ty5 lymphocytes in the presence of monoclonal antibodies of different specificities.
- a strain culture of another mycobacterium it is advantageous to use a fast growing mycobacterium and secreting the desired compounds in the culture medium.
- Mycobacteri um fortui tum biovar fortui tum can be used. In this case, it is not necessary to produce a crude extract, and it suffices to collect the culture medium from which the water-soluble compounds are separated.
- ME extract contains the compounds which are separated by preparative chromatography into fractions capable of activating the T y g 2- lymphocytes.
- the activity of the chromatographic fractions is determined by examining the induced proliferation of a clone of human Tygg2 lymphocytes active on the starting mycobacteria (for example the clone G115) by the method described by F. DAVODEAU et al., Journal of Immunology Flight. 151, P 1214 (1993) (lympho proliferation test).
- the compounds are purified from the ME extract by an anion exchange chromatographic separation, followed by a chromatographic separation of the saline solution by a column of silicic acid, and by an HPLC chromatographic separation on CI8 reverse phase in d pairs. 'ions and / or HPAEC anion exchange chromatographic separation.
- the solutions of the extract ME are passed through an anion exchange column of the diethylaminoethyl (DEAE) type in which the active ingredients are eluted by increasing concentrations of ammonium acetate.
- the stimulatory activity of each eluate is tested on the clone G115 by the lymphoproliferation test.
- the eluents active on the clone G115 are then treated in a chromatographic column of silicic acid to separate them from the saline solution of ammonium acetate.
- the successive eluents used are 1% ethanol, 1% ethanol 90%, propanol 2 100% and propanol H2O (70% - 30%).
- the eluates are dried and subjected to the lymphoproliferation test. It is noted that the last eluate is active, and contains the compounds according to the invention.
- the different active principles of the eluate thus obtained are then subjected to a chromatographic separation on a column C18 for separation of the hydrophilic phase.
- the products eluted in aqueous phase are then subjected to an HPLC chromatographic separation on a preparative column 250/40 C18 such as BISCHOFF (registered trademark) ULTRASEP 10 ⁇ m eluted in pair of ions with 0.1 M ammonium acetate.
- L he absorption at the different wavelengths is controlled by an absorption detector in the ultraviolet. Chromatographic fractions are collected every 30 seconds for a total of 40 min.
- the chromatogram at 260 nm is given by the dotted curve in FIG. 2.
- the white dot curve illustrates the activity of the different chromatographic fractions measured by the lymphoproliferation test. This activity is expressed in Kcpm (thousands of strokes per minute) of the tritiated thymidine incorporated by the clone G115.
- Table I provides the retention times of the four purified compounds and of various standard biomolecules by HPAEC.
- P phosphate
- PF phosphonoformic acid
- PEP phosphoenolpyruvic acid
- TMP5 ' thymidine 5' monophosphate
- TDP5 ' thymidine 5' diphosphate
- TDP5 'Glc thymidine 5' diphosphoryl-1 glucose
- TTP5 ' thymidine 5' triphosphate.
- Detector used conductivity meter preceded by an ionic suppressor with a DIONEX membrane (registered trademark).
- the anionic character of TUBagI and TUBag2 is analogous to that of thymidine 5 'monophosphate (TMP). Also, the anionic character of the isolated antigens TUBag3 and TUBag4 is more important than that of thymidine 5 'diphosphate, but less important than that of thymidine 5' triphosphate.
- the anionic character of the antigenic compounds TUBagI, TUBag2, TUBag3 and TUBag4 is therefore greater than that of phosphonoformic acid and less than that of thymidine 5 'triphosphate.
- ANALYSIS OF THE STIMULATION OF Ty 6 LYMPHOCYTES BY THE TUBag4 COMPOUND The circulating blood lymphocytes were collected from four healthy human donors. These lymphocytes are cultured in the presence of interleukin 2 for 10 days, with or without the compound TUBag4, in a saturating amount. At the end of culture, the numbers of T ⁇ , Ty5 or CD3- (non-T) lymphocytes are compared in each of the two cultures.
- FIG. 3 shows the amplification ratio, ie the ratio of the number of cells in culture with TUBag4 to the number of cells in culture without TUBag4. Each point represents a donor.
- TUBag4 induces in culture a specific polyclonal amplification of the Ty lymphocytes by a factor of between 40 and 500 depending on the individual.
- FIG. 4a and b show the phenotype of the TCR receptor of circulating blood lymphocytes from a healthy human donor cultured in the absence ( Figure 4a) and in the presence (Figure 4b) of TUBag4.
- TUBag4 induces a massive but selective amplification of the Tyg52 lymphocytes.
- FIG. 5 shows the titration of the proliferative effect of TUBag4 on a clone of Tyg52 lymphocytes (G115) measured by the lymphoproliferation test.
- curve with black dots a proliferative effect of TUBag4 has been observed from a concentration of the order of 1 ng / ml.
- the white dot curve reflects the proliferative effect in the absence of TUBag4.
- TUBagI, TUBag2 and TUBag3 confirm the character common antigen of these compounds for lymphocytes
- the aqueous phase of the above-mentioned mycobacterial extract (ME) was first subjected to two experiments.
- the lymphoproliferation test on the clone G115 was carried out on the ME extract alone, on the ME extract in the presence of proteinase K (Merck, 6 mU / mp, 2 h at 37 ° C.), and on the ME extract in the presence of periodate (1 OmM of NaHI04, 2 h at 37 ° C.).
- the test was conducted on the ME extract alone, then on the ME extract in the presence of the phosphoric monoester phosphohydrolase enzyme, in particular alkaline phosphatase.
- the specific lymphoproliferations obtained are expressed in Table II below, after subtracting the spontaneous proliferation (unstimulated) of the clone G115:
- the active antigenic ME extracted G115 T cell clone y g 2-ME This extract is protease resistant, but its activity in the G115 clone is inhibited in the presence of periodate.
- the ME extract therefore undergoes degradation by periodic acid.
- the ME extract is partially degraded under the enzymatic action of an alkaline phosphatase. Consequently, "the antigens are water-soluble, non-peptide in nature, but are acid-labile and organophosphorus.
- the fractions active on the lymphocytes all have a molecular weight less than 1000, and which can be estimated on the order of 500.
- the isolated TUBag4 antigen was subjected to structural analysis by nuclear magnetic resonance.
- the spectrum obtained by the one-dimensional H analysis (FIG. 6) demonstrates the presence of the 2-deoxyribose groups and of thymine.
- two-dimensional, homonuclear H 'H and heteronuclear H ⁇ C magnetic resonance tests make it possible to clearly identify the bond of the thymine group by its first nitrogen to the anomeric carbon of the 2-deoxyribose radical.
- the results make it possible to postulate the binding of the fifth carbon of the 2-dezoxyribose radical to a phosphoryl group.
- the lymphoproliferation test on the clone G115 carried out with thymidine " 5 'monophosphate, thymidine 5' diphosphate and thymidine 5 'triphosphate demonstrates the inactivity of these components on lymphocytes. Consequently, the antigenic compounds according to the invention, do not exclusively consist of these 5 'phosphated thymidines.
- the enzymes used were the phosphoric monoester alkaline phosphohydrolase MP (EC 3.1.3.1), the phosphoric diester phosphohydrolase DP (EC 3.1.4.1) of rattlesnake venom, and the nucleotide pyrophosphatase NPP (EC 3.6.1.9) of rattlesnake venom.
- the phosphoric monoester phosphohydrolase inactivates TUBagI and TUBag2 y but does not inactivate TUBag3 and TUBag4. Consequently, TUBagI and TUBag2 are phosphoric monoesters.
- nucleotide pyrophosphatase does not inactivate TUBagI and TUBag2, but inactivates TUBag3 and cleaves TUBag4. Consequently, the presence of a nucleotide nucleus is confirmed in TUBag3 and TUBag4.
- the activity of antigens in the presence of enzymes was analyzed by C18 reverse-side HPLC chromatography in an ion pair.
- the results are illustrated in FIG. 8 in which the upper curve in dotted lines illustrates the profile of the thymidines TTP, TDP, TDPGlc and TMP.
- the middle curve illustrates the results of the chromatographic separation of TUBag4 previously treated with the phosphoric phosphohydrolase monoester, and the lower curve illustrates the activity of TUBag4 previously treated with the nucleotide pyrophosphatase.
- the compound TUBag4 subjected to the nucleotide pyrophosphatase enzyme provides the compound TUBagI.
- TUBag3 and TUBag4 have a substituent which is a nucleoside group identified in TUBag4 as thymidine linked to the phosphoryl group in the ester by the fifth carbon of its 2-deoxyribose radical.
- TUBagI is the product of the hydrolysis of TUBag4 by the enzymatic action of a nucleotide pyrophosphatase or a phosphoric diester phosphohydrolase.
- TUBagI is a phosphoric monoester which is the active product of the spontaneous or enzymatic hydrolysis of TUBag4;
- TUBag2 is a phosphoric monoester of anionic character similar to that of TUBagI but of higher hydrophobic character;
- TUBag3 is a pyrophosphoric nucleotide diester;
- TUBag4 is a diphosphoric or triphosphoric diester of 5 'thymidine incorporating TUBagI.
- the presence of this antigenic organophosphorus compound can be characterized by subjecting this solution to the chromatographic separation steps mentioned above for the mycobacterial extract ME , in particular at least one preparative separation of the various active principles of the eluate by HPLC chromatography on CI8 reverse phase eluted in ion pair and / or by HPAEC anion exchange chromatography with conductimetric detection operating in chemical suppression mode, each step separation is followed by a measurement of the activity of the various fractions obtained on Tyg lymphocytes.
- CYTOTOXICITY OF Tyg LYMPHOCYTES IN THE PRESENCE OF ISOLATED ORGANO-PHOSPHORUS COMPOUNDS
- the cytolytic activity of a clone of Tyg52 lymphocytes (G115) and of a control clone of non ⁇ ⁇ T lymphocyte. (BH) against target cells is measured in the absence or in the presence of the TUBag compound.
- This activity induced by TUBag4 is determined by the following formula: (% of specific lysis in the presence of TUBag4) - (% of specific lysis without TUBag4).
- This cytotoxicity is estimated at a ratio of killer cells to target cells of 20 to 1.
- TUBag4 induces lysis of all the target cells by the clone G115 only, but does not induce lysis by the control clone BH.
- This lytic activity is not specific to the target cell.
- This cytolytic activity is also not restricted by the MHC complex.
- the antigenic compounds according to the invention activate the cytotoxicity of the Ty ⁇ G115 lymphocyte on different cancer cells originating from different human or mouse donors.
- the compounds according to the invention directly activate the cytotoxic activity of the y5 G115 lymphocyte without interfering with the target cell.
- the invention thus makes it possible to stimulate or inhibit, that is to say to modulate, the proliferation and / or the cytotoxicity of the Ty lymphocytes.
- an organo-phosphorus compound such as TUBagI
- TUBag2, TUBag3, TUBag4, or a compound incorporating these organophosphorus compounds or a similar group to obtain a pharmaceutical composition for the preventive or curative treatment of human or animal diseases in which the Ty5 lymphocytes are involved, in particular infectious diseases (in particular mycobacterials such as leprosy or tuberculosis); human or animal tumor or leukemia disorders; parasitic infections; immuno-deficient pathologies such as AIDS, ...
- a monoester phosphatase and / or a diester phosphatase is used.
- a pyrophosphatase such as a pyrophosphatase nucleotide
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Abstract
Non-peptide hydrosoluble organo-phosphorus containing compound for use as a human T¿η9δ2? cell activator, comprising at least one acidolabile ester bond of phosphoric acid. The activating properties of said compound in relation to lymphocytes are lost when it is placed in the presence of an enzymatic mixture comprising at least one phosphoric phosphohydrolase monoester and at least one phosphoric phosphohydrolase diester. The invention also concerns a method for the preparation, isolation or characterization of such a compound and compositions and pharmaceutical uses thereof.
Description
COMPOSES 0RGAN0-PH0SPH0RES ACTIVATEURS DES LYMPHOCYTES T GAMMA DELTA COMPOUNDS 0RGAN0-PH0SPH0RES ACTIVATORS OF T GAMMA DELTA LYMPHOCYTES
L'invention concerne des composés organo- phosphorés hydrosolubles non-peptidiques stimulant des lymphocytes y humains. L'invention concerne aussi un procédé pour préparer et/ou isoler et/ou caractériser de tels composés organo-phosphorés, une composition pharmaceutique, notamment vaccinante comportant au moins un tel composé organo-phosphoré, diverses utilisations de ces composés pour obtenir des compositions pharmaceutiques, une composition pharmaceutique inhibant l'activité de ces composés sur les lymphocytes y5 et les utilisations de cette composition pour obtenir des médicaments.The present invention relates to non-peptide water-soluble organophosphorus compounds that stimulate human lymphocytes. The invention also relates to a process for preparing and / or isolating and / or characterizing such organophosphorus compounds, a pharmaceutical composition, in particular a vaccinating composition comprising at least one such organophosphorus compound, various uses of these compounds for obtaining pharmaceutical compositions , a pharmaceutical composition inhibiting the activity of these compounds on y5 lymphocytes and the uses of this composition for obtaining medicaments.
On sait déjà que les lymphocytes Ty humains à récepteurs TCR comprenant les régions variables Vy et V (lymphocytes CD3+, TCR αβ-, CD4-, CD8-) jouent un rôle dans le système immunitaire de l'homme et de l'animal. Par exemple, leur présence a été démontrée dans le cadre de diverses maladies telles que les infections bactériennes, notamment mycobactériennes (en particulier la tuberculose et la lèpre) ou à streptocoques, les affections tumorales ou leucémiques, certains parasites (par exemple le Plasmodi um) , le SIDA et les maladies auto-immunes (sclérose en plaques, lupus, ...).It is already known that human Ty lymphocytes with TCR receptors comprising the variable regions Vy and V (CD3 + lymphocytes, TCR αβ-, CD4-, CD8-) play a role in the immune system of humans and animals. For example, their presence has been demonstrated in the context of various diseases such as bacterial, in particular mycobacterial (in particular tuberculosis and leprosy) or streptococcal infections, tumor or leukemic affections, certain parasites (for example Plasmodi um) , AIDS and autoimmune diseases (multiple sclerosis, lupus, ...).
On sait aussi que dans le sang périphérique de l'adulte, les lymphocytes T porteurs de TCR à régions variables Vyg V 2 (lymphocytes Ty952) représentent la majorité (de l'ordre de 80 %) des lymphocytes y5.It is also known that in peripheral adult blood, T lymphocytes carrying variable regions Vyg V 2 TCRs (Ty952 lymphocytes) represent the majority (of the order of 80%) of y 5 lymphocytes.
On cherche ainsi depuis la découverte des lymphocytes Ty5 à comprendre le mécanisme de leur activation et leurs fonctions physiologiques.Since the discovery of Ty5 lymphocytes, we have therefore sought to understand the mechanism of their activation and their physiological functions.
Certains auteurs ont pensé que les lymphocytes Ty5 répondent à un antigène formé d'une protéine telle que HSP60. D'autres auteurs ont cependant contredit cette thèse. Néanmoins, il n'a pas encore été possible d'isoler un antigène des lymphocytes Ty§.
Il a été aussi démontré que les lymphocytes Ty5 reconnaissent et lysent les cellules cancéreuses et les cellules infectées, notamment par les ycobactéries, et s'accumulent sur certains sites infectieux. Or, les affections sus-mentionnées dans lesquelles les lymphocytes Tyg interviennent sont pour la plupart considérées à ce jour comme particulièrement sévères. En conséquence, depuis la découverte des lymphocytes y5, on cherche à mettre en évidence le (les) antigène(s) susceptible(s) de les activer et à expliquer la raison de leur présence en grand nombre dans le sang périphérique de l'adulte.Some authors have thought that Ty5 lymphocytes respond to an antigen formed by a protein such as HSP60. Other authors have, however, contradicted this thesis. However, it has not yet been possible to isolate an antigen from Ty§ lymphocytes. Ty5 lymphocytes have also been shown to recognize and lyse cancer cells and infected cells, including ycobacteria, and accumulate at certain infectious sites. However, the above-mentioned conditions in which Tyg lymphocytes are involved are for the most part considered to date to be particularly severe. Consequently, since the discovery of the y 5 lymphocytes, it is sought to highlight the antigen (s) capable of activating them and to explain the reason for their presence in large numbers in the peripheral blood of the adult.
Par ailleurs, dans certains cas, les lymphocytes Ty5 agissent comme des agents pathogènes qu'il serait souhaitable de pouvoir inhiber. Tel est le cas en particulier dans le cadre des maladies auto-immunes, telles que la sclérose en plaques.Furthermore, in certain cases, the Ty5 lymphocytes act as pathogenic agents which it would be desirable to be able to inhibit. This is particularly the case in the context of autoimmune diseases, such as multiple sclerosis.
L'invention vise donc, de façon générale, à proposer des moyens pour contrôler la prolifération et/ou l'activité cytotoxique des lymphocytes y5, notamment des lymphocytes Tyg§2 humains, que ce soit pour les stimuler en vue de favoriser leur rôle immunitaire, ou pour les inhiber lorsqu'ils sont impliqués dans une pathologie, notamment auto-immune. Ainsi, l'invention vise à proposer des composés isolés activateurs des lymphocytes y5, notamment des lymphocytes Ty952, leur procédé de préparation, leur utilisation dans une composition pharmaceutique, notamment vaccinante, et leurs diverses utilisations thérapeutiques pour le traitement préventif ou curatif de l'homme ou de 1' animal .The invention therefore aims, in general, to propose means for controlling the proliferation and / or cytotoxic activity of y5 lymphocytes, in particular human Tyg§2 lymphocytes, whether it is to stimulate them in order to promote their immune role. , or to inhibit them when they are involved in a pathology, in particular autoimmune. Thus, the invention aims to propose isolated compounds which activate y5 lymphocytes, in particular Ty952 lymphocytes, their preparation process, their use in a pharmaceutical composition, in particular a vaccinating composition, and their various therapeutic uses for the preventive or curative treatment of the man or animal.
L'invention vise en particulier à proposer un procédé et une composition pour stimuler la réponse immunitaire médiée par les lymphocytes Ty . Et l'invention vise à proposer une utilisation de ce procédé ou de cette composition dans le traitement préventif (vaccin) ou curatif (im unomodulateur) : des maladies infectieuses à germes procaryotes ou eucaryotes -notamment
mycobactériennes, à streptocoques ou à plasmodium- ; et/ou des pathologies tumorales ou leucémiques ; et/ou des pathologies d' immunodéficiences telles que le SIDA.The invention aims in particular to provide a method and a composition for stimulating the immune response mediated by Ty lymphocytes. And the invention aims to propose a use of this process or of this composition in the preventive (vaccine) or curative (im unomodulator) treatment: infectious diseases with prokaryotic or eukaryotic germs -in particular mycobacterial, streptococcal or plasmodium-; and / or tumor or leukemic pathologies; and / or immunodeficiency pathologies such as AIDS.
L'invention vise également à proposer un procédé pour isoler et/ou caractériser des composés activant les lymphocytes Ty§, notamment des lymphocytes Ty952 humains.The invention also aims to propose a method for isolating and / or characterizing compounds activating Ty § lymphocytes, in particular human Ty952 lymphocytes.
L'invention vise en outre à proposer une composition destinée à inactiver les lymphocytes Ty5, et plus particulièrement une composition thérapeutique inactivant la cytotoxicité des lymphocytes Ty5 impliqués dans des pathologies auto-immunes telles que la sclérose en plaques.The invention further aims to provide a composition intended for inactivating Ty5 lymphocytes, and more particularly a therapeutic composition inactivating the cytotoxicity of Ty5 lymphocytes involved in autoimmune pathologies such as multiple sclerosis.
Pour ce faire, l'invention concerne un composé organo-phosphoré hydrosoluble de nature non- peptidique, activateur des lymphocytes Tyg humains à récepteurs TCR comprenant les régions variables Vyg et V52, ce composé comprenant au moins une liaison ester acidolabile de l'acide phosphorique, le caractère activateur de ce composé vis-à-vis des lymphocytes y5 disparaissant lorsqu'il est placé en présence d'un mélange enzymatique comprenant au moins une monoester phosphorique phosphohydrolase et au moins une diester phosphorique phosphohydrolase. Selon l'invention, le mélange enzymatique peut comprendre une pyrophosphohydrolase à titre de diester phosphorique phosphohydrolase.To do this, the invention relates to a water-soluble organophosphorus compound of a non-peptide nature, activator of human Tyg lymphocytes with TCR receptors comprising the variable regions Vyg and V52, this compound comprising at least one acidolabile ester bond of phosphoric acid. , the activating character of this compound with respect to the y5 lymphocytes disappearing when it is placed in the presence of an enzyme mixture comprising at least one phosphoric phosphohydrolase monoester and at least one phosphoric phosphohydrolase diester. According to the invention, the enzymatic mixture can comprise a pyrophosphohydrolase as phosphoric diester phosphohydrolase.
Un composé selon l'invention présente un caractère anionique mesuré par chromatographie HPAEC à détection conductimétrique en mode de suppression chimique qui est supérieur à celui de l'acide phosphonoformique et inférieur à celui de la thymidine 5' triphosphate. Egalement, l'invention concerne un composé caractérisé en ce que, après traitement par une enzyme diester phosphorique phosphohydrolase, il présente un caractère anionique mesuré par chromatographie HPAEC à détection conductimétrique en mode de suppression chimique qui est supérieur à celui de l'acide phosphonoformique et inférieur à celui de la thymidine 5' triphosphate. En effet,
l'invention comprend les composés phosphodiester X-Phosphate-R dont la dégradation par l'enzyme diesterase libère le principe actif minimum X-Phosphate.A compound according to the invention has an anionic character measured by HPAEC chromatography with conductometric detection in chemical suppression mode which is higher than that of phosphonoformic acid and lower than that of thymidine 5 'triphosphate. Also, the invention relates to a compound characterized in that, after treatment with a phosphoric diester phosphohydrolase enzyme, it has an anionic character measured by HPAEC chromatography with conductimetric detection in chemical suppression mode which is superior to that of phosphonoformic acid and lower than that of thymidine 5 'triphosphate. Indeed, the invention comprises the phosphodiester X-Phosphate-R compounds, the degradation of which by the enzyme diesterase releases the minimum active principle X-Phosphate.
Egalement, l'invention concerne un composé qui présente un caractère hydrophobe mesuré par chromatographie HPLC sur phase inverse de type C18 éluée en paire d'ions avec l'acétate d'ammonium qui est inférieur à celui de la thymidine 5' monophosphate. Plus particulièrement, un composé selon l'invention est caractérisé en ce que son activation sur les lymphocytes Ty5 est inhibée en présence d'anticorps monoclonaux spécifiques des TCR humains comprenant les régions variables Vyg ou V52-Also, the invention relates to a compound which has a hydrophobic character measured by reverse phase HPLC chromatography of type C18 eluted in ion pair with ammonium acetate which is lower than that of thymidine 5 'monophosphate. More particularly, a compound according to the invention is characterized in that its activation on Ty5 lymphocytes is inhibited in the presence of monoclonal antibodies specific for human TCRs comprising the variable regions Vyg or V52-
Un composé selon 1 ' invention comprend au moins un substituant X de masse moléculaire inférieure à 500, qui est distinct d'un acide nucléique, d'un oligosaccharide, d'un acide gras (c'est-à-dire un acide carboxylique saturé comprenant au moins quatre carbones), d'un protide, d'un alcaloïde et d'un stéroïde, et qui est lié dans le composé à un groupement phosphoryle par une liaison covalente acidolabile susceptible d'être clivée en présence d'une monoester phosphatase. En effet, il apparaît que la présence de la séquence X-Phosphate suffit pour conférer un pouvoir antigénique au composé selon l'invention à l'égard des lymphocytes Tyg.A compound according to the invention comprises at least one substituent X of molecular mass less than 500, which is distinct from a nucleic acid, from an oligosaccharide, from a fatty acid (that is to say a saturated carboxylic acid comprising at least four carbons), a protide, an alkaloid and a steroid, and which is linked in the compound to a phosphoryl group by an acidolabile covalent bond capable of being cleaved in the presence of a monoester phosphatase . Indeed, it appears that the presence of the X-Phosphate sequence is sufficient to confer an antigenic power to the compound according to the invention with regard to Tyg lymphocytes.
Par ailleurs, au moins un des composés organo-phosphorés selon 1 ' invention comporte au moins un groupement nucléotidique. En particulier, l'invention concerne un composé répondant à la formule X-Phosphate—R où R est un hydrogène ou un substituant minéral ou organique, notamment un groupement nucléosidique, et X est un substituant tel que mentionné ci-dessus.Furthermore, at least one of the organophosphorus compounds according to the invention comprises at least one nucleotide group. In particular, the invention relates to a compound corresponding to the formula X-Phosphate — R where R is a hydrogen or an inorganic or organic substituent, in particular a nucleoside group, and X is a substituent as mentioned above.
En particulier, l'invention concerne un composé répondant à la formule :In particular, the invention relates to a compound corresponding to the formula:
X 0 - R
ou :X 0 - R or :
R est un hydrogène ou un substituant minéral ou organique, n est un nombre entier non nul.R is hydrogen or an inorganic or organic substituent, n is a non-zero integer.
L'invention concerne aussi un composé constitué d'un produit d'hydrolyse obtenu à partir d'un composé selon 1 ' invention qui comprend la thymidine liée à un groupement phosphoryle par le cinquième carbone de son radical 2-désoxyribose, cette hydrolyse pouvant notamment être obtenue par l'action enzymatique d'une nucléotide pyrophosphatase et/ou d'une diester phosphorique phosphohydrolase.The invention also relates to a compound consisting of a hydrolysis product obtained from a compound according to the invention which comprises thymidine linked to a phosphoryl group by the fifth carbon of its 2-deoxyribose radical, this hydrolysis possibly in particular be obtained by the enzymatic action of a nucleotide pyrophosphatase and / or a phosphoric diester phosphohydrolase.
Egalement, l'invention concerne un composé constitué d'un monoester phosphorique acidolabile de masse moléculaire inférieure à 500 distinct de la thymidine 5 ' monophosphate, de la thymidine 5' diphosphate, de la thymidine 5 ' diphosphate glucose et de la thymidine 5 ' triphosphate.Also, the invention relates to a compound consisting of an acidolabile phosphoric monoester of molecular mass less than 500 distinct from thymidine 5 'monophosphate, thymidine 5' diphosphate, thymidine 5 'diphosphate glucose and thymidine 5' triphosphate .
Egalement, l'invention concerne un composé constitué d'un diester phosphorique acidolabile de masse moléculaire inférieure à 1 000, distinct de la thymidine 5' monophosphate, de la thymidine 5' diphosphate, de la thymidine 5 ' diphosphate glucose et de la thymidine 5 ' triphosphate.Also, the invention relates to a compound consisting of an acid-labile phosphoric diester with a molecular mass of less than 1000, distinct from thymidine 5 'monophosphate, thymidine 5' diphosphate, thymidine 5 'diphosphate glucose and thymidine 5 'triphosphate.
Un composé selon l'invention constitue un antigène isolé des lymphocytes Tyg humains, notamment des lymphocytes T à récepteurs TCR comprenant les régions variables Vyg et V 2, et peut être utilisé en tant que tel.A compound according to the invention constitutes an antigen isolated from human Tyg lymphocytes, in particular T lymphocytes with TCR receptors comprising the variable regions Vyg and V 2, and can be used as such.
Il est à noter que contrairement au préjugé général de l'art antérieur, et de façon surprenante, un antigène selon l'invention n'est pas peptidique. Il n'est pas non plus constitué d'une protéine telle que hsp60 ou hsp65, ni d'un peptide en dérivant. C'est pourquoi, les procédés traditionnels mis en oeuvre dans l'art antérieur pour isoler ces antigènes à partir d'une solution antigenique, et qui supposent que les antigènes sont de nature protéique, ont échoué.It should be noted that, contrary to the general prejudice of the prior art, and surprisingly, an antigen according to the invention is not peptide. It also does not consist of a protein such as hsp60 or hsp65, nor of a peptide derived therefrom. This is why the traditional methods used in the prior art to isolate these antigens from an antigenic solution, and which assume that the antigens are of a protein nature, have failed.
L'invention concerne aussi une composition pharmaceutique, et plus particulièrement une composition
vaccinante comprenant au moins un composé organo—phosphore selon l'invention.The invention also relates to a pharmaceutical composition, and more particularly to a composition vaccinating comprising at least one organophosphorus compound according to the invention.
Et l'invention concerne l'utilisation d'au moins un composé organo-phosphoré selon 1 ' invention pour le traitement préventif ou curatif des maladies infectieuses bactériennes, notamment mycobactériennes comme la lèpre ou la tuberculose, et/ou des affections tumorales ou leucémiques et/ou des parasitoses et/ou des maladies d' immunodéficiences telles que le SIDA et/ou des maladies parasitaires, notamment du paludisme, de l'homme ou de l'animal. L'invention concerne ainsi l'utilisation d'au moins un composé organo-phosphoré selon 1 ' invention pour obtenir une composition pharmaceutique destinée au traitement préventif ou curatif de ces maladies, notamment par voie générale. Plus particulièrement, la composition pharmaceutique est formulée sous une forme galénique permettant son administration par voie intravasculaire ou intramusculaire, c'est-à-dire directement dans le sang au contact des lymphocytes Ty5 qui doivent être activés. L'invention concerne aussi un procédé pour caractériser un antigène des lymphocytes Ty humains, notamment un composé selon l'invention, caractérisé en ce que :And the invention relates to the use of at least one organophosphorus compound according to the invention for the preventive or curative treatment of bacterial infectious diseases, in particular mycobacterials such as leprosy or tuberculosis, and / or tumor or leukemic diseases and / or parasitic diseases and / or immunodeficiency diseases such as AIDS and / or parasitic diseases, in particular malaria, humans or animals. The invention thus relates to the use of at least one organophosphorus compound according to the invention for obtaining a pharmaceutical composition intended for the preventive or curative treatment of these diseases, in particular by general route. More particularly, the pharmaceutical composition is formulated in a galenical form allowing its administration by intravascular or intramuscular route, that is to say directly into the blood in contact with the Ty5 lymphocytes which must be activated. The invention also relates to a method for characterizing an antigen of human T y lymphocytes, in particular a compound according to the invention, characterized in that:
- l'on vérifie qu'il induit une activation des lymphocytes,- it is verified that it induces activation of the lymphocytes,
- l'on vérifie que cette propriété vis-à- vis des lymphocytes disparaît lorsqu'il est placé en présence d'un mélange enzymatique comprenant au moins une monoester phosphorique phosphohydrolase et au moins une diester phosphorique phosphohydrolase.- It is verified that this property vis-à-vis lymphocytes disappears when it is placed in the presence of an enzyme mixture comprising at least one phosphoric phosphohydrolase monoester and at least one phosphoric phosphohydrolase diester.
L'invention concerne aussi un procédé pour préparer et/ou isoler et/ou caractériser au moins un composé selon l'invention, caractérisé en ce qu'on soumet une solution aqueuse activant les lymphocytes Ty humains à au moins une étape de séparation par chromatographie préparative en différentes fractions, et en ce que l'on teste l'activité de chaque fraction sur des lymphocytes Tyg humains. Le procédé selon l'invention pour isoler au moins
un composé selon 1 ' invention est caractérisé par les étapes suivantes :The invention also relates to a process for preparing and / or isolating and / or characterizing at least one compound according to the invention, characterized in that an aqueous solution activating human Ty lymphocytes is subjected to at least one step of separation by chromatography preparative in different fractions, and in that the activity of each fraction is tested on human Tyg lymphocytes. The method according to the invention for isolating at least a compound according to the invention is characterized by the following stages:
- on cultive une souche d'êtres vivants unicellulaires susceptible d'activer les lymphocytes TγQ humains à récepteurs TCR comprenant les régions variables
- a strain of single-celled living beings capable of activating human TγQ lymphocytes with TCR receptors comprising the variable regions is cultivated
- on réalise un extrait brut de la souche ou on collecte son milieu de culture, et on sépare les composants hydrosolubles de cet extrait ou milieu, - on sépare une solution aqueuse comprenant ces composants hydrosolubles par chromatographie préparative en différentes fractions susceptibles d'activer des lymphocytes Tyg humains à récepteurs TCR comprenant les régions variables Vyg V52. Selon l'invention, on cultive une souche de bactéries, notamment de mycobactéries, on effectue une extraction lipidique, et on sépare la phase aqueuse de l'extrait lipidique brut. .En particulier, on cultive Mycobacteri um tuberculosis souche H37Rv ou H37Ra. En variante, avantageusement et selon l'invention, on cultive une souche de mycobactéries dont la croissance est rapide. Plus particulièrement, on cultive Mycobacterium fortui bum biovar fortui tum apte à sécréter le (les) composé(s) dans le milieu de culture. Selon l'invention, l'étape de séparation par chromatographie comprend une séparation anionique par chromatographie échangeuse d'anions avec une solution saline dont on récupère l'éluat salin qui active les lymphocytes Tyg. Avantageusement, on prévoit une séparation de la solution saline qui active les lymphocytes Tyδ suivie d'un séchage.- a crude extract of the strain is produced or its culture medium is collected, and the water-soluble components are separated from this extract or medium, - an aqueous solution comprising these water-soluble components is separated by preparative chromatography into different fractions capable of activating Human Tyg lymphocytes with TCR receptors comprising the Vyg V52 variable regions. According to the invention, a strain of bacteria, in particular mycobacteria, is cultivated, a lipid extraction is carried out, and the aqueous phase is separated from the crude lipid extract. In particular, Mycobacteri um tuberculosis strain H37Rv or H37Ra is cultivated. As a variant, advantageously and according to the invention, a strain of mycobacteria whose growth is rapid is cultivated. More particularly, Mycobacterium fortui bum biovar fortui tum is cultivated capable of secreting the compound (s) into the culture medium. According to the invention, the separation step by chromatography comprises an anionic separation by anion exchange chromatography with a saline solution from which the saline eluate which activates the Tyg lymphocytes is recovered. Advantageously, provision is made for a separation of the saline solution which activates the Tyδ lymphocytes followed by drying.
Selon l'invention, l'étape de séparation par chromatographie comprend au moins une séparation préparative des différents principes actifs de l'éluat par chromatographie HPLC sur phase inverse CI 8 éluée en paire d'ions et/ou par chromatographie échangeuse d'anions HPAEC avec détection conductimétrique opérant en mode du suppression chimique, chaque étape de séparation étant
suivie d'une mesure de l'activité des différentes fractions obtenues sur les lymphocytes yg.According to the invention, the step of separation by chromatography comprises at least one preparative separation of the different active principles of the eluate by HPLC chromatography on CI 8 reverse phase eluted in ion pair and / or by HPAEC anion exchange chromatography with conductimetric detection operating in chemical suppression mode, each separation step being followed by a measurement of the activity of the various fractions obtained on the yg lymphocytes.
Pour caractériser un composé organo- phosphoré selon l'invention, ou soumet une solution aqueuse de ce composé à un traitement enzymatique en l'incorporant dans un mélange comprenant au moins une monoester phosphorique phosphohydrolase et/ou au moins une diester phosphorique phosphohydrolase, et on mesure l'activité de ce mélange sur des lymphocytes Tyg. Si la solution aqueuse de départ comprend un composé selon l'invention, on constate une disparition de son activité sur les lymphocytes Tγξ.. En outre, selon l'invention, on soumet la solution aqueuse de départ à au moins une analyse chromatographique de ses différents principes actifs par chromatographie HPLC sur phase inverse C18 éluée en paire d'ions et/ou par chromatographie échangeuse d'anions HPAEC avec détection conductimétrique opérant en mode de suppression chimique, chaque étape de séparation étant suivie d'une mesure de l'activité des différentes fractions obtenues sur les lymphocytes Tyg.To characterize an organophosphorus compound according to the invention, or subject an aqueous solution of this compound to an enzymatic treatment by incorporating it into a mixture comprising at least one phosphoric phosphohydrolase monoester and / or at least one phosphoric phosphohydrolase diester, and measures the activity of this mixture on Tyg lymphocytes. If the aqueous starting solution comprises a compound according to the invention, there is a disappearance of its activity on Tγ ξ lymphocytes. In addition, according to the invention, the aqueous starting solution is subjected to at least one chromatographic analysis of its various active ingredients by HPLC chromatography on reverse phase C18 eluted in ion pair and / or by HPAEC anion exchange chromatography with conductimetric detection operating in chemical suppression mode, each separation step being followed by a measurement of the activity of the different fractions obtained on Tyg lymphocytes.
L'invention concerne aussi les composés organo-phosphorés qui peuvent être isolés et/ou caractérisés selon le procédé selon l'invention.The invention also relates to organophosphorus compounds which can be isolated and / or characterized according to the method according to the invention.
Par ailleurs, l'invention concerne aussi une composition thérapeutique destinée à inhiber la prolifération et/ou la cytotoxicité des lymphocytes Tygg2 caractérisée en ce qu'elle contient une proportion pharmaceutiquement acceptable d'au moins un principe présentant une activité enzymatique phosphatase qui est susceptible de cliver au moins un composé activant des lymphocytes Ty5 humains à récepteurs TCR comprenant les régions variables Vyg et V52. Selon l'invention, la composition comporte une monoester phosphorique phosphohydrolase. En variante ou en combinaison, avantageusement et selon l'invention, la composition comporte au moins une nucléotide pyrophosphatase et/ou au moins une diester phosphorique phosphohydrolase. Selon l'invention, la composition comporte au moins une enzyme
phosphatase alcaline.Furthermore, the invention also relates to a therapeutic composition intended to inhibit the proliferation and / or cytotoxicity of Tygg2 lymphocytes, characterized in that it contains a pharmaceutically acceptable proportion of at least one principle having an enzymatic phosphatase activity which is capable of cleave at least one compound activating human Ty5 lymphocytes with TCR receptors comprising the variable regions Vyg and V52. According to the invention, the composition comprises a phosphoric phosphohydrolase monoester. As a variant or in combination, advantageously and according to the invention, the composition comprises at least one nucleotide pyrophosphatase and / or at least one phosphoric diester phosphohydrolase. According to the invention, the composition comprises at least one enzyme alkaline phosphatase.
Selon l'invention, cette composition est formulée sous une forme galénique permettant son administration directement au contact du milieu incorporant les lymphocytes Ty5 auto-immunes pathogènes ou au contact d'un milieu susceptible de transporter la composition au contact des lymphocytes Ty auto-immunes pathogènes.According to the invention, this composition is formulated in a galenic form allowing its administration directly in contact with the medium incorporating the pathogenic autoimmune Ty5 lymphocytes or in contact with a medium capable of transporting the composition in contact with the pathogenic autoimmune Ty lymphocytes .
Ainsi, la composition selon l'invention est avantageusement formulée sous une forme galénique permettant son administration par injection dans le sang circulant par exemple, par voie intravasculaire, notamment intraveineuse, ou intramusculaire, ou intradermique, ou autre.Thus, the composition according to the invention is advantageously formulated in a galenical form allowing its administration by injection into the circulating blood for example, by intravascular route, in particular intravenous, or intramuscular, or intradermal, or other.
L'invention concerne aussi l'utilisation d'une composition selon l'invention pour obtenir un médicament inhibant la cytotoxicité des lymphocytes Tyg52 impliqués dans au moins une pathologie auto-immune, notamment de la sclérose .en plaques. En effet, les lymphocytes T 962 sont impliqués dans les dégradations neurologiques de la sclérose en plaques, et la composition selon l'invention vise à inhiber l'activité cytotoxique in situ des lymphocytes Tyg, c'est-à-dire, leur activité destructrice des cellules du système nerveux central.The invention also relates to the use of a composition according to the invention for obtaining a medicament inhibiting the cytotoxicity of Tyg52 lymphocytes implicated in at least one autoimmune pathology, in particular of multiple sclerosis. Indeed, T 962 lymphocytes are involved in the neurological degradations of multiple sclerosis, and the composition according to the invention aims to inhibit the cytotoxic activity in situ of Tyg lymphocytes, that is to say, their destructive activity. cells of the central nervous system.
D'autres caractéristiques et avantages de l'invention apparaîtront de la description suivante des exemples et essais, qui se réfère aux figures annexées dans lesquelles :Other characteristics and advantages of the invention will appear from the following description of the examples and tests, which refers to the appended figures in which:
- la figure 1 est un chromatogramme par perméation de gel d'un extrait mycobactérien incorporant les composés organo-phosphorés selon l'invention, avec indication des masses moléculaires estimées,FIG. 1 is a gel permeation chromatogram of a mycobacterial extract incorporating the organophosphorus compounds according to the invention, with an indication of the estimated molecular weights,
- la figure 2 est un chromatogramme HPLC C18 en paire d'ions obtenu par une étape de séparation chromatographique d'un procédé de préparation des composés organo-phosphorés selon l'invention,FIG. 2 is an HPLC C18 chromatogram in an ion pair obtained by a chromatographic separation step of a process for the preparation of the organophosphorus compounds according to the invention,
- la figure 3 est un graphe à points illustrant l'amplification des lymphocytes Tyg en culture avec un composé organo-phosphoré selon l'invention,
- les figures 4a et 4b illustrent le profil cytofluori étrique bidimensionnel des lymphocytes y9 2 en culture respectivement sans et avec un composé organo- phosphoré selon l'invention, - la figure 5 est une courbe de titration de l'effet lymphoprolifératif d'un composé organo-phosphoré selon l'invention, pour un clone de lymphocytes Ty952-FIG. 3 is a dot graph illustrating the amplification of the Tyg lymphocytes in culture with an organophosphorus compound according to the invention, - Figures 4a and 4b illustrate the two-dimensional etric cytofluori profile of y9 2 lymphocytes in culture respectively without and with an organophosphorus compound according to the invention, - Figure 5 is a titration curve of the lymphoproliferative effect of a compound organophosphorus according to the invention, for a Ty952- lymphocyte clone
- la figure 6 est un spectre de résonance magnétique nucléaire H d'un composé organo-phosphoré selon l'invention,FIG. 6 is a nuclear magnetic resonance spectrum H of an organophosphorus compound according to the invention,
- la figure 7 est un spectre de masse d'un composé organo-phosphoré selon l'invention,FIG. 7 is a mass spectrum of an organophosphorus compound according to the invention,
- la figure 8 illustre trois chromatogrammes superposés HPLC C18 er paire d'ions respectivement de molécules phosphorylées témoins inactives ; d'un composé organo-phosphoré selon l'invention traité par une monoester phosphohydrolase alcaline ; et une nucléotide pyrophosphatase,- Figure 8 illustrates three superimposed chromatograms HPLC C18 er pair of ions respectively of inactive control phosphorylated molecules; an organophosphorus compound according to the invention treated with an alkaline phosphohydrolase monoester; and a nucleotide pyrophosphatase,
- la figure 9 est un histogramme illustrant la cytotoxicite induite par un composé organo-phosphoré selon l'invention, sur les lymphocytes Ty5 en présence d'anticorps monoclonaux de différentes spécificités.- Figure 9 is a histogram illustrating the cytotoxicity induced by an organophosphorus compound according to the invention, on Ty5 lymphocytes in the presence of monoclonal antibodies of different specificities.
PREPARATION DES COMPOSES ORGANO-PHOSPHORES ISOLES : Pour préparer les composés organo- phosphorés isolés selon l'invention, on part d'une culture d'une souche d'êtres vivants unicellulaires susceptible d'activer des lymphocytes Ty humains. Plus particulièrement, on part d'une culture de Mycobacteri um tuberculosis (Institut Pasteur, PARIS - FRANCE) qui active les lymphocytes -^γ δ2 - On réalise ensuite un extrait brut de la souche dont on sépare la phase aqueuse. Pour ce faire, on recueille le voile mycobactérien de la culture que l'on traite au chloroforme/méthanol de façon répétée afin de tuer les bactéries et d'en extraire les substances lipidiques. On réalise une partition des phases aqueuse et organique obtenues .PREPARATION OF ISOLATED ORGANO-PHOSPHORUS COMPOUNDS: To prepare the isolated organophosphorus compounds according to the invention, one starts with a culture of a strain of single-celled living beings capable of activating human Ty lymphocytes. More particularly, we start from a culture of Mycobacteri um tuberculosis (Institut Pasteur, PARIS - FRANCE) which activates lymphocytes - ^ γ δ2 - A crude extract of the strain is then produced from which the aqueous phase is separated. To do this, the mycobacterial veil from the culture is collected and treated with chloroform / methanol repeatedly in order to kill the bacteria and extract the lipid substances therefrom. A partition is made of the aqueous and organic phases obtained.
En variante, on peut aussi partir d'une
culture de souche d'une autre mycobactérie. En particulier, il est avantageux d'utiliser une mycobactérie à croissance rapide et sécrétant les composés recherchés dans le milieu de culture. Par exemple, on peut utiliser Mycobacteri um fortui tum biovar fortui tum . Dans ce cas, il n'est pas nécessaire de réaliser un extrait brut, et il suffit de collecter le milieu culture dont on sépare les composés hydrosolubles.Alternatively, one can also start from a strain culture of another mycobacterium. In particular, it is advantageous to use a fast growing mycobacterium and secreting the desired compounds in the culture medium. For example, Mycobacteri um fortui tum biovar fortui tum can be used. In this case, it is not necessary to produce a crude extract, and it suffices to collect the culture medium from which the water-soluble compounds are separated.
La phase aqueuse ainsi obtenue, dénommée ci-après "extrait ME", contient les composés que l'on sépare par chromatographie préparative en fractions susceptibles d'activer les lymphocytes Tyg 2-The aqueous phase thus obtained, hereinafter called "ME extract", contains the compounds which are separated by preparative chromatography into fractions capable of activating the T y g 2- lymphocytes.
On détermine l'activité des fractions chro atographiques en examinant la prolifération induite d'un clone de lymphocytes Tygg2 humains actifs sur les mycobactéries de départ (par exemple le clone G115) par la méthode décrite par F. DAVODEAU et al., Journal of Immunology Vol. 151, P 1214 (1993) (test de lympho- prolifération) . Les composés sont purifiés de l'extrait ME par une séparation chromatographique échangeuse d'anions, suivie d'une séparation chromatographique de la solution saline par une colonne d'acide silicique, et d'une séparation chromatographique HPLC sur phase inverse CI8 en paires d'ions et/ou d'une séparation chromatographique échangeuse d'anions HPAEC.The activity of the chromatographic fractions is determined by examining the induced proliferation of a clone of human Tygg2 lymphocytes active on the starting mycobacteria (for example the clone G115) by the method described by F. DAVODEAU et al., Journal of Immunology Flight. 151, P 1214 (1993) (lympho proliferation test). The compounds are purified from the ME extract by an anion exchange chromatographic separation, followed by a chromatographic separation of the saline solution by a column of silicic acid, and by an HPLC chromatographic separation on CI8 reverse phase in d pairs. 'ions and / or HPAEC anion exchange chromatographic separation.
Plus précisément, les solutions de l'extrait ME sont passées dans une colonne échangeuse d'anions de type diéthylaminoéthyl (DEAE) dans laquelle les principes actifs sont élues par des concentrations croissantes de l'acétate d'ammonium. L'activité stimulatrice de chaque éluat est testée sur le clone G115 par le test de lymphoprolifération. Les éluants actifs sur le clone G115 sont traités ensuite dans une colonne chromatographique d'acide silicique pour les séparer de la solution saline d'acétate d'ammonium. Les éluants successifs utilisés sont 1 ' éthanol 100 %, 1 ' éthanol 90 %, le propanol 2 100 % et le propanol H2O (70 % - 30 %). Les
éluats sont séchés et soumis au test de lymphoproliferation. On constate que le dernier éluat est actif, et contient les composés selon l'invention.More specifically, the solutions of the extract ME are passed through an anion exchange column of the diethylaminoethyl (DEAE) type in which the active ingredients are eluted by increasing concentrations of ammonium acetate. The stimulatory activity of each eluate is tested on the clone G115 by the lymphoproliferation test. The eluents active on the clone G115 are then treated in a chromatographic column of silicic acid to separate them from the saline solution of ammonium acetate. The successive eluents used are 1% ethanol, 1% ethanol 90%, propanol 2 100% and propanol H2O (70% - 30%). The eluates are dried and subjected to the lymphoproliferation test. It is noted that the last eluate is active, and contains the compounds according to the invention.
Les différents principes actifs de l'éluat ainsi obtenu sont ensuite soumis à une séparation chromatographique sur une colonne C18 de séparation de la phase hydrophile. Les produits élues en phase aqueuse sont ensuite soumis à une séparation chromatographique HPLC sur une colonne préparative 250/40 C18 telle que BISCHOFF (marque déposée) ULTRASEP 10μm éluée en paire d'ions avec l'acétate d'ammonium 0,1 M. L'absorption aux différentes longueurs d'ondes est contrôlée par un détecteur d'absorption dans l'ultraviolet. On collecte des fractions chromatographiques toutes les 30 secondes pendant une durée totale de 40 min.The different active principles of the eluate thus obtained are then subjected to a chromatographic separation on a column C18 for separation of the hydrophilic phase. The products eluted in aqueous phase are then subjected to an HPLC chromatographic separation on a preparative column 250/40 C18 such as BISCHOFF (registered trademark) ULTRASEP 10 μm eluted in pair of ions with 0.1 M ammonium acetate. L he absorption at the different wavelengths is controlled by an absorption detector in the ultraviolet. Chromatographic fractions are collected every 30 seconds for a total of 40 min.
Le chromatogramme à 260 nm est donné par la courbe en pointillés de la figure 2. La courbe en points blancs illustre l'activité des différentes fractions chromatographiques mesurée par le test de lymphoproliferation. Cette activité est exprimée en Kcpm (milliers de coups par minute) de la thymidine tritiée incorporée par le clone G115. Par cette séparation chromatographique, on constate la présence de quatre fractions comportant respectivement quatre composés selon l'invention désignés TUBagI, TUBag2, TUBag3 et TUBag4 dans leur ordre d'élution croissant. Comme on le voit, figure 2, chaque fraction active est de composition hétérogène. Chaque fraction active est individuellement chromatographiée à nouveau par une séparation chromatographique HPLC conforme à la dernière étape sus- décrite.The chromatogram at 260 nm is given by the dotted curve in FIG. 2. The white dot curve illustrates the activity of the different chromatographic fractions measured by the lymphoproliferation test. This activity is expressed in Kcpm (thousands of strokes per minute) of the tritiated thymidine incorporated by the clone G115. By this chromatographic separation, there is the presence of four fractions comprising respectively four compounds according to the invention designated TUBagI, TUBag2, TUBag3 and TUBag4 in their increasing elution order. As can be seen in FIG. 2, each active fraction is of heterogeneous composition. Each active fraction is individually chromatographed again by an HPLC chromatographic separation in accordance with the last step described above.
Chaque fraction est ensuite soumise à une séparation chromatographique échangeuse d'anions HPAE éluée par un gradient de soude 0,1 M et de méthanol dans l'eau. Par ailleurs, cette chromatographie permet de mesurer le caractère anionique des différents composés selon l'invention révélé par un détecteur conductimétrique opérant en mode de suppression chimique. Cette séparation
chromatographique permet aussi de contrôler l'homogénéité des composés ainsi purifiés.Each fraction is then subjected to an HPAE anion exchange chromatographic separation eluted with a gradient of 0.1 M sodium hydroxide and methanol in water. Furthermore, this chromatography makes it possible to measure the anionic character of the various compounds according to the invention revealed by a conductimetric detector operating in chemical suppression mode. This separation chromatography also makes it possible to control the homogeneity of the compounds thus purified.
Le tableau I fournit les temps de rétention des quatre composés purifiés et de diverses biomolécules étalons par HPAEC.Table I provides the retention times of the four purified compounds and of various standard biomolecules by HPAEC.
TABLEAU ITABLE I
TEMPS DE RETENTION (RT) (exprimés en minutes +/- 0,05 min.)RETENTION TIME (RT) (expressed in minutes +/- 0.05 min.)
COMPOSE RT COMPOSE RT COMPOSE RTCOMPOSE RT COMPOSE RT COMPOSE RT
TUBagI 7,67 Glucose 1P 1,46 TMP5' 7,67TUBagI 7.67 Glucose 1P 1.46 TMP5 '7.67
TUBag2 7,67 Serine P 6,26 TDP5'Glc 7,67TUBag2 7.67 Serine P 6.26 TDP5'Glc 7.67
TUBag3 14,0 PF 7,13 TDP5' 12,66TUBag3 14.0 PF 7.13 TDP5 '12.66
TUBag4 13,0 PEP 7,97 TTP5' 17,27TUBag4 13.0 PEP 7.97 TTP5 '17.27
Abréviations : P : phosphate, PF : acide phosphonoformique, PEP : acide phosphoénolpyruvique, TMP5 ' : thymidine 5' monophosphate, TDP5 ' : thymidine 5' diphosphate, TDP5 ' Glc : thymidine 5' diphosphoryl-1 glucose, TTP5 ' : thymidine 5' triphosphate.Abbreviations: P: phosphate, PF: phosphonoformic acid, PEP: phosphoenolpyruvic acid, TMP5 ': thymidine 5' monophosphate, TDP5 ': thymidine 5' diphosphate, TDP5 'Glc: thymidine 5' diphosphoryl-1 glucose, TTP5 ': thymidine 5' triphosphate.
Colonne utilisée : échangeuse d'anions sur support pelliculaire modèle AS11 Dionex (marque déposée) 250/4 mm, Eluant utilisé : 2 ml/min composé de la séquence suivante :Column used: anion exchanger on film support model AS11 Dionex (registered trademark) 250/4 mm, Eluent used: 2 ml / min composed of the following sequence:
- de 0 à 1 min : isocratique 90 % H2O + 10 % NaOH (0,1 M),- from 0 to 1 min: isocratic 90% H2O + 10% NaOH (0.1 M),
- de 1 à 12,5 min : gradient atteignant 50 % H2O + 50 % NaOH (0,1 M) à 12,5 min, de 12,5 à 22 min : gradient atteignant 5 % H20 + 50 % NaOH (0,1 M) + 45 % Méthanol à 22 min,- from 1 to 12.5 min: gradient reaching 50% H2O + 50% NaOH (0.1 M) at 12.5 min, from 12.5 to 22 min: gradient reaching 5% H 2 0 + 50% NaOH ( 0.1 M) + 45% Methanol at 22 min,
- de 22 à 23 min : gradient atteignant 90 % H20 + 10 % NaOH (0,1 M) à 23 min, - de 23 à 28 min : isocratique 90 % H2O + 10 % NaOH- from 22 to 23 min: gradient reaching 90% H 2 0 + 10% NaOH (0.1 M) at 23 min, - from 23 to 28 min: isocratic 90% H2O + 10% NaOH
(0,1 M). Détecteur utilisé : conductimètre précédé d'un suppresseur ionique à membrane DIONEX (marque déposée).(0.1 M). Detector used: conductivity meter preceded by an ionic suppressor with a DIONEX membrane (registered trademark).
Comme on le voit, le caractère anionique de TUBagI et TUBag2 est analogue à celui de la thymidine 5 ' monophosphaté (TMP) . Egalement, le caractère anionique des antigènes isolés TUBag3 et TUBag4 est plus important que
celui de la thymidine 5' diphosphate, mais moins important que celui de la thymidine 5' triphosphate.As can be seen, the anionic character of TUBagI and TUBag2 is analogous to that of thymidine 5 'monophosphate (TMP). Also, the anionic character of the isolated antigens TUBag3 and TUBag4 is more important than that of thymidine 5 'diphosphate, but less important than that of thymidine 5' triphosphate.
Le caractère anionique des composés antigéniques TUBagI, TUBag2, TUBag3 et TUBag4 est donc supérieur à celui de l'acide phosphonoformique et inférieur à celui de la thymidine 5' triphosphate.The anionic character of the antigenic compounds TUBagI, TUBag2, TUBag3 and TUBag4 is therefore greater than that of phosphonoformic acid and less than that of thymidine 5 'triphosphate.
ANALYSE DE LA STIMULATION DES LYMPHOCYTES Ty 6 PAR LE COMPOSE TUBag4 : On a recueilli les lymphocytes du sang circulant de quatre donneurs humains sains. Ces lymphocytes sont cultivés en présence d' interleukine 2 pendant 10 jours, avec ou sans le composé TUBag4, en quantité saturante. En fin de culture, les nombres de lymphocytes Tαβ, Ty5 ou CD3- (non T) sont comparés dans chacune des deux cultures.ANALYSIS OF THE STIMULATION OF Ty 6 LYMPHOCYTES BY THE TUBag4 COMPOUND: The circulating blood lymphocytes were collected from four healthy human donors. These lymphocytes are cultured in the presence of interleukin 2 for 10 days, with or without the compound TUBag4, in a saturating amount. At the end of culture, the numbers of Tαβ, Ty5 or CD3- (non-T) lymphocytes are compared in each of the two cultures.
La figure 3 présente le rapport d'amplification, soit le rapport du nombre de cellules en culture avec TUBag4 sur le nombre de cellules en culture sans TUBag4. Chaque point représente un donneur. Comme on le voit, TUBag4 induit en culture une amplification spécifique polyclonale des lymphocytes Ty d'un facteur compris entre 40 et 500 selon les individus.FIG. 3 shows the amplification ratio, ie the ratio of the number of cells in culture with TUBag4 to the number of cells in culture without TUBag4. Each point represents a donor. As can be seen, TUBag4 induces in culture a specific polyclonal amplification of the Ty lymphocytes by a factor of between 40 and 500 depending on the individual.
Les figures 4a et b présentent le phénotype du récepteur TCR des lymphocytes du sang circulant d'un donneur humain sain cultivés en l'absence (figure 4a) et en présence (figure 4b) de TUBag4. Comme on le voit, TUBag4 induit une amplification massive mais sélective des lymphocytes Tyg52- La figure 5 présente la titration de l'effet prolifératif de TUBag4 sur un clone de lymphocytes Tyg52 (G115) mesuré par le test de lymphoproliferation. Comme on le voit (courbe à points noirs), on a constaté un effet prolifératif de TUBag4 à partir d'une concentration de l'ordre de 1 ng/ml. La courbe en points blancs traduit l'effet prolifératif en l'absence de TUBag4.Figures 4a and b show the phenotype of the TCR receptor of circulating blood lymphocytes from a healthy human donor cultured in the absence (Figure 4a) and in the presence (Figure 4b) of TUBag4. As can be seen, TUBag4 induces a massive but selective amplification of the Tyg52 lymphocytes. FIG. 5 shows the titration of the proliferative effect of TUBag4 on a clone of Tyg52 lymphocytes (G115) measured by the lymphoproliferation test. As can be seen (curve with black dots), a proliferative effect of TUBag4 has been observed from a concentration of the order of 1 ng / ml. The white dot curve reflects the proliferative effect in the absence of TUBag4.
Des résultats analogues obtenus avec TUBagI , TUBag2 et TUBag3 confirment le caractère
antigenique commun de ces composés pour les lymphocytesSimilar results obtained with TUBagI, TUBag2 and TUBag3 confirm the character common antigen of these compounds for lymphocytes
Tyδ-T y δ-
ANALYSE STRUCTURALE DES COMPOSES ISOLES :STRUCTURAL ANALYSIS OF ISOLATED COMPOUNDS:
On a tout d'abord soumis la phase aqueuse de l'extrait mycobactérien (ME) mentionnée ci-dessus à deux expériences. Dans la première expérience, le test de lymphoproliferation sur le clone G115 a été conduit sur l'extrait ME seul, sur l'extrait ME en présence de protéinase K (Merck, 6 mU/mp, 2 h à 37° C), et sur l'extrait ME en présence de periodate ( 1 OmM de NaHI04, 2 h à 37° C). Dans la seconde expérience, le test a été conduit sur l'extrait ME seul, puis sur l'extrait ME en présence d'enzyme monoester phosphorique phosphohydrolase notamment la phosphatase alcaline. Les lymphoproliférations spécifiques obtenues sont exprimées dans le tableau II suivant, après soustraction de la prolifération spontanée (non stimulée) du clone G115 :The aqueous phase of the above-mentioned mycobacterial extract (ME) was first subjected to two experiments. In the first experiment, the lymphoproliferation test on the clone G115 was carried out on the ME extract alone, on the ME extract in the presence of proteinase K (Merck, 6 mU / mp, 2 h at 37 ° C.), and on the ME extract in the presence of periodate (1 OmM of NaHI04, 2 h at 37 ° C.). In the second experiment, the test was conducted on the ME extract alone, then on the ME extract in the presence of the phosphoric monoester phosphohydrolase enzyme, in particular alkaline phosphatase. The specific lymphoproliferations obtained are expressed in Table II below, after subtracting the spontaneous proliferation (unstimulated) of the clone G115:
TABLEAU IITABLE II
prolifération du clone G 115 (cpm)proliferation of clone G 115 (cpm)
Expérience 1Experiment 1
ME 12 000ME 12,000
ME traité avec protéinase K 12 000ME treated with proteinase K 12,000
ME traité avec periodate 2 000ME treated with periodate 2,000
Expérience 2Experiment 2
ME 15 000ME 15,000
ME traité avec phosphatase 7 000 alcalineME treated with 7,000 alkaline phosphatase
Ainsi, l'extrait antigenique ME active le clone G115 de lymphocytes Tyg 2- Cet extrait ME est résistant aux protéases, mais son activité sur le clone G115 est inhibée en présence de periodate. L'extrait ME subit donc une dégradation par l'acide périodique. De plus, l'extrait ME est partiellement dégradé sous l'action enzymatique d'une phosphatase alcaline. En conséquence," les antigènes sont hydrosolubles, de nature non-peptidique, mais sont acidolabiles et organo-phosphorés.Thus, the active antigenic ME extracted G115 T cell clone y g 2-ME This extract is protease resistant, but its activity in the G115 clone is inhibited in the presence of periodate. The ME extract therefore undergoes degradation by periodic acid. In addition, the ME extract is partially degraded under the enzymatic action of an alkaline phosphatase. Consequently, " the antigens are water-soluble, non-peptide in nature, but are acid-labile and organophosphorus.
A partir de cet extrait ME, on réalise une
séparation chromatographique par filtration de taille avec une colonne permettant la résolution des masses moléculaires comprises entre 400 et 10000, et dans laquelle les principes actifs sont élues par une solution saline d'acétate d'ammonium 0,1 M. Le contenu osidique des fractions recueillies est testé (figure 1, points blancs) par réaction colorimétrique avec 1 ' anthrone sulfurique mesurée à une longueur d'onde de 630 nm. Par ailleurs, l'activité antigenique de chaque fraction obtenue est testée sur le clone G115 (points noirs sur la figure 1) par le test de lymphoproliferation.From this ME extract, we carry out a chromatographic separation by size filtration with a column allowing the resolution of the molecular weights between 400 and 10,000, and in which the active ingredients are eluted with a 0.1 M ammonium acetate saline solution The osidic content of the fractions collected is tested (FIG. 1, white dots) by colorimetric reaction with the sulfuric anthrone measured at a wavelength of 630 nm. Furthermore, the antigenic activity of each fraction obtained is tested on the clone G115 (black dots in FIG. 1) by the lymphoproliferation test.
Il ressort de la figure 1 que les fractions actives sur les lymphocytes ont toutes un poids moléculaire inférieur à 1000, et qui peut être estimé de l'ordre de 500.It appears from FIG. 1 that the fractions active on the lymphocytes all have a molecular weight less than 1000, and which can be estimated on the order of 500.
Par ailleurs, l'antigène TUBag4 isolé a été soumis à une analyse structurale par résonance magnétique nucléaire. Le spectre obtenu par l'analyse H unidimensionnelle (figure 6) démontre la présence des groupes 2-désoxyribose et de la thymine. De plus, des essais de résonance magnétique bidimensionnelle, homonucléaire H 'H et hétéronucléaire H ^C permettent d'identifier de façon claire la liaison du groupement thymine par son premier azote au carbone anomérique du radical 2-désoxyribose. De surcroît, les résultats permettent de postuler la liaison du cinquième carbone du radical 2-dézoxyribose à un groupement phosphoryle.In addition, the isolated TUBag4 antigen was subjected to structural analysis by nuclear magnetic resonance. The spectrum obtained by the one-dimensional H analysis (FIG. 6) demonstrates the presence of the 2-deoxyribose groups and of thymine. In addition, two-dimensional, homonuclear H 'H and heteronuclear H ^ C magnetic resonance tests make it possible to clearly identify the bond of the thymine group by its first nitrogen to the anomeric carbon of the 2-deoxyribose radical. In addition, the results make it possible to postulate the binding of the fifth carbon of the 2-dezoxyribose radical to a phosphoryl group.
Par ailleurs, le spectre obtenu par spectrométrie de masse (figure 7) confirme la présence du groupement phosphoryle dans le composé TUBag4. Sur cette figure, on note également la présence de pics pour des valeurs du rapport masse sur charge de 126 et de 155, qui confirment la présence de la thymidine.Furthermore, the spectrum obtained by mass spectrometry (FIG. 7) confirms the presence of the phosphoryl group in the compound TUBag4. In this figure, we also note the presence of peaks for values of the mass-to-charge ratio of 126 and 155, which confirm the presence of thymidine.
Cependant, le test de lymphoproliferation sur le clone G115 effectué avec la thymidine " 5' monophosphate, la thymidine 5' diphosphate et la thymidine 5' triphosphate démontre l'inactivité de ces composants sur les lymphocytes. En conséquence, les composés antigéniques
selon l'invention, ne sont pas exclusivement constitués de ces thymidines 5' phosphatées.However, the lymphoproliferation test on the clone G115 carried out with thymidine " 5 'monophosphate, thymidine 5' diphosphate and thymidine 5 'triphosphate demonstrates the inactivity of these components on lymphocytes. Consequently, the antigenic compounds according to the invention, do not exclusively consist of these 5 'phosphated thymidines.
Par ailleurs, les quatre composés actifs obtenus par séparation chromatographique ont été soumis a des effets de dégradation enzymatique par des phosphatases. Les enzymes utilisées ont été la monoester phosphorique phosphohydrolase alcaline MP (EC 3.1.3.1), la diester phosphorique phosphohydrolase DP (EC 3.1.4.1 ) de venin de crotale, et la nucléotide pyrophosphatase NPP (EC 3.6.1.9) de venin de crotale.Furthermore, the four active compounds obtained by chromatographic separation were subjected to the effects of enzymatic degradation by phosphatases. The enzymes used were the phosphoric monoester alkaline phosphohydrolase MP (EC 3.1.3.1), the phosphoric diester phosphohydrolase DP (EC 3.1.4.1) of rattlesnake venom, and the nucleotide pyrophosphatase NPP (EC 3.6.1.9) of rattlesnake venom.
L'action de chacun des composés mis au contact respectivement avec chacune de ces enzymes sur le clone G115 est résumée dans le tableau III suivant dans lequel le signe + indique que le composé a conservé une activité alors que le signe - indique la disparition de toute activité à l'égard des lymphocytes.The action of each of the compounds brought into contact respectively with each of these enzymes on the clone G115 is summarized in Table III below in which the sign + indicates that the compound has retained activity while the sign - indicates the disappearance of all activity with regard to lymphocytes.
TABLEAU IIITABLE III
Comme on le voit, la monoester phosphorique phosphohydrolase (AP) inactive TUBagI et TUBag2y mais n' inactive pas TUBag3 et TUBag4. En conséquence, TUBagI et TUBag2 sont des monoesters phosphoriques.As can be seen, the phosphoric monoester phosphohydrolase (AP) inactivates TUBagI and TUBag2 y but does not inactivate TUBag3 and TUBag4. Consequently, TUBagI and TUBag2 are phosphoric monoesters.
Par contre, la nucléotide pyrophosphatase (NPP) n' inactive pas TUBagI et TUBag2, mais inactive TUBag3 et clive TUBag4. En conséquence, la présence d'un noyau nucléotidique est confirmée dans TUBag3 et TUBag4.In contrast, the nucleotide pyrophosphatase (NPP) does not inactivate TUBagI and TUBag2, but inactivates TUBag3 and cleaves TUBag4. Consequently, the presence of a nucleotide nucleus is confirmed in TUBag3 and TUBag4.
L'association d'une monoester phosphorique phosphohydrolase et d'une nucléotide pyrophosphatase sur les a'ntigènes inactive l'intégralité de ces antigènes. Les
mêmes résultats sont obtenus sur TUBag4 en remplaçant la nucléotide pyrophosphatase (NPP) par la diester phosphorique phosphohydrolase (DP) de venin de crotale.The association of a phosphoric phosphohydrolase monoester and a nucleotide pyrophosphatase on the antigens inactivates all of these antigens. The same results are obtained on TUBag4 by replacing the nucleotide pyrophosphatase (NPP) with the phosphoric diester phosphohydrolase (DP) of rattlesnake venom.
Par ailleurs, l'activité des antigènes en présence des enzymes a été analysée par chromatographie HPLC sur face inverse C18 en paire d'ions. Les résultats sont illustrés par la figure 8 sur laquelle la courbe supérieure en traits pointillés illustre le profil des thymidines TTP, TDP, TDPGlc et TMP. La courbe médiane illustre les résultats de la séparation chromatographique de TUBag4 préalablement traité par la monoester phosphorique phosphohydrolase, et la courbe inférieure illustre l'activité de TUBag4 préalablement traité par la nucléotide pyrophosphatase. Comme on le constate sur cette dernière courbe, le composé TUBag4 soumis à l'enzyme nucléotide pyrophosphatase fournit le composé TUBagI .Furthermore, the activity of antigens in the presence of enzymes was analyzed by C18 reverse-side HPLC chromatography in an ion pair. The results are illustrated in FIG. 8 in which the upper curve in dotted lines illustrates the profile of the thymidines TTP, TDP, TDPGlc and TMP. The middle curve illustrates the results of the chromatographic separation of TUBag4 previously treated with the phosphoric phosphohydrolase monoester, and the lower curve illustrates the activity of TUBag4 previously treated with the nucleotide pyrophosphatase. As can be seen on this last curve, the compound TUBag4 subjected to the nucleotide pyrophosphatase enzyme provides the compound TUBagI.
En conséquence, l'ensemble de ces expérimentations démontre que les composés selon l'invention sont des esters phosphoriques structuralement apparentés.Consequently, all of these experiments demonstrate that the compounds according to the invention are structurally related phosphoric esters.
Par ailleurs, TUBag3 et TUBag4 présentent un substituant qui est un groupement nucléosidique identifié dans TUBag4 comme la thymidine liée au groupement phosphoryle dans l'ester par le cinquième carbone de son radical 2-désoxyribose.Furthermore, TUBag3 and TUBag4 have a substituent which is a nucleoside group identified in TUBag4 as thymidine linked to the phosphoryl group in the ester by the fifth carbon of its 2-deoxyribose radical.
Egalement, TUBagI est le produit de l'hydrolyse de TUBag4 par l'action enzymatique d'une nucléotide pyrophosphatase ou d'une diester phosphorique phosphohydrolase.Also, TUBagI is the product of the hydrolysis of TUBag4 by the enzymatic action of a nucleotide pyrophosphatase or a phosphoric diester phosphohydrolase.
Ainsi, TUBagI est un monoester phosphorique qui est le produit actif de l'hydrolyse spontanée ou enzymatique de TUBag4 ; TUBag2 est un monoester phosphorique de caractère anionique analogue à celui de TUBagI mais de caractère hydrophobe supérieur ; TUBag3 est un diester pyrophosphorique nucléotidique ; et TUBag4 est un diester diphosphorique ou triphosphorique de la 5 ' thymidine incorporant TUBagI .
Pour caractériser un composé purifié selon l'invention, il suffit donc de :Thus, TUBagI is a phosphoric monoester which is the active product of the spontaneous or enzymatic hydrolysis of TUBag4; TUBag2 is a phosphoric monoester of anionic character similar to that of TUBagI but of higher hydrophobic character; TUBag3 is a pyrophosphoric nucleotide diester; and TUBag4 is a diphosphoric or triphosphoric diester of 5 'thymidine incorporating TUBagI. To characterize a purified compound according to the invention, it is therefore sufficient to:
- démontrer qu'il induit une activation des lymphocytes τy952' Par exemple par un test de lymphoproliferation,- demonstrate that induces activation of τ y952 lymphocytes F or example by a lymphoproliferation assay,
- démontrer que cette activité vis—à-vis des lymphocytes Ty962 disparaît soit en présence d'une monoester phosphorique phosphohydrolase (sa structure s'apparente donc à celle de TUBagI ou de TUBag2), soit en présence d'une part d'une monoester phosphorique phosphohydrolase et, d'autre part, d'une diester phosphorique phosphohydrolase et/ou d'une pyrophosphohydrolase (sa structure s'apparente donc à celle de TUBag3 ou de TUBag4). Pour identifier précisément la nature du composé, il suffit en outre d'observer son ordre d'élution à la chromatographie HPLC de type CI8 éluée en paire d'ions avec l'acétate d'ammonium 0^1 M en le comparant à ceux obtenus avec une solution contenant les composés selon l'invention isolés à partir de l'extrait mycobacterien ME (figure 2), et de déterminer son temps de rétention en chromatographie HPAEC et le comparer au tableau I ci- dessus.- demonstrate that this activity vis-à-vis the Ty962 lymphocytes disappears either in the presence of a phosphoric monoester phosphohydrolase (its structure is therefore similar to that of TUBagI or TUBag2), or in the presence of a part of a monoester phosphoric phosphohydrolase and, on the other hand, a phosphoric phosphohydrolase diester and / or a pyrophosphohydrolase (its structure is therefore similar to that of TUBag3 or TUBag4). To precisely identify the nature of the compound, it is also sufficient to observe its order of elution by HPLC chromatography of type CI8 eluted in pair of ions with ammonium acetate 0 ^ 1 M by comparing it with those obtained. with a solution containing the compounds according to the invention isolated from the mycobacterial extract ME (FIG. 2), and to determine its retention time in HPAEC chromatography and compare it to table I above.
Par ailleurs, si on dispose d'une solution hétérogène incorporant un composé selon l'invention, on peut caractériser la présence de ce composé organo- phosphoré antigenique en soumettant cette solution aux étapes de séparation chromatographique mentionées ci-dessus pour l'extrait mycobacterien ME, notamment au moins une séparation préparative des différents principes actifs de l'éluat par chromatographie HPLC sur phase inverse CI8 éluée en paire d'ions et/ou par chromatographie échangeuse d'anions HPAEC avec détection conductimétrique opérant en mode de suppression chimique, chaque étape de séparation étant suivie d'une mesure de l'activité des différentes fractions obtenues sur des lymphocytes Tyg.
CYTOTOXICITE DES LYMPHOCYTES Tyg EN PRESENCE DES COMPOSES ORGANO-PHOSPHORES ISOLES :Furthermore, if there is a heterogeneous solution incorporating a compound according to the invention, the presence of this antigenic organophosphorus compound can be characterized by subjecting this solution to the chromatographic separation steps mentioned above for the mycobacterial extract ME , in particular at least one preparative separation of the various active principles of the eluate by HPLC chromatography on CI8 reverse phase eluted in ion pair and / or by HPAEC anion exchange chromatography with conductimetric detection operating in chemical suppression mode, each step separation is followed by a measurement of the activity of the various fractions obtained on Tyg lymphocytes. CYTOTOXICITY OF Tyg LYMPHOCYTES IN THE PRESENCE OF ISOLATED ORGANO-PHOSPHORUS COMPOUNDS:
L'activité cytolytique d'un clone de lymphocytes Tyg52 (G115) et d'un clone contrôle de lymphocyte T non γξ. (BH) contre des cellules cibles est mesurée en 1 ' absence ou en présence du composé TUBag .The cytolytic activity of a clone of Tyg52 lymphocytes (G115) and of a control clone of non γ ξ T lymphocyte. (BH) against target cells is measured in the absence or in the presence of the TUBag compound.
Cette activité induite par TUBag4 est déterminée par la formule suivante : (% de lyse spécifique en présence de TUBag4) - (% de lyse spécifique sans TUBag4).This activity induced by TUBag4 is determined by the following formula: (% of specific lysis in the presence of TUBag4) - (% of specific lysis without TUBag4).
Cette cytotoxicite est estimée à un rapport de cellules tueuses sur cellules cibles de 20 pour 1.This cytotoxicity is estimated at a ratio of killer cells to target cells of 20 to 1.
Les résultats sont illustrés par le tableau IV suivant :The results are illustrated by the following Table IV:
TABLEAU IVTABLE IV
CELLULES CIBLES LYSE INDUITE PAR TUBag4TUBag4-INDUCED LYSE TARGET CELLS
ORIGINE NOM NATURE G115 BHORIGIN NAME NATURE G115 BH
Humaine DAB BLCL 32 0Human DAB BLCL 32 0
BL70 B Lymphoma 21 0BL70 B Lymphoma 21 0
BL2195 B Lymphoma 37 3BL2195 B Lymphoma 37 3
RJ225 B Lymphoma 25 0RJ225 B Lymphoma 25 0
RAJI B Lymphoma 32 0RAJI B Lymphoma 32 0
BL30195 B Lymphoma 20 0BL30195 B Lymphoma 20 0
BL 30 B Lymphoma 34 5BL 30 B Lymphoma 34 5
KGI T Lymphoma 16 0KGI T Lymphoma 16 0
THPI T Lymphoma 12 0THPI T Lymphoma 12 0
KE6TG T Lymphoma 26 0KE6TG T Lymphoma 26 0
HSB2 T Lymphoma 21 0HSB2 T Lymphoma 21 0
MRC5 Fibroblast 39 0MRC5 Fibroblast 39 0
Murine DA2 Inconnue 14 6Murine DA2 Unknown 14 6
BW5147 Thymoma 12 0BW5147 Thymoma 12 0
A20 B Lymphoma 22 0A20 B Lymphoma 22 0
SP20 Myeloma 9 0SP20 Myeloma 9 0
P815 Mastocytoma 32 0P815 Mastocytoma 32 0
Comme on le voit, TUBag4 induit une lyse de toutes les cellules cibles par le clone G115 seulement, mais n'induit pas de lyse par le clone contrôle BH.As can be seen, TUBag4 induces lysis of all the target cells by the clone G115 only, but does not induce lysis by the control clone BH.
Cette activité lytique n'est pas spécifique de la cellule cible. Cette activité cytolytique n'est pas non plus restreinte par le complexe MHC. En particulier,
les composés antigéniques selon 1 ' invention activent la cytotoxicite du lymphocyte Ty§ G115 sur différentes cellules cancéreuses issues de donneurs différents humains ou de souris. De plus, il a été démontré que les composés selon l'invention activent directement l'activité cytotoxique du lymphocyte y5 G115 sans interférer avec la cellule cible.This lytic activity is not specific to the target cell. This cytolytic activity is also not restricted by the MHC complex. In particular, the antigenic compounds according to the invention activate the cytotoxicity of the Ty § G115 lymphocyte on different cancer cells originating from different human or mouse donors. In addition, it has been demonstrated that the compounds according to the invention directly activate the cytotoxic activity of the y5 G115 lymphocyte without interfering with the target cell.
Cette démonstration a été effectuée par des essais de pré-incubation dont les résultats indiquent que TUBag4 active directement le clone Tyg52-This demonstration was carried out by pre-incubation tests, the results of which indicate that TUBag4 directly activates the clone Tyg52-
Cette interaction directe implique TUBag4 et le TCR Vy V52 comme l'indique l'inhibition totale de cette activité cytotoxique lorsque l'expérience est effectuée en présence d'anticorps monoclonaux dirigés contre le TCR Vyg§2 (figure 9). Par contre, les anticorps anti-TCR V 1 , HLA DR, HLA DP/DR et HLA 1 n'inhibent pas cette activité cytotoxique.. Cela confirme l'absence de restriction par le MHC de cette réponse lymphocytaire. L'efficacité thérapeutique des composés et compositions selon l'invention n'est donc pas limitée par les caractéristiques tissulaires du patient traité, et ce contrairement à la plupart des vaccins connus.This direct interaction involves TUBag4 and TCR Vy V52 as indicated by the total inhibition of this cytotoxic activity when the experiment is carried out in the presence of monoclonal antibodies directed against TCR Vyg§2 (FIG. 9). On the other hand, the anti-TCR V 1, HLA DR, HLA DP / DR and HLA 1 antibodies do not inhibit this cytotoxic activity. This confirms the absence of restriction by the MHC of this lymphocyte response. The therapeutic efficacy of the compounds and compositions according to the invention is therefore not limited by the tissue characteristics of the patient treated, unlike most of the known vaccines.
L'invention permet ainsi de stimuler ou d'inhiber, c'est-à-dire de moduler, la prolifération et/ou la cytotoxcité des lymphocytes Ty . A ce titre, on peut donc utiliser un composé organo-phosphoré tel que TUBagI ,The invention thus makes it possible to stimulate or inhibit, that is to say to modulate, the proliferation and / or the cytotoxicity of the Ty lymphocytes. As such, one can therefore use an organo-phosphorus compound such as TUBagI,
TUBag2, TUBag3, TUBag4, ou un composé incorporant ces composés organo-phosphorés ou un groupement similaire, pour obtenir une composition pharmaceutique pour le traitement préventif ou curatif des maladies de l'homme ou de l'animal dans lesquelles les lymphocytes Ty5 sont impliqués, notamment les maladies infectieuses (en particulier mycobactériennes telles que la lèpre ou la tuberculose) ; les affections tumorales ou leucémiques de l'homme ou de l'animal ; les parasitoses ; les pathologies immuno- déficiences telles que le SIDA, ...TUBag2, TUBag3, TUBag4, or a compound incorporating these organophosphorus compounds or a similar group, to obtain a pharmaceutical composition for the preventive or curative treatment of human or animal diseases in which the Ty5 lymphocytes are involved, in particular infectious diseases (in particular mycobacterials such as leprosy or tuberculosis); human or animal tumor or leukemia disorders; parasitic infections; immuno-deficient pathologies such as AIDS, ...
A l'inverse, on peut aussi inhiber
l'activité des lymphocytes Ty in situ en utilisant une enzyme dégradant les antigènes formés des composés organo- phosphorés selon l'invention. Selon l'antigène concerné, on utilise une monoester phosphatase et/ou une diester phosphatase (y compris une pyrophosphatase telle qu'une nucléotide pyrophosphatase).
Conversely, we can also inhibit the activity of Ty lymphocytes in situ using an enzyme degrading the antigens formed from the organophosphorus compounds according to the invention. Depending on the antigen concerned, a monoester phosphatase and / or a diester phosphatase (including a pyrophosphatase such as a pyrophosphatase nucleotide) is used.
Claims
REVENDICATIONS
1 / - Composé organo-phosphoré hydrosoluble de nature non-peptidiqύe, activateur des lymphocytes Ty humains à récepteurs TCR comprenant les régions variables Vyg et V 2, ce composé comprenant au moins une liaison ester acidolabile de l'acide phosphorique, le caractère activateur de ce composé vis-à-vis des lymphocytes Ty5 disparaissant lorsqu'il est placé en présence d'un mélange enzymatique comprenant au moins une monoester phosphorique phosphohydrolase et au moins une diester phosphorique phosphohydrolase.1 / - Water-soluble organophosphorus compound of a non-peptidic nature, activator of human Ty lymphocytes with TCR receptors comprising the Vyg and V 2 variable regions, this compound comprising at least one acidolabile ester bond of phosphoric acid, the activator character of this compound vis-à-vis the Ty5 lymphocytes disappearing when it is placed in the presence of an enzymatic mixture comprising at least one phosphoric phosphohydrolase monoester and at least one phosphoric phosphohydrolase diester.
2/ - Composé selon la revendication 1 , caractérisé en ce qu'il présente un caractère anionique mesuré par chromatographie HPAEC à détection conductimétrique en mode de suppression chimique qui est supérieur à celui de l'acide phosphonoformique et inférieur à celui de la thymidine 5' triphosphate.2 / - Compound according to claim 1, characterized in that it has an anionic character measured by HPAEC chromatography with conductimetric detection in chemical suppression mode which is higher than that of phosphonoformic acid and lower than that of thymidine 5 ' triphosphate.
3/ - Composé selon l'une des revendications3 / - Compound according to one of claims
1 et 2 caractérisé en ce que, après traitement par une enzyme diester phosphorique phosphohydrolase, il présente un caractère anionique mesuré par chromatographie HPAEC à détection conductimétrique en mode de suppression chimique qui est supérieur à celui de l'acide phosphonoformique et inférieur à celui de la thymidine 5' triphosphate -. 4/ - Composé selon l'une des revendications1 and 2 characterized in that, after treatment with a phosphoric diester phosphohydrolase enzyme, it has an anionic character measured by HPAEC chromatography with conductimetric detection in chemical suppression mode which is higher than that of phosphonoformic acid and lower than that of thymidine 5 'triphosphate -. 4 / - Compound according to one of claims
1 à 3, caractérisé en ce qu'il présente un caractère hydrophobe mesuré par chromatographie HPLC sur phase inverse de type C18 éluée en paire d'ions avec l'acétate d'ammonium, inférieur à celui de la thymidine 5' monophosphate.1 to 3, characterized in that it has a hydrophobic character measured by HPLC chromatography on reverse phase of type C18 eluted in pair of ions with ammonium acetate, lower than that of thymidine 5 'monophosphate.
5/ - Composé selon l'une des revendications5 / - Compound according to one of claims
1 à 4, caractérisé en ce que son effet d' activation sur les lymphocytes Ty§ est inhibé en présence d'anticorps monoclonaux spécifiques de TCR humains comprenant les régions variables Vy et V 2.1 to 4, characterized in that its activation effect on the Ty§ lymphocytes is inhibited in the presence of monoclonal antibodies specific for human TCR comprising the variable regions Vy and V 2.
6/ - Composé selon l'une des revendications 1 à 5, caractérisé en ce qu'il comprend au moins un substituant X d'un atome de phosphore de masse moléculaire
inférieure à 500, qui est distinct d'un acide nucléique, d'un oligosaccharide, d'un acide gras, d'un protide, d'un alcaloïde et d'un stéroïde, et qui est lié dans le composé à un groupement phosphoryle par une liaison covalente acidolabile susceptible d'être clivée en présence d'une monoester phosphatase.6 / - Compound according to one of claims 1 to 5, characterized in that it comprises at least one substituent X of a phosphorus atom of molecular mass less than 500, which is distinct from a nucleic acid, an oligosaccharide, a fatty acid, a protide, an alkaloid and a steroid, and which is linked in the compound to a phosphoryl group by an acidolabile covalent bond capable of being cleaved in the presence of a monoester phosphatase.
7/ - Composé selon la revendication 6, caractérisé en ce qu'il répond à la formule :7 / - Compound according to claim 6, characterized in that it corresponds to the formula:
R est un hydrogène ou un substituant minéral ou organique, n est un nombre entier non nul.R is hydrogen or an inorganic or organic substituent, n is a non-zero integer.
8/ - Composé selon l'une des revendications 1 à 7, caractérisé en ce qu'il comporte au moins un groupement nucléosidique.8 / - Compound according to one of claims 1 to 7, characterized in that it comprises at least one nucleoside group.
9/ - Composé selon la revendication 8, caractérisé en ce que le groupement nucléosidique est la thymidine liée à un groupement phosphoryle par le cinquième carbone de son radical 2-désoxyribose. 10/ - Composé selon l'une des revendications 1 à 7, caractérisé en ce qu'il est constitué d'un produit de l'hydrolyse d'un composé selon la revendication 9 par l'action enzymatique d'une nucléotide pyrophosphatase et/ou d'une diester phosphorique phosphohydrolase.9 / - Compound according to claim 8, characterized in that the nucleoside group is thymidine linked to a phosphoryl group by the fifth carbon of its 2-deoxyribose radical. 10 / - Compound according to one of claims 1 to 7, characterized in that it consists of a product of the hydrolysis of a compound according to claim 9 by the enzymatic action of a nucleotide pyrophosphatase and / or a phosphoric phosphohydrolase diester.
11/ - Composé selon l'une des revendications 1 à 10, caractérisé en ce qu'il est constitué d'un monoester phosphorique acidolabile de masse moléculaire inférieure à 500 distinct de la thymidine 5' monophosphate, de la thymidine 5' diphosphate, de la thymidine 5 ' diphosphate glucose et de la thymidine- 5 ' triphosphate.11 / - Compound according to one of claims 1 to 10, characterized in that it consists of an acidolabile phosphoric monoester of molecular mass less than 500 distinct from thymidine 5 'monophosphate, thymidine 5' diphosphate, thymidine 5 'diphosphate glucose and thymidine-5' triphosphate.
12/ - Composé selon l'une des revendications 1 à 10, caractérisé en ce qu'il est constitué d'un diester phosphorique acidolabile de masse
moléculaire inférieure à 1 000, distinct de la thymidine 5' monophosphate, de la thymidine 5' diphosphate, de la thymidine 5 ' diphosphate glucose et de la thymidine 5 ' triphosphate. 13/ - Composé selon l'une des revendications 1 à 12 pour utilisation comme antigène des lymphocytes Ty5 humains.12 / - Compound according to one of claims 1 to 10, characterized in that it consists of an acidolabile phosphoric diester of mass molecular mass less than 1000, distinct from thymidine 5 'monophosphate, thymidine 5' diphosphate, thymidine 5 'diphosphate glucose and thymidine 5' triphosphate. 13 / - Compound according to one of claims 1 to 12 for use as an antigen of human Ty5 lymphocytes.
14/ - Procédé pour caractériser un antigène des lymphocytes Tyg humains caractérisé en ce que : - l'on vérifie qu'il induit une activation des lymphocytes,14 / - Method for characterizing an antigen of human Tyg lymphocytes characterized in that: - it is verified that it induces activation of the lymphocytes,
- l'on vérifie que cette propriété vis-à- vis des lymphocytes disparaît lorsqu'il est placé en présence d'un mélange enzymatique comprenant au moins une monoester phosphorique phosphohydrolase et au moins une diester phosphorique phosphohydrolase.- It is verified that this property vis-à-vis lymphocytes disappears when it is placed in the presence of an enzyme mixture comprising at least one phosphoric phosphohydrolase monoester and at least one phosphoric phosphohydrolase diester.
15/ - Procédé pour préparer et/ou isoler et/ou caractériser au moins, un composé organo-phosphoré selon l'une des revendications 1 à 13, caractérisé en ce qu'on soumet une solution aqueuse activant les lymphocytes Ty§ humains à au moins une étape de séparation par chromatographie préparative en différentes fractions, et en ce que l'on teste l'activité de chaque fraction sur des lymphocytes Ty5 humains. 16/ - Procédé selon la revendication 15 pour isoler au moins un composé selon l'une des revendications 1 à 13, caractérisé par les étapes suivantes :15 / - Process for preparing and / or isolating and / or characterizing at least one organophosphorus compound according to one of claims 1 to 13, characterized in that an aqueous solution is subjected which activates human Ty§ lymphocytes to at minus a separation step by preparative chromatography into different fractions, and in that the activity of each fraction is tested on human Ty5 lymphocytes. 16 / - Method according to claim 15 for isolating at least one compound according to one of claims 1 to 13, characterized by the following steps:
- on cultive une souche d'êtres vivants unicellulaires susceptible d'activer des lymphocytes y humains à récepteurs TCR comprenant les régions variables- a strain of single-celled living beings capable of activating human lymphocytes y with TCR receptors comprising the variable regions is cultivated
V 9 Vδ2,V 9 V δ2 ,
- on réalise un extrait brut de la souche ou on collecte son milieu de culture, dont on sépare les composants hydrosolubles,- a crude extract of the strain is produced or its culture medium is collected, from which the water-soluble components are separated,
- on sépare une solution aqueuse comprenant ces composants hydrosolubles par chromatographie préparative en différentes fractions susceptibles d'activer
les lymphocytes y humains à récepteurs TCR comprenant les régions variables Vy V 2-- An aqueous solution comprising these water-soluble components is separated by preparative chromatography into different fractions capable of activating human y lymphocytes with TCR receptors comprising the Vy V 2- variable regions
17/ - Procédé selon la revendication 16, caractérisé en ce qu'on cultive une souche de mycobactéries, on effectue une extraction lipidique, et on sépare la phase aqueuse de l'extrait lipidique brut.17 / - Process according to claim 16, characterized in that a strain of mycobacteria is cultivated, a lipid extraction is carried out, and the aqueous phase is separated from the crude lipid extract.
18/ - Procédé selon la revendication 17, caractérisé en ce qu'on cultive Mycobacterium tuberculosis.18 / - Method according to claim 17, characterized in that cultivates Mycobacterium tuberculosis.
19/ - Procédé selon l'une des revendications 15 et 16, caractérisé en ce qu'on cultive une souche de mycobactéries aptes à sécréter le (les) composé(s) dans le milieu de culture.19 / - Method according to one of claims 15 and 16, characterized in that cultivates a strain of mycobacteria capable of secreting (the) compound (s) in the culture medium.
20/ - Procédé selon la revendication 19, caractérisé en ce qu'on cultive Mycobacteri um fortui tum biovar fortui tum.20 / - A method according to claim 19, characterized in that one cultivates Mycobacteri um fortui tum biovar fortui tum.
21 / - Procédé selon l'une des revendications 15 à 20, caractérisé en ce que l'étape de séparation par chromatographie comprend une séparation anionique par chromatographie échangeuse d'anions avec une solution saline dont on récupère l'éluat qui active les lymphocytes Tyg.21 / - Method according to one of claims 15 to 20, characterized in that the separation step by chromatography comprises an anionic separation by anion exchange chromatography with a saline solution from which the eluate is recovered which activates the Tyg lymphocytes .
22/ - Procédé selon la revendication 21 , caractérisé en ce qu'on effectue ensuite une séparation de la solution saline qui active les lymphocytes Ty5 suivie d'un séchage.22 / - Method according to claim 21, characterized in that one then performs a separation of the saline solution which activates the Ty5 lymphocytes followed by drying.
23/ - Procédé selon l'une des revendications 15 à 22, caractérisé en ce que l'étape de séparation par chromatographie comprend au moins une séparation préparative des différents principes actifs de l'éluat par chromatographie HPLC sur phase inverse C18 éluée en paire d'ions et/ou par chromatographie échangeuse d'anions HPAEC avec détection conductimétrique opérant en mode de suppression chimique, chaque étape de séparation étant suivie d'une mesure de l'activité des différentes fractions obtenues sur des lymphocytes T.,5.23 / - Method according to one of claims 15 to 22, characterized in that the separation step by chromatography comprises at least one preparative separation of the different active ingredients of the eluate by HPLC chromatography on reverse phase C18 eluted in pair d ion and / or by HPAEC anion exchange chromatography with conductimetric detection operating in chemical suppression mode, each separation step being followed by a measurement of the activity of the various fractions obtained on T lymphocytes, 5.
24/ - Composé activant les lymphocytes humains à récepteurs TCR comprenant les régions variables Vy et V5 -notamment Vy et V 2- caractérisé en ce qu'il est
obtenu par un procédé selon l'une des revendications 15 à 23.24 / - Compound activating human lymphocytes with TCR receptors comprising the variable regions Vy and V5-notably Vy and V 2- characterized in that it is obtained by a process according to one of claims 15 to 23.
25/ - Composition pharmaceutique caracté¬ risée en ce qu'elle comporte au moins un composé organo- phosphore selon l'une des revendications 1 à 13 ou 24.25 / - Pharmaceutical composition caracté¬ ized in that it comprises at least one organophosphorus compound according to one of claims 1 to 13 or 24.
26/ - Composition vaccinante caractérisée en ce qu'elle comporte au moins un antigène selon l'une des revendications 1 à 13 ou 24.26 / - Vaccinating composition characterized in that it comprises at least one antigen according to one of claims 1 to 13 or 24.
27/ - Utilisation d'au moins un composé selon l'une des revendications 1 à 13 ou 24, pour obtenir une composition pharmaceutique pour le traitement préventif ou curatif des maladies infectieuses de l'homme ou de 1'animal.27 / - Use of at least one compound according to one of claims 1 to 13 or 24, to obtain a pharmaceutical composition for the preventive or curative treatment of infectious diseases of humans or animals.
28/ - Utilisation d'au moins un composé selon l'une des revendications 1 à 13 ou 24 pour obtenir une composition pharmaceutique pour le traitement préventif ou curatif des maladies infectieuses mycobactériennes -notamment la lèpre ou la tuberculose- de 1'homme ou de 1'animal. 29/ - Utilisation d'au moins un composé selon l'une des revendications 1 à 13 ou 24 pour obtenir une composition pharmaceutique pour le traitement préventif ou curatif des affections tumorales ou leucémiques de 1'homme ou de 1'animal. 30/ - Utilisation d'au moins un composé selon l'une des revendications 1 à 13 ou 24 pour obtenir une composition pharmaceutique pour le traitement préventif ou curatif des parasitoses de l'homme ou de l'animal.28 / - Use of at least one compound according to one of claims 1 to 13 or 24 to obtain a pharmaceutical composition for the preventive or curative treatment of mycobacterial infectious diseases - notably leprosy or tuberculosis - of humans or The animal. 29 / - Use of at least one compound according to one of claims 1 to 13 or 24 to obtain a pharmaceutical composition for the preventive or curative treatment of tumor or leukemic diseases of humans or animals. 30 / - Use of at least one compound according to one of claims 1 to 13 or 24 to obtain a pharmaceutical composition for the preventive or curative treatment of parasitic diseases in humans or animals.
31/ - U.tilisation d'au moins un composé selon l'une des revendications 1 à 13 ou 24 pour obtenir une composition pharmaceutique pour le traitement préventif ou curatif des pathologies d' immunodéficiences telles que le SIDA.31 / - U. use of at least one compound according to one of claims 1 to 13 or 24 to obtain a pharmaceutical composition for the preventive or curative treatment of immunodeficiency pathologies such as AIDS.
32/ - Utilisation d'au moins un composé selon l'une des revendications 1 à 13 ou 24 pour obtenir une composition pharmaceutique pour le traitement préventif ou curatif des maladies parasitaires, notamment du paludisme.
33/ - Composition thérapeutique caracté¬ risée en ce qu'elle contient une proportion pharmaceutiquement acceptable d'au moins un principe présentant une activité enzymatique phosphatase susceptible de dégrader au moins un composé activant des lymphocytes Ty5 humains à récepteurs TCR comprenant les régions variables Vyg et V 2.32 / - Use of at least one compound according to one of claims 1 to 13 or 24 to obtain a pharmaceutical composition for the preventive or curative treatment of parasitic diseases, in particular malaria. 33 / - Therapeutic composition characterized in that it contains a pharmaceutically acceptable proportion of at least one principle having an enzymatic phosphatase activity capable of degrading at least one compound activating human Ty5 lymphocytes with TCR receptors comprising the variable regions y g and V 2.
34/ - Composition selon la revendication 33, caractérisée en , ce qu'elle comporte au moins une monoester phosphorique phosphohydrolase.34 / - Composition according to claim 33, characterized in, that it comprises at least one phosphoric phosphohydrolase monoester.
35/ - Composition selon l'une des revendications 33 et 34, caractérisée en ce qu'elle comporte au moins une nucléotide pyrophosphatase.35 / - Composition according to one of claims 33 and 34, characterized in that it comprises at least one nucleotide pyrophosphatase.
36/ - Composition selon l'une des revendications 33 à 35, caractérisée en ce qu'elle comporte au moins une diester phosphorique phosphohydrolase.36 / - Composition according to one of claims 33 to 35, characterized in that it comprises at least one phosphoric phosphohydrolase diester.
37/ - Composition selon l'une des revendications 33 à 36, caractérisée en ce qu'elle comporte au moins une enzyme phosphatase alcaline. 38/ - Composition selon l'une des revendications 25, 26 ou 33 à 37, caractérisée en ce qu'elle est formulée sous une forme galénique destinée à son administration par injection dans le sang circulant.37 / - Composition according to one of claims 33 to 36, characterized in that it comprises at least one alkaline phosphatase enzyme. 38 / - Composition according to one of claims 25, 26 or 33 to 37, characterized in that it is formulated in a galenical form intended for its administration by injection into the circulating blood.
39/ - Utilisation d'une composition selon l'une des revendications 33 à 38 pour obtenir un médicament inhibant la cytotoxicite des lymphocytes Ty impliqués dans au moins une pathologie auto-immune.39 / - Use of a composition according to one of claims 33 to 38 for obtaining a medicament inhibiting the cytotoxicity of the Ty lymphocytes involved in at least one autoimmune pathology.
40/ - Utilisation d'une composition selon l'une des revendications 33 à 38 pour obtenir un médicament destiné au traitement de la sclérose en plaques.
40 / - Use of a composition according to one of claims 33 to 38 to obtain a medicament intended for the treatment of multiple sclerosis.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9401170A FR2715660A1 (en) | 1994-01-28 | 1994-01-28 | Organophosphorus activating compounds of Tgammadelta lymphocytes, process for preparing and / or isolating and / or characterizing these compounds, compositions and pharmaceutical uses. |
| FR94/01170 | 1994-01-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1995020673A1 true WO1995020673A1 (en) | 1995-08-03 |
Family
ID=9459692
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/FR1995/000092 WO1995020673A1 (en) | 1994-01-28 | 1995-01-26 | Organo-phosphorus compounds as activators of gamma delta t cells |
Country Status (2)
| Country | Link |
|---|---|
| FR (1) | FR2715660A1 (en) |
| WO (1) | WO1995020673A1 (en) |
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| US6015796A (en) * | 1998-03-11 | 2000-01-18 | Zeria Pharmaceutical Co., Ltd. | Method for treating AIDS |
| US6020325A (en) * | 1999-03-09 | 2000-02-01 | Zeria Pharmaceutical Co., Ltd. | Method for inhibiting replication of HIV |
| FR2782721A1 (en) * | 1998-09-01 | 2000-03-03 | Inst Nat Sante Rech Med | NEW PHOSPHOHALOHYDRIN COMPOUNDS, MANUFACTURING PROCESS AND APPLICATIONS |
| FR2782722A1 (en) * | 1998-09-01 | 2000-03-03 | Inst Nat Sante Rech Med | NOVEL PHOSPHOEPOXIDE COMPOUNDS, MANUFACTURING METHOD AND APPLICATIONS |
| WO2000059916A1 (en) * | 1999-04-06 | 2000-10-12 | Institut National De La Sante Et De La Recherche Medicale | Compounds selectively inhibiting gamma 9 delta 2 t lymphocytes |
| WO2005077411A2 (en) * | 2004-02-10 | 2005-08-25 | Innate Pharma | Composition and method for the treatment of carcinoma |
| WO2006067635A2 (en) * | 2004-12-20 | 2006-06-29 | Innate Pharma S.A. | USE OF Ϝδ T LYMPHOCYTE ACTIVATORS AS VACCINE ADJUVANT |
| WO2007039635A2 (en) * | 2005-10-06 | 2007-04-12 | Innate Pharma | Phosphoantigen salts of organic bases and methods for their crystallization |
| WO2008006895A2 (en) | 2006-07-13 | 2008-01-17 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for increasing the efficiency of therapeutic antibodies using gamma delta t cell activators |
| WO2008059052A1 (en) | 2006-11-17 | 2008-05-22 | Innate Pharma | Improved methods of using phosphoantigen for the treatment of cancer |
| US7399756B2 (en) | 2001-07-20 | 2008-07-15 | Bioagency Ag | Organo-phosphorous compounds for activating gamma/delta T cells |
| EP2123285A1 (en) * | 2008-05-21 | 2009-11-25 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Nucleosidic phosphoantigens for use in VGAMMA9DELTA2 T cell-mediated therapy |
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|---|---|---|---|---|
| WO2000043403A1 (en) * | 1999-01-21 | 2000-07-27 | Chugai Seiyaku Kabushiki Kaisha | 2-methyl-3-butenyl-1-pyrophosphoric acid salts and agents for treating lymphocytes |
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1994
- 1994-01-28 FR FR9401170A patent/FR2715660A1/en active Pending
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1995
- 1995-01-26 WO PCT/FR1995/000092 patent/WO1995020673A1/en active Application Filing
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| FR2782722A1 (en) * | 1998-09-01 | 2000-03-03 | Inst Nat Sante Rech Med | NOVEL PHOSPHOEPOXIDE COMPOUNDS, MANUFACTURING METHOD AND APPLICATIONS |
| US7109183B2 (en) | 1998-09-01 | 2006-09-19 | Institut National de la Sante et de la Recherchik Medicale | Phosphohalohydrins, process for the production thereof and use thereof |
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| JP2002523513A (en) * | 1998-09-01 | 2002-07-30 | アンスティテュ ナシオナル ド ラ サンテ エ ド ラ ルシェルシュ メディカル | Phosphohalohydrins, their preparation and use |
| WO2000012516A1 (en) * | 1998-09-01 | 2000-03-09 | Institut National De La Sante Et De La Recherche Medicale | Phosphohalohydrins, method for making same and uses |
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| FR2791981A1 (en) * | 1999-04-06 | 2000-10-13 | Inst Nat Sante Rech Med | COMPOUNDS SELECTIVELY INHIBITING TGAMMA9DELTA2 LYMPHOCYTES, AND THEIR APPLICATIONS |
| WO2000059916A1 (en) * | 1999-04-06 | 2000-10-12 | Institut National De La Sante Et De La Recherche Medicale | Compounds selectively inhibiting gamma 9 delta 2 t lymphocytes |
| US6624151B1 (en) | 1999-04-06 | 2003-09-23 | Institut National De La Sante Et De La Recherche Medicale | Compounds selectively inhibiting gamma 9 delta 2 T lymphocytes |
| US7871992B2 (en) | 2001-07-20 | 2011-01-18 | Bioagency Ag | Organophosphorous compounds for the activation of gamma/delta T cells |
| US7399756B2 (en) | 2001-07-20 | 2008-07-15 | Bioagency Ag | Organo-phosphorous compounds for activating gamma/delta T cells |
| WO2005077411A3 (en) * | 2004-02-10 | 2006-04-13 | Innate Pharma | Composition and method for the treatment of carcinoma |
| WO2005077411A2 (en) * | 2004-02-10 | 2005-08-25 | Innate Pharma | Composition and method for the treatment of carcinoma |
| WO2006067635A3 (en) * | 2004-12-20 | 2006-08-24 | Innate Pharma Sa | USE OF Ϝδ T LYMPHOCYTE ACTIVATORS AS VACCINE ADJUVANT |
| WO2006067635A2 (en) * | 2004-12-20 | 2006-06-29 | Innate Pharma S.A. | USE OF Ϝδ T LYMPHOCYTE ACTIVATORS AS VACCINE ADJUVANT |
| WO2007039635A3 (en) * | 2005-10-06 | 2007-06-21 | Innate Pharma Sa | Phosphoantigen salts of organic bases and methods for their crystallization |
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| WO2008006895A2 (en) | 2006-07-13 | 2008-01-17 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for increasing the efficiency of therapeutic antibodies using gamma delta t cell activators |
| WO2008059052A1 (en) | 2006-11-17 | 2008-05-22 | Innate Pharma | Improved methods of using phosphoantigen for the treatment of cancer |
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| FR2715660A1 (en) | 1995-08-04 |
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