EP1305332A2 - Molecule of pharmaceutical interest comprising at its n-terminal a glutamic acid or a glutamine in the form of a physiologically acceptable strong acid - Google Patents
Molecule of pharmaceutical interest comprising at its n-terminal a glutamic acid or a glutamine in the form of a physiologically acceptable strong acidInfo
- Publication number
- EP1305332A2 EP1305332A2 EP01919544A EP01919544A EP1305332A2 EP 1305332 A2 EP1305332 A2 EP 1305332A2 EP 01919544 A EP01919544 A EP 01919544A EP 01919544 A EP01919544 A EP 01919544A EP 1305332 A2 EP1305332 A2 EP 1305332A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- molecule
- seq
- mhc
- glutamine
- strong acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 title claims abstract description 35
- 235000013922 glutamic acid Nutrition 0.000 title claims abstract description 34
- 239000004220 glutamic acid Substances 0.000 title claims abstract description 34
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 title claims abstract description 26
- 239000002253 acid Substances 0.000 title claims description 36
- 239000003446 ligand Substances 0.000 claims abstract description 61
- 229960005486 vaccine Drugs 0.000 claims abstract description 30
- 150000003839 salts Chemical class 0.000 claims abstract description 25
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 98
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 35
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 27
- 230000000694 effects Effects 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 150000001875 compounds Chemical class 0.000 claims description 15
- 235000018102 proteins Nutrition 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 15
- VFRSADQPWYCXDG-LEUCUCNGSA-N ethyl (2s,5s)-5-methylpyrrolidine-2-carboxylate;2,2,2-trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.CCOC(=O)[C@@H]1CC[C@H](C)N1 VFRSADQPWYCXDG-LEUCUCNGSA-N 0.000 claims description 10
- 239000002671 adjuvant Substances 0.000 claims description 9
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 238000000338 in vitro Methods 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 8
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- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 3
- 229910002651 NO3 Inorganic materials 0.000 claims description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 3
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- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 claims description 2
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- UZQJVUCHXGYFLQ-AYDHOLPZSA-N [(2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-4-[(2r,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-6-(hy Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2[C@@]1(C=O)C)C)(C)CC(O)[C@]1(CCC(CC14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O[C@H]4[C@@H]([C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)[C@H](O)[C@@H](CO)O4)O)[C@H](O)[C@@H](CO)O3)O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UZQJVUCHXGYFLQ-AYDHOLPZSA-N 0.000 claims description 2
- 229940037003 alum Drugs 0.000 claims description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 2
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 claims description 2
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 18
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 18
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 13
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- POVNCJSPYFCWJR-USZUGGBUSA-N (4s)-4-[[(2s)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-4-methylpentanoyl]amino]-5-[(2s)-2-[[2-[(2s)-2-[[(2s)-1-[[(2s,3r)-1-[[(1s)-1-carboxy-2-methylpropyl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamoyl]pyrrolidin-1- Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N1[C@@H](CCC1)C(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O)C1=CC=C(O)C=C1 POVNCJSPYFCWJR-USZUGGBUSA-N 0.000 description 6
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to a molecule of pharmaceutical interest, preferably a ligand of the Complex Histocompatibility major (MHC), comprising a glutamic acid or a glutamine at its N-terminal end, which is in the form of a physiologically acceptable strong acid addition salt, as well as a vaccine comprising such a ligand.
- MHC Complex Histocompatibility major
- Vaccination is an effective way to prevent or reduce viral or bacterial infections.
- Vaccine antigens administered alone in the host are often not immunogenic enough to induce an immune response, and must therefore be combined with an adjuvant or coupled to a carrier protein to induce (or increase) their immunogenicity. Under these conditions, only a humoral type immune response can be induced.
- CTL cytotoxic T lymphocytes
- CTL responses and T auxiliary have also been well described for vaccines against parasites such as Plasmodium falciparum, the causative agent of malaria (Le et al, Vaccine, 1998. 16, 305-312).
- the primordial role of CTL responses has also been well documented in anti-tumor responses, in particular those directed against melanoma cells (reviewed in Rivoltini et al., Crit. Rev. Immunol, 1998, 18, 55-63).
- the CTL epitope (s) peptide sequences interacting with class I molecules and presented to CD8 + T lymphocytes have been defined for several antigens.
- MHC ligands The interest of these MHC ligands is confirmed by the increasing number of clinical studies in humans of these compounds as vaccine candidates against various pathologies and in particular as anti-melanoma vaccines (epitopes m27-25 MART 1, g209- 217, g280-288, gplOO, MAGE 3), as an anti-HIN vaccine (Klinguer et al, Vaccine, 2000, 18, 259-267) or also as anti-HBN vaccines of anti-HBN lipopeptide types (Livingston et al, J. Immunol., 1999, 162, 3088-3095).
- the difficulty of these studies lies in the fact that the peptides used are difficult to keep before their administration to patients, which can lead to a reduction in their vaccine power, and to more rapid degradation in vivo.
- the peptides having an amino acid of glutamic acid (Glu, E) or glutamine (Gln, Q) type at their N-terminal end cyclize with the free ⁇ -carboxylic acid function to form a pyroglutamate according to the reaction defined above below:
- This acetylation reaction is however a minor modification of the structure of the peptide conventionally used by a person skilled in the art to improve the stability of a peptide (Brinckerhoff et al, Int. J. Cancer, 1999, 83, 326): it it is the replacement of one of the protons of the N-terminal NH function with an acetyl group CH 3 CO with a slight increase in mass (42 Da over 985 Da), all the rest of the structure remaining unchanged.
- peptides obtained by chemical synthesis are purified by reverse phase HPLC using eluents containing trifluoroacetic acid (TFA) before being lyophilized.
- the purified peptides obtained are positively charged and are in the form of trifluoroacetate salt (RNH 3 + , CF 3 CO " ).
- the amount of trifluoroacetate and residual trifluoroacetic acid is generally proportional to the number of basic amino acids (Lysine, Arginine and Histidine) contained in the sequence as well as in the amino function of the N-terminal amino acid.
- Peptides in the form of trifluoroacetate are commonly used for pre-clinical in vitro and in vivo experiments in animals.
- acetic acid is a weak acid, which gives increased instability to the peptide. This forces the investigators to keep the peptide at -80 ° C (liquid nitrogen) in lyophilized form and to resolubilize it extemporaneously just before injection. which involves a very restrictive cold chain.
- the present invention proposes to solve these problems of structural instability, conservation over time, toxicity and biological activity.
- molecules of pharmaceutical interest in particular MHC ligands, having a glutamic acid or a glutamine at their N-terminal end can be stabilized in the form of addition salt of a strong acid, and that this makes it possible both to maintain the biological activity and to obtain an easy conservation of the peptide or the like in a stable form, which allows its therapeutic use in humans.
- molecule of pharmaceutical interest in particular the MHC ligands, the natural or synthetic molecules having an epitope for the generation of antibodies, the molecules derived from receptor ligands, and having an agonist or antagonist activity with respect to these receptors, or having antibiotic, antifungal, or antiviral activity.
- the molecules of therapeutic interest according to the invention are all characterized in that they have a glutamic acid or a -glutamine at their N-terminal end.
- the molecules of pharmaceutical interest preferred according to the present invention are the ligands of the MHC.
- the present invention thus in particular relates to a MHC ligand comprising at its N-terminal end a glutamic acid or a glutamine, characterized in that it is in the form of addition salt of a strong physiologically acceptable acid.
- the physiologically acceptable strong acid addition salt can in particular be chosen from addition salts with strong mineral or organic acids.
- methanesulfonate or mesilate
- hydrochloride hydrobromide
- sulfate nitrate
- phosphate phosphate
- MHC ligands within the meaning of the present invention are in particular MHC class I and IL ligands MHC is an important group of proteins involved in the presentation of antigens to T lymphocytes.
- MHC class I molecules are membrane proteins found on all nucleated cells and platelets.
- MHC class II molecules are expressed on B cells, macrophages, monocytes, antigen-presenting cells and certain T cells.
- B cells are lymphocytes, which in mature form have immunoglobulin on their surface making "antigen receptor” function.
- T cells are lymphocytes that express their receptor for the antigen (TcR) and differentiate into 2 subpopulations: helper T cells (Th or T helper) and cytotoxic T cells (CTL).
- Th cells help B cells to divide, differentiate and produce antibodies.
- the majority of Th are CD4 + (specific surface marker) and recognize the antigen presented on the surface of cells presenting the antigen, in association with MHC class II molecules.
- Cytotoxic T cells are capable of destroying target cells infected by viruses or allogenic cells.
- the majority are CD 8+ and recognize the antigen associated with MHC class I molecules on the surface of the target cell.
- the antigen is recognized by the formation of a complex comprising in particular the MHC molecule having a MHC ligand, and the T cell receptor (TCR).
- the molecules of pharmaceutical interest in particular the MHC ligands according to the present invention, can be chosen from natural or synthetic molecules, and inter alia, from proteins, peptides, multi-epitopic polypeptide constructs, or analogs of peptides of the pseudopeptide type, retro-inverso, peptoids, peptido-mimetics, lipopeptides. These molecules can also consist in part of a peptide chain, with the replacement of certain amino acids by analogues of amino acids, or having ramifications. These molecules can also present the various modifications which are observed on natural proteins or peptides (for example O- or N-glycosylation).
- the MHC ligands according to the present invention are chosen from CTL epitopes, that is to say which. allow the generation of cytotoxic T lymphocytes and in particular among those which are in the form of octapeptide, nonapeptide or decapeptide.
- the MHC ligand can also be chosen from the ligands described in the SYFPEITHI or MHCPEP databases, previously cited, and which comprise at their N-terminal end a glutamic acid or a glutamine.
- This ligand can in particular be chosen from MHC ligands (ligands of MHC molecules of class I or II) included in the group consisting of peptides corresponding to the sequences SEQ ID No. 1 to SEQ ID No. 694.
- ELA MART-1 26-35 EAAGIGILTN A2 112 MAGE-1 161-169 EADPTGHSY Al 2
- the ligands according to the invention can also be chosen from multiepitopic polypeptide constructions having an amino acid of glutamic acid (Glu, E) or glutamine (Gln, Q) type at the N-terminal end such as the following peptide (SEQ ID N ° 695): NEF 117 EWRFDSRLAFHHVAREHPEYFNKNK (PaIm) NH 2
- anti-HIV lipopeptide in clinical phase I Klinguer, et al, Vaccine, 1999, 18, 259-267.
- the peptide analogs can be chosen from those described in application FR276307 which comprise at their N-terminal end a glutamic acid or a glutamine.
- the invention relates to the MHC ligand of sequence ELAGIGILTV, in sulphate form or, even more preferably, in hydrochloride form.
- the invention also relates to a pharmaceutical composition comprising at least one molecule of pharmaceutical interest according to the invention.
- compositions can in particular be intended for the treatment of various immunopathologies: immunodeficiency, autoimmune diseases, hypersensitivities, allergies or to avoid rejection of grafts.
- a composition according to the invention can also be used for an antibiotic, antiviral or antifungal purpose, or can be intended for the treatment of diseases linked to hormonal disorders, or to diseases of the central nervous system.
- compositions according to the invention can also be used in the veterinary field. Indeed, the same problems of structural instability, conservation over time, toxicity and activity which arise for the preparation of veterinary preparations comprising a peptide or a molecule having a glutamic acid or a glutamine at their N- end terminal, can be resolved using strong acid addition salts to stabilize said peptides or molecules.
- a preferred composition consists of a vaccine characterized in that it comprises at least one MHC ligand according to the invention, which is in the form of a physiologically acceptable strong acid addition salt , as defined above.
- This vaccine can also comprise at least one adjuvant, in particular chosen from Aluminum (Alum) or Calcium salts, OmpA enterobacterial proteins, tetanus toxoid (TT), diphtheria toxoid (DT), CRM 197 (cross-reactive material), PLGA, ISCOM, Montanide ISA 720, aliphatic quaternary ammoniums, MPL-A, Quil-A, CpG, Leif, cholera toxin (CT), LT (LT for "Heat labile enterotoxin "heat labile enterotoxin) or detoxified versions of CT or LT.
- the vaccine further comprises a carrier compound mixed or coupled to said ligand.
- said carrier compound is chosen from the group of peptides comprising toxoids, in particular diphtheria toxoid (DT) or tetanus toxoid (TT), proteins derived from streptococcus (such as the protein for binding to human serum albumin, called “ BB “described in WO96 / 14415), OmpA membrane proteins (for” Outer Membrane Protein type A ”) and external membrane protein complexes (OMPC), external membrane vesicles (OMV) or heat shock proteins ("Heat Shock Protein” or HSP).
- said carrier compound is covalently coupled with the ligand.
- the term “coupling” is intended to denote both a coupling carried out chemically between the two compounds, and a biological coupling, by genetic recombination, as defined below.
- the covalent coupling of the antigen or hapten can be carried out at the N or C terminus of the carrier compound.
- the bifunctional reagents allowing this coupling are determined according to the end of the chosen carrier compound and the nature of the antigen or the hapten to be coupled. These coupling techniques are well known to those skilled in the art.
- the conjugates resulting from a coupling of peptides can also be prepared by genetic recombination.
- the hybrid (conjugated) peptide can in fact be produced by recombinant DNA techniques by insertion or addition to the DNA sequence coding for the carrier compound, of a sequence coding for the antigen, immunogenic or hapten peptides. These techniques for preparing hybrid peptide by genetic recombination are well known to those skilled in the art (cf. for example Makrides, 1996, Microbiologicals Reviews, 60, 512-538).
- said carrier compound is a protein derived from streptococcus or an OmpA membrane protein from enterobacteria, in particular from Klebsiella pneumoniae, or one of its fragments.
- the ligand according to the invention optionally associated with a carrier compound can be incorporated into vectors chosen from liposomes, virosomes, nanospheres, microspheres, microcapsules or biovectors.
- a person skilled in the art knows how to choose the appropriate vector according to the aim sought (protection of the ligand possibly associated with a carrier compound or an adjuvant from degradation, targeting of cells of interest, search for penetration of the material contained in the vector inside target cells ).
- One embodiment of the invention relates in particular to an anti-melanoma vaccine characterized in that it comprises at least one ELAGIGILTV peptide (SEQ ID No. 81) in the form of the hydrochloride or sulphate.
- Another form relates to an anti-melanoma vaccine characterized in that it comprises at least one ELAGIGILTV peptide (SEQ ID No. 81) in the form of the hydrochloride or sulfate and in addition an enterobacterium protein OmpA.
- Vaccines according to the invention can also be developed for use in the veterinary field, the identical problems of structural instability, storage over time, toxicity and activity being able to be solved in the same way.
- the subject of the invention is also a method of in vitro diagnosis of pathologies associated with the presence in the body of a patient, of MHC ligands which can interact with MHC molecules, and which may be directly or indirectly involved.
- MHC ligands which can interact with MHC molecules, and which may be directly or indirectly involved.
- it comprises the steps of: bringing a biological sample from a patient into contact, in particular blood or any biological sample susceptible to contain lymphocytes, with a MHC ligand according to the invention, under conditions allowing the formation of a binary complex between said MHC ligand and the MHC molecules present in said sample, and the reaction between said binary complex and the T cell receptors that may be present in said biological sample.
- the diagnostic methods according to the invention are advantageously carried out as follows: incubation of said biological sample with MHC ligands according to the invention, said MHC ligands being fixed on a solid support, in particular inside wells of plates microtitration of the type usually used for the implementation of detection or assay techniques well known under the name of ELISA (Enzyme Linked Immuno Sorbent Assay),
- Rinsing steps are advantageously carried out between the different steps of this process.
- Those skilled in the art know how to define the different incubation conditions, as well as the methods for detecting MHC complexes - MHC ligand - T receptor, the use of antibodies being only one method among others.
- a subject of the invention is also the kits or kits for implementing in vitro diagnostic methods as described above, comprising:
- reagents for detecting the ternary complex according to the invention which was produced at the end of the immunological reaction, said reagents possibly containing a marker or being capable of being recognized in turn by a labeled reagent, more particularly in the case where the peptide analog is not labeled.
- the use of the peptides ELAGIGILTV (SEQ ID No. 81), EAAGIGILTV (SEQ ID No. 112), EADPTGHSY (SEQ ID No. 2), or EVDPIGHLY (SEQ ID No. 273) is preferred in a method of diagnosis of melanoma.
- the peptides QVPLRPMTYK SEQ ID No. 567
- EIYKRWIIL SEQ ID No. 10
- EIKDTKEAL SEQ ID No. 692
- a ligand according to the invention for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of viral, bacterial, parasitic or fungal infections is another object of the invention.
- the invention also relates to the use of a ligand according to the invention, for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of cancers and preferably for inhibiting the growth of tumors.
- the present invention also relates to the use of a strong physiologically acceptable acid for stabilizing and maintaining the biological activity of a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its " N-terminal " end.
- the activity which one seeks to maintain is an activity of stimulation and of interaction with the cells of the immune system.
- the invention also relates to the use of a strong acid to reduce and / or suppress the formation of the pyroglutamic derivative of a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its N-terminal end.
- the present invention relates to a process for the stabilization of a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its N-terminal end, characterized in that said molecule is reacted with a strong acid in conditions making it possible to obtain said molecule in the form of a physiologically acceptable strong acid addition salt.
- the reaction with the strong acid is carried out in particular according to a process as defined below, the strong acid can be chosen from the strong acids defined above, and preferably makes it possible to obtain a hydrochloride.
- the invention also relates to a process for the preparation of a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its N-terminal end in the form of a physiologically acceptable strong acid addition salt according to the invention.
- This process may in particular comprise a step of purification by RP-HPLC of said molecule from the corresponding trifluoroacetate salt using an eluent based on said strong acid, optionally followed by a step of lyophilization of the solution thus obtained.
- An alternative method comprises a step of dissolving a trifluoroacetate salt of said molecule in an excess solution of said strong acid, optionally followed by a step of lyophilization of the solution thus obtained.
- a MHC ligand is preferred, in particular SEQ ID No. 81, 112, 2, 273, 567, 10, 692, 11, 464, 466, 106, 257, or 568. More preferably, it this is SEQ ID No. 81 and the strong acid salt is a hydrochloride.
- Figure 1 Difference in cell lysis of EL-4 A2 / Kb cells pre-prepursed with the peptide ELA, by lymphocytes obtained after immunization of mice with the peptides ELA (diamonds) or AcELA (squares) in the presence of the adjuvant protein rP40, according to the protocol of Example III ..
- FIG 3 Chromatogram of the ELA peptide in acetate (3. A) or hydrochloride (3.B) form stored at 37 ° C for two months.
- Figure 4 Chromatogram of the ELA peptide in hydrochloride form initially (4.A) or after one month of storage at 4 ° C (4.B).
- ELA peptide the ELA peptide (SEQ ID No. 81) is synthesized in solid phase from the C-terminal amino acid to the N-terminal amino acid (glutamic acid) in FMOC or tBOC chemistry. After cleavage of the resin and of the protective groups of the reactive side chains, the peptide is purified in a conventional manner with eluents based on trifluoroacetic acid / water and trifluoroacetic acid / acetonitrile before being lyophilized. The purity of the peptide is checked by reverse phase liquid chromatography. The amino acid composition is checked after hydrolysis and determination of the derived amino acids obtained. The exact mass is measured by mass spectrometry.
- Peptide PyrELA PyrELA the peptide is synthesized in the same manner as the ELA peptide to the single "difference of the coupling of the last amino acid the N-terminal: glutamic acid is replaced by a pyroglutamic acid.
- AcELA peptide the AcELA peptide is synthesized in the same way as the ELA peptide with the only difference of a covering (capping) of glutamic acid using acetic anhydride.
- This reaction is carried out using the corresponding trifluoroacetate salt, using ion exchange chromatography.
- Ion exchange resins commercially available in the form of hydrochloride (Dowex resin 1X4, Amberlite IRA 416) are used, which can be used as they are once regenerated.
- a) Regeneration of the resin the resin to be regenerated is introduced into a wide column equipped with a high porosity frit (1 or 2). The resin is then washed successively with ultra pure water (pH 5-6), IN sodium hydroxide solution (pH 14), ultra pure water (pH 7), IN HCl (pH 1) and a again with ultra pure water (pH 5-6).
- the resin is stored in an acetonitrile / HCl 10 "4 N (20/80) mixture at room temperature for at least one year.
- the amount of hydrochloride can be determined by anion exchange chromatography.
- the amount of trifluoroacetic acid can be determined by gas chromatography.
- HLA-A * 0201 / Kb (A2 / Kb) transgenic mice of strain C57B1 / 6 x BDA / 2 were used in this study (Vitiello et al., 1991, J. Exp. Med., 173,
- the MHC class I molecule expressed in these mice is a chimeric molecule formed from the al and a2 domains of the human molecule HLA-A0201
- A2 / Kb mice received 300 ⁇ g of rP40 mixed with 50 ⁇ g of ELA or 300 ⁇ g of rP40 mixed with 50 ⁇ g of AcELA. a) Generation of effector cytotoxic cells:
- lymphocytes of the draining ganglia are recovered to be stimulated in vitro with the peptide falling.
- These lymphocytes (4-5 10 6 ) are cultured in a 24-well plate in DMEM plus 10 mM HEPES, 10% FCS and 50 ⁇ M ⁇ -2-mercaptoefhanol with 2-5 10 5 EL-4 A2 / Kb cells (murine cells transfected with the HLA-A * 0201 / Kb) gene irradiated (10 kRads) pre-pulsed for 1 h at 37 ° C with 1 ⁇ M of the relevant peptide. After two weekly stimulations, the cells are tested for their cytotoxic activity.
- b) Measurement of cytotoxic activity is a measure the cytotoxic activity:
- the A2 / Kb EL-4 cells are incubated for 1 h with 51 Cr in the presence or not of ELA, washed and then co-incubated with the effector cells at different ratio in 96-well plate in a volume of 200 ⁇ l for 4 to 6 h at 37 ° C. The cells are then centrifuged and the release of 51 Cr is measured in 100 ⁇ l of supernatant. The percentage of specific lysis is calculated as follows:
- % specific lysis % lysis with cells pulsed by the peptide -% lysis with cells not pulsed by the peptide.
- Comparative example IN CTL activity of the peptides ELA, PyrELA and AcELA
- mice received:
- lymphocytes (4-5 10 6 ) are cultured in a 24-well plate in DMEM plus lOmM HEPES, 10% FCS and 50 ⁇ M ⁇ -2-mercaptoethanol with 2-5 10 5 EL- 4 A2 / Kb cells (murine cells transfected with the HLA-A * 0201 / Kb gene) irradiated (10 kRads) 1 h at 37 ° C with 1 ⁇ M of the relevant peptide. After two weekly stimulations, the cells are tested for their cytotoxic activity.
- cytotoxic activity a measurement of cytotoxic activity:
- the A2 / Kb EL-4 cells are incubated for 1 h with 51 Cr in the presence or not of ELA, washed and then co-incubated with the effector cells at different ratio in 96-well plate in a volume of 200 ⁇ l for 4 to 6 h at 37 ° C. The cells are then centrifuged and the release of 51 Cr is measured in 100 ⁇ l of supernatant. The percentage of specific lysis is calculated as follows:
- Example V Studies of accelerated stability of the acetate and hydrochloride forms of the peptide ELA. Peptides are analyzed by reverse phase HPLC using an eluent
- FIG. 3 shows the chromatograms of the ELA peptide in the form of acetate (3.A) or hydrochloride (3.B) stored at 37 ° C for 2 months.
- Example NI Stability of the ELA peptide in the form of the hydrochloride stored at 4 ° C.
- the ELA peptide in the hydrochloride form is extremely stable at 4 ° C. It can therefore be easily handled and stored at a temperature of 4 or -20 ⁇ ° C. This is not the case for an equivalent peptide (MART 3), prepared in the form of acetate which must be stored at -80 ° C (M. Marchand et al, Int.
- the strong acid saline form therefore allows a much easier conservation at 4 ° C (refrigerator) or at -20 ° C (freezer) with total physicochemical stability, as shown in the examples above.
Abstract
The invention concerns a molecule of pharmaceutical interest, preferably a major histocompatibility complex (MHC) ligand, comprising a glutamic acid or a glutamine at its N-terminal, in the form of a physiologically acceptable addition salt, and a vaccine comprising such a ligand.
Description
MOLECULE D'INTERET PHARMACEUTIQUE COMPORTANT EN SON EXTREMITE N-TERMINALE UN ACIDE GLUTAMIQUE OU UNE GLUTAMINE SOUS FORME DE SEL D'ADDITION D'ACIDE FORT PHYSIOLOGIQUEMENT ACCEPTABLE La présente invention a pour objet une molécule d'intérêt pharmaceutique, de préférence un ligand du Complexe Majeur d'Histocompatibilité (CMH), comportant un acide glutamique ou une glutamine à son extrémité N-terminale, qui se présente sous forme de sel d'addition d'acide fort physiologiquement acceptable, ainsi qu'un vaccin comprenant un tel ligand. La vaccination est un moyen efficace de prévenir ou de réduire les infections virales ou bactériennes. Les antigènes vaccinaux administrés seuls chez l'hôte ne sont souvent pas assez immunogéniques pour induire une réponse immunitaire, et doivent donc être associés à un adjuvant ou couplés à une protéine porteuse pour induire (ou augmenter) leur immunogénicité. Dans ces conditions, seules une réponse immune de type humorale peut être induite. Or, dans le cadre d'une thérapie antivirale, la génération de lymphocytes T cytotoxique (CTL) capables de reconnaître et de détruire le virus est de toute importance (Bachmann et al., Eur. J. Immunol, 199A, 24, 2228-2236 ; Borrow P., J Virol. Hepat, 1997, 4, 16-24), comme l'attestent de nombreuses études montrant in vivo, le rôle protecteur des réponses dirigées contre les épitopes viraux (Arvin AM, J. Inf. Dis. , 1992, 166, S35- S41 ; Koszinowski et al., Immunol. Lett., 1987, 16, 185-192).PHARMACEUTICAL MOLECULE OF INTEREST COMPRISING AT ITS N-TERMINAL A GLUTAMIC ACID OR A GLUTAMINE IN THE FORM OF A PHYSIOLOGICALLY ACCEPTABLE ACID ADDED SALT The present invention relates to a molecule of pharmaceutical interest, preferably a ligand of the Complex Histocompatibility major (MHC), comprising a glutamic acid or a glutamine at its N-terminal end, which is in the form of a physiologically acceptable strong acid addition salt, as well as a vaccine comprising such a ligand. Vaccination is an effective way to prevent or reduce viral or bacterial infections. Vaccine antigens administered alone in the host are often not immunogenic enough to induce an immune response, and must therefore be combined with an adjuvant or coupled to a carrier protein to induce (or increase) their immunogenicity. Under these conditions, only a humoral type immune response can be induced. However, in the context of antiviral therapy, the generation of cytotoxic T lymphocytes (CTL) capable of recognizing and destroying the virus is of great importance (Bachmann et al., Eur. J. Immunol, 199A, 24, 2228- 2236; Borrow P., J Virol. Hepat, 1997, 4, 16-24), as attested by numerous studies showing in vivo, the protective role of responses directed against viral epitopes (Arvin AM, J. Inf. Dis ., 1992, 166, S35-S41; Koszinowski et al., Immunol. Lett., 1987, 16, 185-192).
L'importance des réponses CTL et T auxiliaire a également été bien décrite pour des vaccins contre les parasites comme Plasmodium falciparum, l'agent responsable de la Malaria (Le et al, Vaccine, 1998, .16, 305-312). Le rôle primordial des réponses CTL a aussi été fortement documenté dans les réponses antitumorales notamment celles dirigées contre les cellules de mélanome (revue dans Rivoltini et al., Crit. Rev. Immunol, 1998, 18, 55-63). Le ou les épitopes CTL (séquences peptidiques interagissant avec les molécules de classe I et présentés aux lymphocytes T CD8+) ont été définis pour plusieurs antigènes. Cependant, la difficulté réside dans la génération de CTL in vivo, due à la faible immunogénicité de ces peptides (Melief, Adv. Cancer Res., 1992, 58, 143-175 ; Nandaz et Sercaz, Cell, 1995, 82, 13-17).
De nombreux ligands du CMH (Classe I et II) et notamment des peptides épitopes CTL ont été identifiés (HG Rammensee et al, Immunogenetics, 1999, 50, 213) et certaines de leurs séquences sont accessibles sur internet dans des bases de données publiques. On peut citer notamment les bases SYFPEITHI (http://www.uni-tuebingen.de/uni/kxi/) et MHCPEPThe importance of CTL responses and T auxiliary has also been well described for vaccines against parasites such as Plasmodium falciparum, the causative agent of malaria (Le et al, Vaccine, 1998. 16, 305-312). The primordial role of CTL responses has also been well documented in anti-tumor responses, in particular those directed against melanoma cells (reviewed in Rivoltini et al., Crit. Rev. Immunol, 1998, 18, 55-63). The CTL epitope (s) (peptide sequences interacting with class I molecules and presented to CD8 + T lymphocytes) have been defined for several antigens. However, the difficulty lies in the generation of CTL in vivo, due to the low immunogenicity of these peptides (Melief, Adv. Cancer Res., 1992, 58, 143-175; Nandaz and Sercaz, Cell, 1995, 82, 13- 17). Numerous MHC ligands (Class I and II) and in particular CTL epitope peptides have been identified (HG Rammensee et al, Immunogenetics, 1999, 50, 213) and some of their sequences are accessible on the Internet in public databases. These include the SYFPEITHI (http://www.uni-tuebingen.de/uni/kxi/) and MHCPEP databases
(http://wehih.wehi.edu.au/mhcpep/). De même, des super-types des principaux HLA ont été décrits (Sette et al, Immunogenetics, 1999, 50, 201-212).(Http://wehih.wehi.edu.au/mhcpep/). Similarly, super-types of the main HLAs have been described (Sette et al, Immunogenetics, 1999, 50, 201-212).
L'intérêt de ces ligands du CMH est confirmé par le nombre croissant d'études cliniques chez l'homme de ces composés en tant que candidats vaccins contre différentes pathologies et notamment comme vaccins anti-mélanome (épitopes m27-25 MART 1, g209-217, g280-288, gplOO, MAGE 3), comme vaccin anti-HIN (Klinguer et al, Vaccine, 2000, 18, 259-267) ou encore comme vaccins anti-HBN de types lipopeptides anti-HBN (Livingston et al, J. Immunol., 1999, 162, 3088-3095). Toutefois, la difficulté de ces études réside dans le fait que les peptides utilisés sont difficiles à conserver avant leur administration- aux patients, ce qui peut mener à une réduction de leur pouvoir vaccinal, et à une dégradation plus rapide in vivo.The interest of these MHC ligands is confirmed by the increasing number of clinical studies in humans of these compounds as vaccine candidates against various pathologies and in particular as anti-melanoma vaccines (epitopes m27-25 MART 1, g209- 217, g280-288, gplOO, MAGE 3), as an anti-HIN vaccine (Klinguer et al, Vaccine, 2000, 18, 259-267) or also as anti-HBN vaccines of anti-HBN lipopeptide types (Livingston et al, J. Immunol., 1999, 162, 3088-3095). However, the difficulty of these studies lies in the fact that the peptides used are difficult to keep before their administration to patients, which can lead to a reduction in their vaccine power, and to more rapid degradation in vivo.
Pour stabiliser un peptide à usage pharmaceutique ayant un acide glutamique ou une glutamine en Ν-terminal sous une forme de sel compatible avec une administration chez l'homme, la stratégie habituellement utilisée par l'homme du métier est de synthétiser le dérivé pyroglutamique de ce peptide, comme l'illustrent les deux exemples ci-dessous de la Buséréline et de la Gonadoréline (analogues du LH-RH, Pharmacopée européenne, 1999) :To stabilize a peptide for pharmaceutical use having a glutamic acid or a Ν-terminal glutamine in a salt form compatible with administration in humans, the strategy usually used by those skilled in the art is to synthesize the pyroglutamic derivative of this peptide, as illustrated by the two examples below of Buserelin and Gonadorelin (analogues of LH-RH, European Pharmacopoeia, 1999):
Buséréline
buserelin
Gonadorélinegonadoréline
Ceci permet par ailleurs d'augmenter la demi-vie du peptide en limitant sa dégradation protéolytique par des N-aminopeptidases.This also makes it possible to increase the half-life of the peptide by limiting its proteolytic degradation by N-aminopeptidases.
Toutefois, lorsque cette méthode est utilisée pour stabiliser un ligand du CMH comme le décapeptide ELA (épitope CTL de séquence ELAGIGILTV et de formule C45H8oN1oOι4 = 985 Da), le dérivé PyrELA obtenu (de séquence PyrELAGIGILTV et de formule C 5H78N10O13 = 967 Da), ne présente plus l'activité vaccinale recherchée et est notamment quasiment inactif d'un point de vue réponse CTL. Cette modification de structure est pourtant mineure : il s'agit de la cyclisation de la fonction α-amino N-terminale de l'acide glutamique avec sa propre fonction γ-carboxylique et perte d'une molécule d'eau. En effet, les peptides présentant un acide aminé de type acide glutamique (Glu, E) ou glutamine (Gin, Q) à leur extrémité N-terminale se cyclisent avec la fonction acide γ-carboxylique libre pour former un pyroglutamate selon la réaction définie ci-dessous :However, when this method is used to stabilize a MHC ligand such as the decapeptide ELA (CTL epitope of sequence ELAGIGILTV and of formula C 45 H 8 oN 1 oOι 4 = 985 Da), the PyrELA derivative obtained (of sequence PyrELAGIGILTV and of formula C 5 H 7 8N 1 0O1 3 = 967 Da), no longer exhibits the desired vaccine activity and is in particular almost inactive from a CTL response point of view. This structural modification is however minor: it involves the cyclization of the N-terminal α-amino function of glutamic acid with its own γ-carboxylic function and loss of a water molecule. Indeed, the peptides having an amino acid of glutamic acid (Glu, E) or glutamine (Gln, Q) type at their N-terminal end cyclize with the free γ-carboxylic acid function to form a pyroglutamate according to the reaction defined above below:
Acide glutamique : X = OH pyr oglutamateGlutamic acid: X = OH pyr oglutamate
Glutamine : X≈NH,Glutamine: X≈NH,
L'absence d'activité vaccinale pour ces peptides est d'autant surprenante que la diminution de masse entre le décapeptide ELA et le dérivé PyrELA obtenu est de 18 Daltons seulement, tout le reste de la structure demeurant inchangé :
PeptideThe absence of vaccine activity for these peptides is all the more surprising since the reduction in mass between the ELA decapeptide and the PyrELA derivative obtained is only 18 Daltons, all the rest of the structure remaining unchanged: peptide
Peptide
peptide
Peptide Ac-ELAPeptide Ac-ELA
Il a aussi été constaté que la synthèse d'un autre dérivé du peptide ELA acétylé sur la fonction aminé de l'acide glutamique de manière à empêcher la cyclisation en pyroglutamate (peptide AcELA, de séquence AcELAGIGILTV et de formule C47H82Nι0Oι5 = 1027 Da, voir ci-dessus) permet de résoudre le problème de stabilité mais fait perdre toute activité vaccinale, et notamment les activités de génération de cellules CTL, au dérivé AcELA ainsi obtenu.It has also been found that the synthesis of another derivative of the acetylated ELA peptide on the amino function of glutamic acid so as to prevent cyclization into pyroglutamate (AcELA peptide, of sequence AcELAGIGILTV and of formula C 47 H 82 Nι 0 Oι 5 = 1027 Da, see above) solves the stability problem but causes all vaccine activity, and in particular CTL cell generation activities, to be lost to the AcELA derivative thus obtained.
Cette réaction d'acétylation est pourtant une modification mineure de la structure du peptide classiquement utilisé par l'homme du métier pour améliorer la stabilité d'un peptide (Brinckerhoff et al, Int. J. Cancer, 1999, 83, 326) : il s'agit du remplacement d'un des protons de la fonction NH N-terminale par un groupe acétyle CH3CO avec une faible augmentation de masse (42 Da sur 985 Da), tout le reste de la structure demeurant inchangé.This acetylation reaction is however a minor modification of the structure of the peptide conventionally used by a person skilled in the art to improve the stability of a peptide (Brinckerhoff et al, Int. J. Cancer, 1999, 83, 326): it it is the replacement of one of the protons of the N-terminal NH function with an acetyl group CH 3 CO with a slight increase in mass (42 Da over 985 Da), all the rest of the structure remaining unchanged.
De même, Elliott et al (Vaccine, 1999, 17, 2009-2019)) ont décrit des problèmes de stabilité d'épitopes CTL contenant des méthionines (oxydation en sulfoxide) ou des acides glutamiques en position N-terminale (peptide EEGAIVGEI, dérivé de la protéine Influenza NSP-1 du virus de la grippe (acides aminés 152-160) et correspondant à un épitope CTL de souris H-2Kk restreint). Il a été constaté que ce peptide se cyclise spontanément en pyroglutamate (30 % en 2 mois) lorsque qu'il est formulé avec une solution adjuvante de type Montanide ISA 720. Les auteurs soulèvent le problème que cette dégradation pose par rapport à l'activité vaccinale recherchée, sans y apporter de solution.Similarly, Elliott et al (Vaccine, 1999, 17, 2009-2019)) described stability problems of CTL epitopes containing methionines (oxidation to sulfoxide) or glutamic acids in N-terminal position (peptide EEGAIVGEI, derivative of the influenza NSP-1 protein of the influenza virus (amino acids 152-160) and corresponding to a CTL epitope of mouse H-2Kk restricted). It has been found that this peptide spontaneously cyclizes to pyroglutamate (30% in 2 months) when it is formulated with an adjuvant solution of the Montanide ISA 720 type. The authors raise the problem that this degradation poses in relation to the activity vaccine sought, without providing a solution.
En outre, la quasi totalité des peptides obtenus par synthèse chimique sont purifiés par HPLC en phase inverse à l'aide d'éluants contenant de l'acide trifluoroacétique (TFA) avant d'être lyophilisées. Les peptides purifiés obtenus sont
chargés positivement et se trouvent sous forme de sel de trifluoroacétate (RNH3 +,CF3CO "). La quantité de trifluoroacétate et d'acide trifluoroacétique résiduel est en général proportionnelle au nombre d'acides aminés basiques (Lysine, Arginine et Histidine) contenus dans la séquence ainsi que de la fonction aminé de l'acide aminé N-terminal. Les peptides sous forme de trifluoroacétate sont couramment utilisés pour des expérimentations pré-cliniques in vitro et in vivo chez l'animal. Pour un usage pharmaceutique chez l'homme, cette forme de sel n'est toutefois pas acceptée en particulier lors des dernières étapes de purifications parce que l'acide trifluoroacétique fait partie d'une classe de solvant (classe IV) dont la toxicologie n'est pas parfaitement documentée (Leblanc et al, STP Pharma, 1999, 9, 334-341). Ainsi, aucun des peptides ayant obtenu une autorisation de mise sur le marché (Somatostatine, Tétracoside, Desmopressine, Calcitonine, Buséréline, Gonadoreline, etc..) ne l'a été sous forme de trifluoroacétate, comme on peut le constater dans les monographies de la pharmacopée européenne (Ph. Eur. 1999), mais plutôt sous forme d'acétate. La quantité d'acide trifluoroacétique résiduel tolérée dans ces peptides est d'ailleurs extrêmement limitée.In addition, almost all of the peptides obtained by chemical synthesis are purified by reverse phase HPLC using eluents containing trifluoroacetic acid (TFA) before being lyophilized. The purified peptides obtained are positively charged and are in the form of trifluoroacetate salt (RNH 3 + , CF 3 CO " ). The amount of trifluoroacetate and residual trifluoroacetic acid is generally proportional to the number of basic amino acids (Lysine, Arginine and Histidine) contained in the sequence as well as in the amino function of the N-terminal amino acid. Peptides in the form of trifluoroacetate are commonly used for pre-clinical in vitro and in vivo experiments in animals. , however, this form of salt is not accepted in particular during the final stages of purification because trifluoroacetic acid is part of a solvent class (class IV) whose toxicology is not fully documented (Leblanc et al, STP Pharma, 1999, 9, 334-341). Thus, none of the peptides having obtained a marketing authorization (Somatostatin, Tetracoside, Desmopressin, Calcitonin, Buserelin, Gonadoreline, etc.) was not in the form of trifluoroacetate, as can be seen in the monographs of the European Pharmacopoeia (Ph. Eur. 1999), but rather in the form of acetate. The amount of residual trifluoroacetic acid tolerated in these peptides is, moreover, extremely limited.
Par ailleurs une étude récente (Cornish et al., Am. J. Physiol. Endocrinol. Metab., 1999, 277, E779-E783) a montré que plusieurs peptides synthétiques (Amyline, Calcitonine) sous forme de trifluoroacétate sont toxiques pour des cellules en culture (ostéoblastes et chondrocytes).Furthermore, a recent study (Cornish et al., Am. J. Physiol. Endocrinol. Metab., 1999, 277, E779-E783) has shown that several synthetic peptides (Amyline, Calcitonin) in the form of trifluoroacetate are toxic for cells. in culture (osteoblasts and chondrocytes).
Une solution pour résoudre ces différents problèmes de toxicité de l'acide trifluoroacétique a été proposée par Marchand et al (Int. J. Cancer, 1999, 80, 219- 230), qui rapportent les résultats d'une étude clinique démontrant une régression tumorale chez des patients atteints d'un mélanome. Le principe actif utilisé est le nonapeptide MAGE-3 de séquence EVDPIGHLY (SEQ ID N° 273), qui possède un acide glutamique en N-terminal. Le peptide a été utilisé chez les patients sous forme d'acétate qui est la forme utilisée dans la quasi totalité des peptides administrés à l'homme.A solution to solve these different toxicity problems of trifluoroacetic acid has been proposed by Marchand et al (Int. J. Cancer, 1999, 80, 219-230), who report the results of a clinical study demonstrating tumor regression in patients with melanoma. The active principle used is the nonapeptide MAGE-3 of sequence EVDPIGHLY (SEQ ID No. 273), which has an N-terminal glutamic acid. The peptide has been used in patients in the form of acetate which is the form used in almost all peptides administered to humans.
Néanmoins, l'acide acétique est un acide faible, qui confère une instabilité accrue au peptide. Ceci oblige les investigateurs à conserver le peptide à -80°C (azote liquide) sous forme lyophilisée et à le resolubiliser extemporanément juste avant l'injection, ce. qui implique une chaîne du froid très contraignante.
La présente invention se propose de résoudre ces problèmes d'instabilité structurelle, de conservation dans le temps, de toxicité et d'activité biologique.However, acetic acid is a weak acid, which gives increased instability to the peptide. This forces the investigators to keep the peptide at -80 ° C (liquid nitrogen) in lyophilized form and to resolubilize it extemporaneously just before injection. which involves a very restrictive cold chain. The present invention proposes to solve these problems of structural instability, conservation over time, toxicity and biological activity.
En effet, il a été constaté que, de façon surprenante, les molécules d'intérêt pharmaceutique, en particulier les ligands du CMH, possédant un acide glutamique ou une glutamine à leur extrémité N-terminale peuvent être stabilisées sous forme de sel d'addition d'un acide fort, et que ceci permet à la fois de maintenir l'activité biologique, d'obtenir une conservation aisée du peptide ou analogue sous une forme stable, qui permet son utilisation thérapeutique chez l'homme.Indeed, it has been found that, surprisingly, molecules of pharmaceutical interest, in particular MHC ligands, having a glutamic acid or a glutamine at their N-terminal end can be stabilized in the form of addition salt of a strong acid, and that this makes it possible both to maintain the biological activity and to obtain an easy conservation of the peptide or the like in a stable form, which allows its therapeutic use in humans.
Par « molécule d'intérêt pharmaceutique », on entend en particulier les ligands du CMH, les molécules naturelles ou synthétiques présentant un épitope pour la génération d'anticorps, les molécules dérivées de ligands de récepteurs, et présentant une activité agoniste ou antagoniste par rapport à ces récepteurs, ou possédant une activité antibiotique, antifongique, ou antiviral. Les molécules d'intérêt thérapeutique selon l'invention sont toutes caractérisées en ce qu'elles possèdent un acide glutamique ou une -glutamine à leur extrémité N-terminale.- Les molécules d'intérêt pharmaceutique préférées selon la-présente invention sont les ligands du CMH.By “molecule of pharmaceutical interest” is meant in particular the MHC ligands, the natural or synthetic molecules having an epitope for the generation of antibodies, the molecules derived from receptor ligands, and having an agonist or antagonist activity with respect to these receptors, or having antibiotic, antifungal, or antiviral activity. The molecules of therapeutic interest according to the invention are all characterized in that they have a glutamic acid or a -glutamine at their N-terminal end. The molecules of pharmaceutical interest preferred according to the present invention are the ligands of the MHC.
La présente invention a ainsi en particulier pour objet un ligand du CMH comportant en son extrémité N-terminale un acide glutamique ou une glutamine, caractérisé en ce qu'il se présente sous forme de sel d'addition d'un acide fort physiologiquement acceptable.The present invention thus in particular relates to a MHC ligand comprising at its N-terminal end a glutamic acid or a glutamine, characterized in that it is in the form of addition salt of a strong physiologically acceptable acid.
Le sel d'addition d'acide fort physiologiquement acceptable peut notamment être choisi parmi les sels d'addition avec les acides forts minéraux ou organiques.The physiologically acceptable strong acid addition salt can in particular be chosen from addition salts with strong mineral or organic acids.
Il est préférentiellement choisi parmi le méthanesulfonate (ou mésilate), le chlorhydrate, le bromhydrate, le sulfate, le nitrate et le phosphate et plus préférentiellement parmi le chlorhydrate, le sulfate, le nitrate et le méthanesulfonate.It is preferably chosen from methanesulfonate (or mesilate), hydrochloride, hydrobromide, sulfate, nitrate and phosphate and more preferably from hydrochloride, sulfate, nitrate and methanesulfonate.
Ces sels d'addition d'acide fort sont physiologiquement acceptables pour une utilisation thérapeutique chez l'homme. Par exemple, la Protamine (obtenue par extraction du sperme ou de la laitance de poisson et qui nécessite un sel d'acide fort pour être solubilisée) est enregistrée sous forme de chlorhydrate d'une part et sous forme de sulfate d'autre part (Ph Eur, 1999).
Les ligands du CMH au sens de la présente invention sont notamment les ligands du CMH de classe I et IL Le CMH est un groupe important de protéines impliquées dans la présentation des antigènes aux lymphocytes T. Les molécules du CMH de classe I sont des protéines membranaires intégrales que l'on trouve sur toutes les cellules nucléées et les plaquettes. Les molécules du CMH de classe II, sont exprimées sur les cellules B, les macrophages, les monocytes, les cellules présentatrices d'antigène et certaines cellules T. Les cellules B sont des lymphocytes, qui sous forme mature présentent à leur surface des immunoglobùlines faisant fonction de « récepteur pour l'antigène ». Les cellules T sont des lymphocytes qui expriment leur récepteur pour l'antigène (TcR) et se différencient en 2 sous-populations : les cellules T auxiliaires (Th ou T helper) et les cellules T cytotoxiques (CTL). Les cellules Th aident les cellules B à se diviser, à se différencier et à produire des anticorps. La majorité des Th sont CD4+ (marqueur de surface spécifique) et reconnaissent l'antigène présenté à la surface des cellules présentant l'antigène, en association avec les molécules de classe II du CMH. Les cellules T cytotoxiques sont capable de détruire les cellules cibles infectées par des virus ou des cellules allogéniques. La majorité sont CD 8+ et reconnaissent l'antigène associé avec les molécules du CMH de classe I à la surface de la cellule cible. La reconnaissance de l'antigène s'effectue par formation d'un complexe comprenant en particulier la molécule du CMH présentant un ligand du CMH, et le récepteur de la cellule T (TCR).These strong acid addition salts are physiologically acceptable for therapeutic use in humans. For example, Protamine (obtained by extraction of semen or milt from fish and which requires a strong acid salt to be dissolved) is registered as hydrochloride on the one hand and as sulphate on the other hand ( Ph Eur, 1999). MHC ligands within the meaning of the present invention are in particular MHC class I and IL ligands MHC is an important group of proteins involved in the presentation of antigens to T lymphocytes. MHC class I molecules are membrane proteins found on all nucleated cells and platelets. MHC class II molecules are expressed on B cells, macrophages, monocytes, antigen-presenting cells and certain T cells. B cells are lymphocytes, which in mature form have immunoglobulin on their surface making "antigen receptor" function. T cells are lymphocytes that express their receptor for the antigen (TcR) and differentiate into 2 subpopulations: helper T cells (Th or T helper) and cytotoxic T cells (CTL). Th cells help B cells to divide, differentiate and produce antibodies. The majority of Th are CD4 + (specific surface marker) and recognize the antigen presented on the surface of cells presenting the antigen, in association with MHC class II molecules. Cytotoxic T cells are capable of destroying target cells infected by viruses or allogenic cells. The majority are CD 8+ and recognize the antigen associated with MHC class I molecules on the surface of the target cell. The antigen is recognized by the formation of a complex comprising in particular the MHC molecule having a MHC ligand, and the T cell receptor (TCR).
Les molécules d'intérêt pharmaceutiques, en particulier les ligands du CMH selon la présente invention, peuvent être choisies parmi les molécules naturelles ou synthétiques, et entre autres, parmi les protéines, les peptides, les constructions polypeptidiques multi-épitopiques, ou des analogues de peptides du type pseudopeptides, rétro-inverso, peptoïdes, les peptido-mimétiques, les lipopeptides. Ces molécules peuvent également être constituées en partie d'une chaîne peptidique, avec le remplacement de certains acide aminés par des analogues d'acides aminés, ou présentant des ramifications. Ces molécules peuvent également présenter les diverses modifications qui sont observées sur les protéines ou peptides naturels (par exemple O- ou N-glycosylation).The molecules of pharmaceutical interest, in particular the MHC ligands according to the present invention, can be chosen from natural or synthetic molecules, and inter alia, from proteins, peptides, multi-epitopic polypeptide constructs, or analogs of peptides of the pseudopeptide type, retro-inverso, peptoids, peptido-mimetics, lipopeptides. These molecules can also consist in part of a peptide chain, with the replacement of certain amino acids by analogues of amino acids, or having ramifications. These molecules can also present the various modifications which are observed on natural proteins or peptides (for example O- or N-glycosylation).
Dans une forme de réalisation préférée de l'invention, les ligands du CMH selon la présente invention sont choisis parmi les épitopes CTL, c'est-à-dire qui.
permettent la génération de lymphocytes T cytotoxiques et notamment parmi ceux qui se présentent sous forme d'octapeptide, de nonapeptide ou de décapeptide.In a preferred embodiment of the invention, the MHC ligands according to the present invention are chosen from CTL epitopes, that is to say which. allow the generation of cytotoxic T lymphocytes and in particular among those which are in the form of octapeptide, nonapeptide or decapeptide.
Le ligand du CMH peut aussi être choisi parmi les ligands décrits dans les bases de données SYFPEITHI ou MHCPEP, précédemment citées, et qui comportent en leur extrémité N-terminale un acide glutamique ou une glutamine.The MHC ligand can also be chosen from the ligands described in the SYFPEITHI or MHCPEP databases, previously cited, and which comprise at their N-terminal end a glutamic acid or a glutamine.
Ce ligand peut notamment être choisi parmi les ligands du CMH (ligands des molécules du CMH de classe I ou II) compris dans le groupe constitué des peptides correspondant aux séquences SEQ ID N° 1 à SEQ ID N° 694.This ligand can in particular be chosen from MHC ligands (ligands of MHC molecules of class I or II) included in the group consisting of peptides corresponding to the sequences SEQ ID No. 1 to SEQ ID No. 694.
Dans une forme de réalisation de l'invention encore plus particulièrement préférée , il est choisi parmi les peptides suivants :In an even more particularly preferred embodiment of the invention, it is chosen from the following peptides:
Noms Séquences HLA SEQ ID NC Names Sequences HLA SEQ ID N C
ELA MART-1 26-35 A27L ELAGIGILTV A2 81ELA MART-1 26-35 A27L ELAGIGILTV A2 81
ELA MART-1 26-35 EAAGIGILTN A2 112 MAGE-1 161-169 EADPTGHSY Al 2ELA MART-1 26-35 EAAGIGILTN A2 112 MAGE-1 161-169 EADPTGHSY Al 2
MAGE-3 168-176 EVDPIGHLY Al 273MAGE-3 168-176 EVDPIGHLY Al 273
HER-2/neu 950-958 ELVSEFSRM A2 110HER-2 / neu 950-958 ELVSEFSRM A2 110
HCV-1 env E 66-75 QLRRHIDLLV A2 464HCV-1 env E 66-75 QLRRHIDLLV A2 464
ΝY-ESO-1 155-163 QLSLLMWIT A2 466 HIV nef 73-82 QVPLRPMTYK A3 567ΝY-ESO-1 155-163 QLSLLMWIT A2 466 HIV nef 73-82 QVPLRPMTYK A3 567
Influenza NP 380-388 ELRSRYWAI B8 106Influenza NP 380-388 ELRSRYWAI B8 106
HIV gag p24262-270 EIYKRWIIL B8 10HIV gag p24262-270 EIYKRWIIL B8 10
HIV gag pl7 93-101 EIKDTKEAL B8 692HIV gag pl7 93-101 EIKDTKEAL B8 692
InfluenzaNP 339-347 EDLRVLSFI B*3701 257 EBNA 6 130-139 EENLLDFVRF B*4403 568InfluenzaNP 339-347 EDLRVLSFI B * 3701 257 EBNA 6 130-139 EENLLDFVRF B * 4403 568
Les ligands selon l'invention peuvent aussi être choisis parmi les constructions polypeptidiques multiépitopiques présentant un acide aminé de type acide glutamique (Glu, E) ou glutamine (Gin, Q) à l'extrémité N-terminale tel que le peptide suivant (SEQ ID N° 695) : NEF 117 EWRFDSRLAFHHVAREHPEYFNKNK(PaIm)NH2 The ligands according to the invention can also be chosen from multiepitopic polypeptide constructions having an amino acid of glutamic acid (Glu, E) or glutamine (Gln, Q) type at the N-terminal end such as the following peptide (SEQ ID N ° 695): NEF 117 EWRFDSRLAFHHVAREHPEYFNKNK (PaIm) NH 2
(lipopeptide anti-HIV en phase clinique I : Klinguer, et al, Vaccine, 1999, 18, 259-267).
Les analogues de peptides peuvent être choisis parmi ceux décrits dans la demande FR276307 qui comportent en leur extrémité N-terminale un acide glutamique ou une glutamine.(anti-HIV lipopeptide in clinical phase I: Klinguer, et al, Vaccine, 1999, 18, 259-267). The peptide analogs can be chosen from those described in application FR276307 which comprise at their N-terminal end a glutamic acid or a glutamine.
De façon la plus préférée, l'invention concerne le ligand du CMH de séquence ELAGIGILTV, sous forme sulfate ou, de façon encore plus préférée, sous forme chlorhydrate.Most preferably, the invention relates to the MHC ligand of sequence ELAGIGILTV, in sulphate form or, even more preferably, in hydrochloride form.
L'invention concerne encore une composition pharmaceutique comprenant au moins une molécule d'intérêt pharmaceutique selon l'invention.The invention also relates to a pharmaceutical composition comprising at least one molecule of pharmaceutical interest according to the invention.
Cette composition pharmaceutique peut notamment être destinée au traitement de différentes immunopathologies : l'immunodéficience, les maladies auto-immunes, les hypersensibilités, les allergies ou pour éviter les rejets de greffes. Selon la molécule utilisée, une composition selon l'invention peut également être utilisée dans un but antibiotique, antiviral ou antifongique, ou peut être destinée au traitement de maladies liées à des dérèglements hormonaux, ou à des maladies du système nerveux central.This pharmaceutical composition can in particular be intended for the treatment of various immunopathologies: immunodeficiency, autoimmune diseases, hypersensitivities, allergies or to avoid rejection of grafts. Depending on the molecule used, a composition according to the invention can also be used for an antibiotic, antiviral or antifungal purpose, or can be intended for the treatment of diseases linked to hormonal disorders, or to diseases of the central nervous system.
-Les compositions selon l'invention peuvent aussi être utilisées dans le domaine vétérinaire. En effet, les mêmes problèmes d'instabilité structurelle, de conservation dans le temps, de toxicité et d'activité qui se posent pour la préparation de préparations vétérinaires comprenant un peptide ou une molécule possédant un acide glutamique ou une glutamine à leur extrémité N-terminale, peuvent être résolus en utilisant des sels d'addition d'acides forts pour stabiliser lesdits peptides ou molécules.The compositions according to the invention can also be used in the veterinary field. Indeed, the same problems of structural instability, conservation over time, toxicity and activity which arise for the preparation of veterinary preparations comprising a peptide or a molecule having a glutamic acid or a glutamine at their N- end terminal, can be resolved using strong acid addition salts to stabilize said peptides or molecules.
Parmi les compositions pharmaceutiques selon l'invention, une composition préférée consiste en un vaccin caractérisé en ce qu'il comprend au moins un ligand du CMH selon l'invention, se présentant sous la forme de sel d'addition d'acide fort physiologiquement acceptable, tel que défini ci-dessus.Among the pharmaceutical compositions according to the invention, a preferred composition consists of a vaccine characterized in that it comprises at least one MHC ligand according to the invention, which is in the form of a physiologically acceptable strong acid addition salt , as defined above.
Ce vaccin peut comprendre en outre au moins un adjuvant, notamment choisi parmi les sels d'Aluminium (Alum) ou de Calcium, les protéines OmpA d'entérobactérie, le toxoïde tétanique (TT), le toxoïde diphtérique (DT), le CRM 197 (matériel à réactivité croisée), PLGA, ISCOM, Montanide ISA 720, les ammoniums quaternaires aliphatiques, le MPL-A, le Quil-A, les CpG, la Leif, la toxine cholérique (CT), la LT (LT pour « Heat labile enterotoxin » entérotoxine labile à la chaleur) ou les versions détoxifïées de la CT ou la LT.
Dans une forme préférée de l'invention, le vaccin comprend en outre, un composé porteur mélangé ou couplé audit ligand.This vaccine can also comprise at least one adjuvant, in particular chosen from Aluminum (Alum) or Calcium salts, OmpA enterobacterial proteins, tetanus toxoid (TT), diphtheria toxoid (DT), CRM 197 (cross-reactive material), PLGA, ISCOM, Montanide ISA 720, aliphatic quaternary ammoniums, MPL-A, Quil-A, CpG, Leif, cholera toxin (CT), LT (LT for "Heat labile enterotoxin "heat labile enterotoxin) or detoxified versions of CT or LT. In a preferred form of the invention, the vaccine further comprises a carrier compound mixed or coupled to said ligand.
De préférence, ledit composé porteur est choisi dans le groupe de peptide comprenant les anatoxines, notamment le toxoïde diphtérique (DT) ou le toxoïde tétanique (TT), les protéines dérivées du streptocoque (comme la protéine de liaison à la séralbumine humaine, appelée "BB" décrite dans WO96/14415), les protéines membranaires OmpA (pour "Outer Membrane Protein de type A") et les complexes de protéines de membranes externes (OMPC), les vésicules de membranes externes (OMV) ou les protéines de choc thermique (« Heat Shock Protein » ou HSP). Avantageusement, ledit composé porteur est couplé de façon covalente avec le ligand. On entend désigner par « couplage », aussi bien un couplage effectué par voie chimique entre les deux composés, qu'un couplage biologique, par recombinaison génétique, tel que défini ci-dessous.Preferably, said carrier compound is chosen from the group of peptides comprising toxoids, in particular diphtheria toxoid (DT) or tetanus toxoid (TT), proteins derived from streptococcus (such as the protein for binding to human serum albumin, called " BB "described in WO96 / 14415), OmpA membrane proteins (for" Outer Membrane Protein type A ") and external membrane protein complexes (OMPC), external membrane vesicles (OMV) or heat shock proteins ("Heat Shock Protein" or HSP). Advantageously, said carrier compound is covalently coupled with the ligand. The term “coupling” is intended to denote both a coupling carried out chemically between the two compounds, and a biological coupling, by genetic recombination, as defined below.
Ainsi, selon l'invention, il est possible d'introduire un ou plusieurs éléments de liaison, notamment des acides aminés pour faciliter les réactions de couplage entre le composé porteur et l'antigène ou l'haptène, en-particulier lorsqu'ils sont de nature peptidique, le couplage covalent de l'antigène ou l'haptène pouvant être réalisé à l'extrémité N ou C terminale du composé porteur.Thus, according to the invention, it is possible to introduce one or more linking elements, in particular amino acids to facilitate the coupling reactions between the carrier compound and the antigen or the hapten, in particular when they are peptide in nature, the covalent coupling of the antigen or hapten can be carried out at the N or C terminus of the carrier compound.
Les réactifs bifonctionnels permettant ce couplage sont déterminés en fonction de l'extrémité du composé porteur choisi et de la nature de l'antigène ou l'haptène à coupler. Ces techniques de couplage sont bien connues de l'homme du métier.The bifunctional reagents allowing this coupling are determined according to the end of the chosen carrier compound and the nature of the antigen or the hapten to be coupled. These coupling techniques are well known to those skilled in the art.
Les conjugués issus d'un couplage de peptides peuvent être également préparés par recombinaison génétique. Le peptide hybride (conjugué) peut en effet être produit par des techniques d'ADN recombinant par insertion ou addition à la séquence d'ADN codant pour le composé porteur, d'une séquence codant pour le ou les peptides antigènes, immunogènes ou haptènes. Ces techniques de préparation de peptide hybride par recombinaison génétique sont bien connues de l'homme du métier (cf. par exemple Makrides, 1996, Microbiologicals Reviews, 60, 512-538). De préférence, ledit composé porteur est une protéine dérivée du streptocoque ou une protéine de membrane OmpA d'entérobactérie, notamment de Klebsiella pneumoniae, ou l'un de ses fragments.
Le ligand selon l'invention associé éventuellement à un composé porteur peut être incorporé dans des vecteurs choisis parmi les liposomes, les virosomes, les nanosphères, les microsphères, les microcapsules ou les biovecteurs. L'homme du métier sait choisir le vecteur approprié en fonction du but recherché (protection du ligand éventuellement associé à un composé porteur ou un adjuvant de la dégradation, ciblage de cellules d'intérêt, recherche d'une pénétration du matériel contenu dans le vecteur à l'intérieur de cellules cibles...).The conjugates resulting from a coupling of peptides can also be prepared by genetic recombination. The hybrid (conjugated) peptide can in fact be produced by recombinant DNA techniques by insertion or addition to the DNA sequence coding for the carrier compound, of a sequence coding for the antigen, immunogenic or hapten peptides. These techniques for preparing hybrid peptide by genetic recombination are well known to those skilled in the art (cf. for example Makrides, 1996, Microbiologicals Reviews, 60, 512-538). Preferably, said carrier compound is a protein derived from streptococcus or an OmpA membrane protein from enterobacteria, in particular from Klebsiella pneumoniae, or one of its fragments. The ligand according to the invention optionally associated with a carrier compound can be incorporated into vectors chosen from liposomes, virosomes, nanospheres, microspheres, microcapsules or biovectors. A person skilled in the art knows how to choose the appropriate vector according to the aim sought (protection of the ligand possibly associated with a carrier compound or an adjuvant from degradation, targeting of cells of interest, search for penetration of the material contained in the vector inside target cells ...).
Une forme de réalisation de l'invention concerne notamment un vaccin anti- mélanome caractérisé en ce qu'il comprend au moins un peptide ELAGIGILTV (SEQ ID N° 81) sous forme de chlorhydrate ou de sulfate.One embodiment of the invention relates in particular to an anti-melanoma vaccine characterized in that it comprises at least one ELAGIGILTV peptide (SEQ ID No. 81) in the form of the hydrochloride or sulphate.
Une autre forme a pour objet un vaccin anti-mélanome caractérisé en ce qu'il comprend au moins un peptide ELAGIGILTV (SEQ ID N° 81) sous forme de chlorhydrate ou de sulfate et en outre une protéine OmpA d'entérobactérie.Another form relates to an anti-melanoma vaccine characterized in that it comprises at least one ELAGIGILTV peptide (SEQ ID No. 81) in the form of the hydrochloride or sulfate and in addition an enterobacterium protein OmpA.
On peut également développer des vaccins selon l'invention pour une utilisation dans le domaine vétérinaire, les problèmes identiques d'instabilité structurelle, de conservation dans le temps, de toxicité et d'activité pouvant être résolus de la même façon.Vaccines according to the invention can also be developed for use in the veterinary field, the identical problems of structural instability, storage over time, toxicity and activity being able to be solved in the same way.
L'invention a encore pour objet, une méthode de diagnostic in vitro de pathologies associées à la présence dans l'organisme d'un patient, de ligands du CMH pouvant interagir avec des molécules du CMH, et susceptibles d'être directement ou indirectement impliqués dans le processus de développement de ces pathologies chez l'homme ou l'animal, caractérisée en ce qu'elle comprend les étapes de : mise en contact d'un échantillon biologique provenant d'un patient, notamment du sang ou tout échantillon biologique susceptible de contenir des lymphocytes, avec un ligand du CMH selon l'invention, dans des conditions permettant la formation d'un complexe binaire entre ledit ligand du CMH et les molécules de CMH présentes dans ledit échantillon, et la réaction entre ledit complexe binaire et les récepteurs des cellules T susceptibles d'être présentes dans ledit échantillon biologique. détection in vitro du complexe ternaire CMH - ligand du CMH - récepteur T, susceptible d'être formé à l'étape précédente.
Les méthodes de diagnostic selon l'invention sont avantageusement réalisées de la façon suivante : incubation dudit échantillon biologique avec des ligands de CMH selon l'invention, lesdits ligands de CMH étant fixés sur un support solide, notamment à l'intérieur de puits de plaques de microtitration de type de celles habituellement utilisées pour la mise en œuvre de techniques de détection ou dosage bien connues sous le nom d'ELISA (Enzyme Linked Immuno Sorbent Assay),The subject of the invention is also a method of in vitro diagnosis of pathologies associated with the presence in the body of a patient, of MHC ligands which can interact with MHC molecules, and which may be directly or indirectly involved. in the process of development of these pathologies in humans or animals, characterized in that it comprises the steps of: bringing a biological sample from a patient into contact, in particular blood or any biological sample susceptible to contain lymphocytes, with a MHC ligand according to the invention, under conditions allowing the formation of a binary complex between said MHC ligand and the MHC molecules present in said sample, and the reaction between said binary complex and the T cell receptors that may be present in said biological sample. in vitro detection of the MHC - MHC ligand - T receptor ternary complex, capable of being formed in the previous step. The diagnostic methods according to the invention are advantageously carried out as follows: incubation of said biological sample with MHC ligands according to the invention, said MHC ligands being fixed on a solid support, in particular inside wells of plates microtitration of the type usually used for the implementation of detection or assay techniques well known under the name of ELISA (Enzyme Linked Immuno Sorbent Assay),
- incubation des éléments fixés sur le support solide, après une éventuelle étape de rinçage, avec un milieu contenant des anticorps, notamment des anticorps anti -complexe ternaire selon l'invention, marqués (notamment de manière radioactive, enzymatique ou fluorescente), ou susceptibles d'être reconnus à leur tour par un réactif marqué, détection des anticorps marqués restés respectivement liés aux complexes ternaires lors de l'étape d'incubation précédente.incubation of the elements fixed on the solid support, after a possible rinsing step, with a medium containing antibodies, in particular anti-ternary complex antibodies according to the invention, marked (in particular in a radioactive, enzymatic or fluorescent manner), or susceptible to be recognized in turn by a labeled reagent, detection of the labeled antibodies which remained respectively linked to the ternary complexes during the previous incubation step.
Des étapes de rinçage-sont- avantageusement effectuées entre les différentes étapes de ce procédé. L'homme du métier sait définir les différentes conditions d'incubation, ainsi que les méthodes de détection des complexes CMH - ligand du CMH - récepteur T, l'utilisation d'anticorps n'étant qu'une méthode parmi d'autres. L'invention a également pour objet les nécessaires ou kits pour la mise en œuvre de méthodes de diagnostic in vitro telles que décrites ci-dessus, comprenant :Rinsing steps are advantageously carried out between the different steps of this process. Those skilled in the art know how to define the different incubation conditions, as well as the methods for detecting MHC complexes - MHC ligand - T receptor, the use of antibodies being only one method among others. A subject of the invention is also the kits or kits for implementing in vitro diagnostic methods as described above, comprising:
- un ligand du CMH selon l'invention ;- a MHC ligand according to the invention;
- éventuellement des réactifs pour permettre la formation d'une réaction immunologique entre ledit ligand, les molécules du CMH et les récepteurs des cellules T éventuellement présents dans l'échantillon biologique ;- optionally reagents to allow the formation of an immunological reaction between said ligand, MHC molecules and T cell receptors possibly present in the biological sample;
- éventuellement des réactifs permettant de détecter le complexe ternaire selon l'invention, qui a été produit à l'issue de la réaction immunologique, lesdits réactifs contenant éventuellement un marqueur ou étant susceptibles d'être reconnus à leur tour par un réactif marqué, plus particulièrement dans le cas où l'analogue peptidique n'est pas marqué.
En particulier, on préfère l'utilisation des peptides ELAGIGILTV (SEQ ID N° 81), EAAGIGILTV (SEQ ID N° 112), EADPTGHSY (SEQ ID N° 2), ou EVDPIGHLY (SEQ ID N° 273) dans une méthode de diagnostic d'un mélanome. Les peptides QVPLRPMTYK (SEQ ID N° 567), EIYKRWIIL (SEQ ID N° 10), et EIKDTKEAL (SEQ ID N° 692) peuvent être utilisés dans une méthode de diagnostic d'une infection par le VIH.- optionally reagents for detecting the ternary complex according to the invention, which was produced at the end of the immunological reaction, said reagents possibly containing a marker or being capable of being recognized in turn by a labeled reagent, more particularly in the case where the peptide analog is not labeled. In particular, the use of the peptides ELAGIGILTV (SEQ ID No. 81), EAAGIGILTV (SEQ ID No. 112), EADPTGHSY (SEQ ID No. 2), or EVDPIGHLY (SEQ ID No. 273) is preferred in a method of diagnosis of melanoma. The peptides QVPLRPMTYK (SEQ ID No. 567), EIYKRWIIL (SEQ ID No. 10), and EIKDTKEAL (SEQ ID No. 692) can be used in a method of diagnosing HIV infection.
L'utilisation d'un ligand selon l'invention, pour la préparation d'un vaccin destiné au traitement prophylactique ou thérapeutique des infections virales, bactériennes, parasitaires ou fongiques est un autre objet de l'invention. L'invention concerne encore l'utilisation d'un ligand selon l'invention, pour la préparation d'un vaccin destiné au traitement prophylactique ou thérapeutique des cancers et préférentiellement pour inhiber la croissance de tumeurs.The use of a ligand according to the invention, for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of viral, bacterial, parasitic or fungal infections is another object of the invention. The invention also relates to the use of a ligand according to the invention, for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of cancers and preferably for inhibiting the growth of tumors.
La présente invention concerne aussi l'utilisation d'un acide fort physiologiquement acceptable pour stabiliser et maintenir l'activité biologique d'une molécule d'intérêt pharmaceutique comportant un acide glutamique ou une glutamine à son extrémité "N-terminale.The present invention also relates to the use of a strong physiologically acceptable acid for stabilizing and maintaining the biological activity of a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its " N-terminal " end.
Dans le cas préféré où la molécule d'intérêt pharmaceutique est un ligand du CMH, l'activité que l'on cherche à maintenir est une activité de stimulation et d'interaction avec les cellules du système immunitaire. L'invention concerne également l'utilisation d'un acide fort pour diminuer et/ou supprimer la formation du dérivé pyroglutamique d'une molécule d'intérêt pharmaceutique comportant un acide glutamique ou une glutamine à son extrémité N-terminale.In the preferred case where the pharmaceutical molecule of interest is a ligand of the MHC, the activity which one seeks to maintain is an activity of stimulation and of interaction with the cells of the immune system. The invention also relates to the use of a strong acid to reduce and / or suppress the formation of the pyroglutamic derivative of a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its N-terminal end.
De même, la présente invention concerne un procédé pour la stabilisation d'une molécule d'intérêt pharmaceutique comportant un acide glutamique ou une glutamine à son extrémité N-terminale, caractérisé en ce que l'on fait réagir ladite molécule avec un acide fort dans des conditions permettant d'obtenir ladite molécule sous la forme d'un sel d'addition d'acide fort physiologiquement acceptable. La réaction avec l'acide fort est effectuée en particulier selon un procédé tel que défini ci-dessous, l'acide fort pouvant être choisi parmi les acides forts définis ci-dessus, et permet d'obtenir de préférence un chlorhydrate.Likewise, the present invention relates to a process for the stabilization of a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its N-terminal end, characterized in that said molecule is reacted with a strong acid in conditions making it possible to obtain said molecule in the form of a physiologically acceptable strong acid addition salt. The reaction with the strong acid is carried out in particular according to a process as defined below, the strong acid can be chosen from the strong acids defined above, and preferably makes it possible to obtain a hydrochloride.
En effet, l'invention concerne aussi un procédé de préparation d'une molécule d'intérêt pharmaceutique comportant un acide glutamique ou une
glutamine à son extrémité N-terminale sous forme de sel d'addition d'acide fort physiologiquement acceptable selon l'invention.Indeed, the invention also relates to a process for the preparation of a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its N-terminal end in the form of a physiologically acceptable strong acid addition salt according to the invention.
Ce procédé peut notamment comprendre une étape de purification par RP- HPLC de ladite molécule à partir du sel de trifluoroacétate correspondant en utilisant un éluant à base dudit acide fort, suivie de façon optionnelle d'une étape de lyophilisation de la solution ainsi obtenue.This process may in particular comprise a step of purification by RP-HPLC of said molecule from the corresponding trifluoroacetate salt using an eluent based on said strong acid, optionally followed by a step of lyophilization of the solution thus obtained.
Un procédé alternatif comporte une étape de dissolution d'un sel de trifluoroacétate de ladite molécule dans une solution en excès dudit acide fort, suivie de façon optionnelle d'une étape de lyophilisation de la solution ainsi obtenue.An alternative method comprises a step of dissolving a trifluoroacetate salt of said molecule in an excess solution of said strong acid, optionally followed by a step of lyophilization of the solution thus obtained.
On peut également mettre en œuvre le un procédé selon l'invention qui comporte une étape de chromatographie par échange ion à partir du sel de trifluoroacétate correspondant de ladite molécule d'intérêt pharmaceutique, après dissolution dudit sel dans une solution contenant ledit acide fort. La lyophilisation du produit obtenu est également optionnelle.One can also implement the a method according to the invention which comprises a step of ion exchange chromatography from the corresponding trifluoroacetate salt of said molecule of pharmaceutical interest, after dissolution of said salt in a solution containing said strong acid. Lyophilization of the product obtained is also optional.
Dans toutes ces applications, on préfère un ligand du CMH, notamment SEQ ID N° 81, 112, 2, 273, 567, 10, 692, 11, 464, 466, 106, 257, ou 568. De façon plus préférée, il s'agit de SEQ ID N° 81 et le sel d'acide fort est un chlorhydrate.In all of these applications, a MHC ligand is preferred, in particular SEQ ID No. 81, 112, 2, 273, 567, 10, 692, 11, 464, 466, 106, 257, or 568. More preferably, it this is SEQ ID No. 81 and the strong acid salt is a hydrochloride.
Les exemples qui suivent sont destinés à illustrer certains modes de réalisation de l'invention et ne doivent pas être considérés comme limitant le champ de l'invention.The examples which follow are intended to illustrate certain embodiments of the invention and should not be considered as limiting the scope of the invention.
DESCRIPTION DES FIGURESDESCRIPTION OF THE FIGURES
Figure 1 : Différence de lyse cellulaire des cellules EL-4 A2/Kb prépuisées avec le peptide ELA, par des lymphocytes obtenus après immunisation de souris avec les peptides ELA (losanges) ou AcELA (carrés) en présence de la protéine adjuvante rP40, selon le protocole de l'exemple III..Figure 1: Difference in cell lysis of EL-4 A2 / Kb cells pre-prepursed with the peptide ELA, by lymphocytes obtained after immunization of mice with the peptides ELA (diamonds) or AcELA (squares) in the presence of the adjuvant protein rP40, according to the protocol of Example III ..
Figure 2 : Génération de CTL après immunisation par les peptides ELAFigure 2: CTL generation after immunization with ELA peptides
(trifluoroacétate, 2.A), ELA (chlorhydrate, 2.B) ou PyrELA (trifluoroacétate, 2.C) en présence de la protéine adjuvante rP40, selon le protocole del'exmple IV.(trifluoroacetate, 2.A), ELA (hydrochloride, 2.B) or PyrELA (trifluoroacetate, 2.C) in the presence of the adjuvant protein rP40, according to the protocol of Example IV.
Figure 3 : Chromatogramme du peptide ELA sous forme acétate (3. A) ou chlorhydrate (3.B) conservé à 37°C pendant deux mois.
Figure 4 : Chromatogramme du peptide ELA sous forme chlorhydrate initialement (4.A) ou après un mois de conservation à 4°C (4.B).Figure 3: Chromatogram of the ELA peptide in acetate (3. A) or hydrochloride (3.B) form stored at 37 ° C for two months. Figure 4: Chromatogram of the ELA peptide in hydrochloride form initially (4.A) or after one month of storage at 4 ° C (4.B).
EXEMPLES Exemple I : Synthèse des peptides ELA, PyrELA et AcELAEXAMPLES Example I: Synthesis of the peptides ELA, PyrELA and AcELA
Peptide ELA : le peptide ELA (SEQ ID N° 81) est synthétisé en phase solide à partir de l'acide aminé C-terminale vers l'acide aminé N-terminal (acide glutamique) en chimie FMOC ou tBOC. Après clivage de la résine et des groupes protecteurs des chaîne latérales réactives, le peptide est purifié de façon classique avec des éluants à base d'acide trifluoroacétique/eau et d'acide trifluoroacétique/acétonitrile avant d'être lyophilisé. Le pureté du peptide est vérifiée par chromatographie liquide en phase inverse. La composition en acide aminés est vérifiée après hydrolyse et dosage des acides aminés dérivés obtenus. La masse exacte est mesurée par spectrométrie de masse. Peptide PyrELA : le peptide PyrELA est synthétisé de la même manière que le peptide ELA à la seule "différence du couplage du dernier acide aminé N- terminal : l'acide glutamique est remplacé par un acide pyroglutamique.ELA peptide: the ELA peptide (SEQ ID No. 81) is synthesized in solid phase from the C-terminal amino acid to the N-terminal amino acid (glutamic acid) in FMOC or tBOC chemistry. After cleavage of the resin and of the protective groups of the reactive side chains, the peptide is purified in a conventional manner with eluents based on trifluoroacetic acid / water and trifluoroacetic acid / acetonitrile before being lyophilized. The purity of the peptide is checked by reverse phase liquid chromatography. The amino acid composition is checked after hydrolysis and determination of the derived amino acids obtained. The exact mass is measured by mass spectrometry. Peptide PyrELA: PyrELA the peptide is synthesized in the same manner as the ELA peptide to the single "difference of the coupling of the last amino acid the N-terminal: glutamic acid is replaced by a pyroglutamic acid.
Peptide AcELA : le peptide AcELA est synthétisé de la même manière que le peptide ELA à la seule différence d'un recouvrement (capping) de l'acide glutamique à l'aide d'anhydride acétique.AcELA peptide: the AcELA peptide is synthesized in the same way as the ELA peptide with the only difference of a covering (capping) of glutamic acid using acetic anhydride.
Exemple II : Préparation d'un sel de chlorhydrateExample II: Preparation of a hydrochloride salt
IL A : méthode AIT A: method A
A partir du sel de trifluoroacétate correspondant, on effectue une purification par RP-HPLC à l'aide d'un éluant A composé d'eau à 0.1 % d'HCl et d'un éluant B composé de 80% d'acétonitrile et de 20 % d'eau à 0.1 % d'HCl.From the corresponding trifluoroacetate salt, purification is carried out by RP-HPLC using an eluent A composed of water containing 0.1% HCl and an eluent B composed of 80% acetonitrile and 20% water to 0.1% HCl.
On procède ensuite à une étape classique de lyophilisation.Then a conventional lyophilization step is carried out.
IL B : méthode BIL B: method B
A partir du sel de trifluoroacétate correspondant, on dissout dans une solution en excès d'HCl et on laisse agiter pendant 2 heures. On peut utiliser également une solution aqueuse organique du peptide dans laquelle on fait buller de l'HCl sous forme gazeuse.From the corresponding trifluoroacetate salt, it is dissolved in an excess solution of HCl and allowed to stir for 2 hours. It is also possible to use an organic aqueous solution of the peptide in which HCl is bubbled in gaseous form.
On procède ensuite à une étape classique de lyophilisation.
IL C : méthode CThen a conventional lyophilization step is carried out. IL C: method C
On effectue cette réaction à partir du sel de trifluoroacétate correspondant, à l'aide d'une chromatographie par échange d'ion.This reaction is carried out using the corresponding trifluoroacetate salt, using ion exchange chromatography.
On utilise des résines échangeuses d'ions disponibles commercialement sous forme de chlorhydrate (Résine Dowex 1X4, Amberlite IRA 416), utilisables telles quelles une fois régénérées. a) Régénération de la résine : la résine à régénérer est introduite dans une colonne large équipée d'un fritte de grande porosité (1 ou 2). La résine est lavée ensuite successivement à l'eau ultra pure (pH 5-6), à la soude IN (pH 14), à l'eau ultra pure (pH 7), à l'HCl IN (pH 1) et une nouvelle fois à l'eau ultra pure (pH 5-6). La résine est conservée dans un mélange acétonitrile / HC1 10"4 N (20/80) à température ambiante pendant au moins un an. b) Echange d'anion (trifluoroacétate => chlorure) : le peptide est dissous dans une solution d'HCl 10"4 N / acétonitrile, avec une proportion en acétonitrile pouvant varier de 0 à 80%). La solution est injectée en tête de colonne. Le peptide est élue" avec la solution de dissolution. Les fractions contenant le produit sont rassemblées avant d'être lyophilisé.Ion exchange resins commercially available in the form of hydrochloride (Dowex resin 1X4, Amberlite IRA 416) are used, which can be used as they are once regenerated. a) Regeneration of the resin: the resin to be regenerated is introduced into a wide column equipped with a high porosity frit (1 or 2). The resin is then washed successively with ultra pure water (pH 5-6), IN sodium hydroxide solution (pH 14), ultra pure water (pH 7), IN HCl (pH 1) and a again with ultra pure water (pH 5-6). The resin is stored in an acetonitrile / HCl 10 "4 N (20/80) mixture at room temperature for at least one year. B) Anion exchange (trifluoroacetate => chloride): the peptide is dissolved in a solution of HCl 10 "4 N / acetonitrile, with a proportion of acetonitrile which can vary from 0 to 80%). The solution is injected at the top of the column. The peptide is eluted "with the dissolution solution. The fractions containing the product are combined before being lyophilized.
La quantité de chlorhydrate peut être dosée par une chromatographie d'échange anionique. La quantité d'acide trifluoroacétique peut être dosée par chromatographie en phase gazeuse.The amount of hydrochloride can be determined by anion exchange chromatography. The amount of trifluoroacetic acid can be determined by gas chromatography.
Exemple III : Génération de CTL anti-Melan-A après immunisation par rP40 mélangée à ELA ou AcELAExample III Generation of Anti-Melan-A CTL After Immunization with rP40 Mixed with ELA or AcELA
Des souris transgéniques HLA-A* 0201/Kb (A2/Kb) de souche C57B1/6 x BDA/2 ont été utilisées dans cette étude (Vitiello et al., 1991, J. Exp. Med., 173,HLA-A * 0201 / Kb (A2 / Kb) transgenic mice of strain C57B1 / 6 x BDA / 2 were used in this study (Vitiello et al., 1991, J. Exp. Med., 173,
1007). La molécule MHC de classe I exprimée chez ces souris est une molécule chimérique formée des domaines al et a2 de la molécule humaine HLA-A02011007). The MHC class I molecule expressed in these mice is a chimeric molecule formed from the al and a2 domains of the human molecule HLA-A0201
(allotype le plus fréquemment retrouvé) et du domaine a3 de la molécule murine Kb (most commonly found allotype) and of the a3 domain of the murine molecule K b
Des souris A2/Kb ont reçu 300μg de rP40 mélangée à 50μg de ELA ou 300μg de rP40 mélangée à 50μg de AcELA. a) Génération de cellules cytotoxiques effectrices :A2 / Kb mice received 300 μg of rP40 mixed with 50 μg of ELA or 300 μg of rP40 mixed with 50 μg of AcELA. a) Generation of effector cytotoxic cells:
10 jours après l'immunisation, les souris sont sacrifiées et les lymphocytes des ganglions drainant sont récupérés pour être stimulés in vitro avec le peptide
relevant. Ces lymphocytes (4-5 106) sont cultivés en plaque 24 puits en DMEM plus lOmM HEPES, 10% SVF et 50 μM β-2-mercaptoefhanol avec 2-5 105 cellules EL- 4 A2/Kb (cellules murines transfectées avec le gène HLA-A* 0201/Kb) irradiées (10 kRads) pré-pulsées 1 h à 37°C avec 1 μM du peptide relevant. Après deux stimulations hebdomadaires, les cellules sont testées pour leur activité cytotoxique. b) Mesure de l'activité cytotoxique :10 days after immunization, the mice are sacrificed and the lymphocytes of the draining ganglia are recovered to be stimulated in vitro with the peptide falling. These lymphocytes (4-5 10 6 ) are cultured in a 24-well plate in DMEM plus 10 mM HEPES, 10% FCS and 50 μM β-2-mercaptoefhanol with 2-5 10 5 EL-4 A2 / Kb cells (murine cells transfected with the HLA-A * 0201 / Kb) gene irradiated (10 kRads) pre-pulsed for 1 h at 37 ° C with 1 μM of the relevant peptide. After two weekly stimulations, the cells are tested for their cytotoxic activity. b) Measurement of cytotoxic activity:
Les cellules EL-4 A2/Kb sont incubées 1 h avec du 51Cr en présence ou non de ELA, lavées puis co-incubées avec les cellules effectrices à différents ratio en plaque 96 puits dans un volume de 200μl pendant 4 à 6h à 37°C. Les cellules sont ensuite centrifugées et le relargage de 51Cr est mesuré dans lOOμl de surnageant. Le pourcentage de lyse spécifique est calculé comme suit :The A2 / Kb EL-4 cells are incubated for 1 h with 51 Cr in the presence or not of ELA, washed and then co-incubated with the effector cells at different ratio in 96-well plate in a volume of 200 μl for 4 to 6 h at 37 ° C. The cells are then centrifuged and the release of 51 Cr is measured in 100 μl of supernatant. The percentage of specific lysis is calculated as follows:
% lyse = (relargage expérimental - relargage spontané)/ (relargage total - relargage spontané) x 100% lysis = (experimental release - spontaneous release) / (total release - spontaneous release) x 100
% lyse spécifique = % lyse avec cellules puisées par le peptide - % lyse avec cellules non puisées par le peptide. _ - _% specific lysis =% lysis with cells pulsed by the peptide -% lysis with cells not pulsed by the peptide. _ - _
"La différence de lyse cellulaire observée pour les peptides ELA (losanges) et AcELA (carrés) en présence de la protéine adjuvante rP40 (I. Rauly et al, Infect. Immun., 1999, 67, 5547) est représentée par la figure 1. c) conclusion : Alors qu'une activité CTL anti-ELA est observée après immunisation de souris avec P40/ELA, aucune activité CTL n'est mesurée lorsque les souris ont été immunisées par P40/AcELA. Ces résultats indiquent que les CTL générées par AcELA ne reconnaissent pas le peptide natif ELA. " The difference in cell lysis observed for the peptides ELA (diamonds) and AcELA (squares) in the presence of the adjuvant protein rP40 (I. Rauly et al, Infect. Immun., 1999, 67, 5547) is represented by FIG. 1 c) conclusion: While anti-ELA CTL activity is observed after immunization of mice with P40 / ELA, no CTL activity is measured when the mice have been immunized with P40 / AcELA These results indicate that the CTLs generated by AcELA do not recognize the native ELA peptide.
Exemple comparatif IN : Activité CTL des peptides ELA, PyrELA et AcELAComparative example IN: CTL activity of the peptides ELA, PyrELA and AcELA
Des souris A2/Kb ont reçu :A2 / Kb mice received:
- 300 μg de rP40 mélangée à 50 μg de ELA (Trifluoroacétate)- 300 μg of rP40 mixed with 50 μg of ELA (Trifluoroacetate)
- 300 μg de rP40 mélangée à 50 μg de ELA (Chlorhydrate)- 300 μg of rP40 mixed with 50 μg of ELA (hydrochloride)
- 300 μg de rP40 mélangée à 50 μg de PyrELA (Trifluoroacétate) a) Génération de cellules cytotoxiques effectrices :- 300 μg of rP40 mixed with 50 μg of PyrELA (Trifluoroacetate) a) Generation of effector cytotoxic cells:
10 jours après l'immunisation, les souris sont sacrifiées et les lymphocytes des ganglions drainant sont récupérés pour être stimulés in vitro avec le peptide relevant. Ces lymphocytes (4-5 106) sont cultivés en plaque 24 puits en DMEM plus
lOmM HEPES, 10% SVF et 50 μM β-2-mercaptoethanol avec 2-5 105 cellules EL- 4 A2/Kb (cellules murines transfectées avec le gène HLA-A* 0201/Kb) irradiées (10 kRads) pré-pulsées 1 h à 37°C avec 1 μM du peptide relevant. Après deux stimulations hebdomadaires, les cellules sont testées pour leur activité cytotoxique. b) Mesure de l'activité cytotoxique :10 days after immunization, the mice are sacrificed and the lymph nodes of the draining ganglia are recovered to be stimulated in vitro with the relevant peptide. These lymphocytes (4-5 10 6 ) are cultured in a 24-well plate in DMEM plus lOmM HEPES, 10% FCS and 50 μM β-2-mercaptoethanol with 2-5 10 5 EL- 4 A2 / Kb cells (murine cells transfected with the HLA-A * 0201 / Kb gene) irradiated (10 kRads) 1 h at 37 ° C with 1 μM of the relevant peptide. After two weekly stimulations, the cells are tested for their cytotoxic activity. b) Measurement of cytotoxic activity:
Les cellules EL-4 A2/Kb sont incubées 1 h avec du 51Cr en présence ou non de ELA, lavées puis co-incubées avec les cellules effectrices à différents ratio en plaque 96 puits dans un volume de 200μl pendant 4 à 6h à 37°C. Les cellules sont ensuite centrifugées et le relargage de 51Cr est mesuré dans lOOμl de surnageant. Le pourcentage de lyse spécifique est calculé comme suit :The A2 / Kb EL-4 cells are incubated for 1 h with 51 Cr in the presence or not of ELA, washed and then co-incubated with the effector cells at different ratio in 96-well plate in a volume of 200 μl for 4 to 6 h at 37 ° C. The cells are then centrifuged and the release of 51 Cr is measured in 100 μl of supernatant. The percentage of specific lysis is calculated as follows:
% lyse = (relargage expérimental-relargage spontané)/ (relargage total - relargage spontané) X 100% lysis = (experimental release-spontaneous release) / (total release - spontaneous release) X 100
% lyse spécifique = %lyse avec cellules puisées par le peptide - % lyse avec cellules non puisées par le peptide ELA. c) La génération de CTL anti-Melan-A après immunisation par rP40 mélangée avec les peptides ELA (Trifluoroacétate), ELA (Chlorydrate) ou PyrELA (Trifluoacétate) est représentée par la figure 2. d) Conclusions :% specific lysis =% lysis with cells pulsed by the peptide -% lysis with cells not pulsed by the peptide ELA. c) The generation of anti-Melan-A CTL after immunization with rP40 mixed with the peptides ELA (Trifluoroacetate), ELA (Chlorydrate) or PyrELA (Trifluoacetate) is represented by FIG. 2. d) Conclusions:
1. Alors qu'une activité CTL anti-ELA est observée après immunisation de souris avec P40/ELA (Trifluoroacétate), aucune activité CTL n'est mesurée lorsque les souris ont été immunisées par P40/PyrELA (Trifluoroacétate). Ces résultats indiquent que les CTL générées par PyrELA ne reconnaissent pas le peptide natif ELA.1. While anti-ELA CTL activity is observed after immunization of mice with P40 / ELA (Trifluoroacetate), no CTL activity is measured when the mice have been immunized with P40 / PyrELA (Trifluoroacetate). These results indicate that the CTLs generated by PyrELA do not recognize the native ELA peptide.
2. De manière surprenante, l'immunisation par P40/ELA (Chlorhydrate) est aussi efficace que celle par P40/ELA (Trifluoroacétate) pour générer une réponse2. Surprisingly, immunization with P40 / ELA (hydrochloride) is as effective as that with P40 / ELA (Trifluoroacetate) in generating a response
CTL anti-ELA.CTL anti-ELA.
Exemple V : Etudes de stabilité accélérée des formes acétate et chlorhydrate du peptide ELA. Les peptides sont analysés par HPLC en phase inverse à l'aide d'un éluantExample V: Studies of accelerated stability of the acetate and hydrochloride forms of the peptide ELA. Peptides are analyzed by reverse phase HPLC using an eluent
A composé d'eau à 0.1 % de TFA et d'un éluant B composé de 80% d' acétonitrile et de 20 % d'eau à 0.1 % de TFA.
La figure 3 montre les chromatogrammes du peptide ELA sous forme d'acétate (3.A) ou de chlorhydrate (3.B) conservé à 37°C pendant 2 mois.A composed of water with 0.1% TFA and an eluent B composed of 80% acetonitrile and 20% water with 0.1% TFA. Figure 3 shows the chromatograms of the ELA peptide in the form of acetate (3.A) or hydrochloride (3.B) stored at 37 ° C for 2 months.
Conclusion :Conclusion:
Sous forme d'acétate, la dégradation du peptide ELA en peptide cyclisé PyrELA inactif après 2 mois à 37°C est de 53%. De façon surprenante, sous forme de Chlorhydrate, elle n'est que de 10 %.In acetate form, the degradation of the ELA peptide into an inactive pyrELA cyclized peptide after 2 months at 37 ° C. is 53%. Surprisingly, in the form of hydrochloride, it is only 10%.
Exemple NI : Stabilité du peptide ELA sous forme de chlorhydrate conservé à 4°C. La figure 4 montre un chromatogramme du peptide ELA sous forme de chlorhydrate à t=0 : (98,9 % d'ELA et 0,4 % de PyrELA ; figure 4. A) et après un mois de conservation à 4 °C (98,8 % d'ELA et 0,5 % de PyrELA ; figure 4.B). Conclusion :Example NI: Stability of the ELA peptide in the form of the hydrochloride stored at 4 ° C. FIG. 4 shows a chromatogram of the peptide ELA in the form of hydrochloride at t = 0: (98.9% of ELA and 0.4% of PyrELA; FIG. 4. A) and after one month of storage at 4 ° C. ( 98.8% ELA and 0.5% PyrELA; Figure 4.B). Conclusion:
De façon surprenante, le peptide ELA sous forme de Chlorhydrate est extrêmement stable- à 4°C. Il peut donc être facilement manipulé et conservé à une température de 4 ou -20~°C. Ce n'est pas le cas d'un peptide équivalent (MART 3), préparé sous forme d'acétate qui doit être conservé à -80°C (M. Marchand et al, Int.Surprisingly, the ELA peptide in the hydrochloride form is extremely stable at 4 ° C. It can therefore be easily handled and stored at a temperature of 4 or -20 ~ ° C. This is not the case for an equivalent peptide (MART 3), prepared in the form of acetate which must be stored at -80 ° C (M. Marchand et al, Int.
J. Cancer, 1999, 80, 219).J. Cancer, 1999, 80, 219).
La forme saline acide fort permet donc une conservation beaucoup plus facile à 4°C (réfrigérateur) ou à -20°C (congélateur) avec une stabilité physicochimique totale, comme le montrent les exemples ci-dessus.
The strong acid saline form therefore allows a much easier conservation at 4 ° C (refrigerator) or at -20 ° C (freezer) with total physicochemical stability, as shown in the examples above.
Claims
1. Molécule d'intérêt pharmaceutique comportant en son extrémité N-terminale un acide glutamique ou une glutamine, caractérisé en ce qu'elle se présente sous forme de sel d'addition d'acide fort physiologiquement acceptable.1. Molecule of pharmaceutical interest comprising at its N-terminal end a glutamic acid or a glutamine, characterized in that it is in the form of a physiologically acceptable strong acid addition salt.
2. Molécule d'intérêt pharmaceutique selon la revendication 1 , caractérisée en ce qu'il s'agit d'un ligand du CMH comportant en son extrémité N-terminale un acide glutamique ou une glutamine.2. Molecule of pharmaceutical interest according to claim 1, characterized in that it is a MHC ligand comprising at its N-terminal end a glutamic acid or a glutamine.
3. Molécule d'intérêt pharmaceutique selon la revendication 1 ou 2, caractérisée en ce que le sel d'addition d'acide fort physiologiquement acceptable est choisi parmi les sels d'addition avec les acides minéraux ou organiques préférentiellement parmi les méthanesulfonate, chlorhydrate, bromhydrate, sulfate, nitrate et phosphate.3. Molecule of pharmaceutical interest according to claim 1 or 2, characterized in that the physiologically acceptable strong acid addition salt is chosen from addition salts with mineral or organic acids, preferably from methanesulfonate, hydrochloride, hydrobromide, sulfate, nitrate and phosphate.
4. Molécule d'intérêt pharmaceutique selon l'une des revendications 1 à 3, caractérisée en ce qu'elle- est -choisi parmi les molécules naturelles ou synthétiques. ~ 4. Molecule of pharmaceutical interest according to one of claims 1 to 3, characterized in that it is chosen from natural or synthetic molecules. ~
5. Molécule d'intérêt pharmaceutique selon l'une des revendications 1 à 4, caractérisé en ce qu'elle est choisie dans le groupe constitué des protéines, peptides, constructions polypeptidiques multi-épitopiques, pseudopeptides, rétro-inverso, peptoïdes, peptido-mimétiques et lipopeptides.5. Molecule of pharmaceutical interest according to one of claims 1 to 4, characterized in that it is chosen from the group consisting of proteins, peptides, multi-epitopic polypeptide constructs, pseudopeptides, retro-inverso, peptoids, peptido- mimetics and lipopeptides.
6. Ligand du MHC selon l'une des revendications 2 à 4, caractérisé en ce qu'il est choisi parmi les épitopes CTL.6. MHC ligand according to one of claims 2 to 4, characterized in that it is chosen from CTL epitopes.
7. Ligand du MHC selon la revendication 6, caractérisé en ce qu'il est choisi parmi les épitopes CTL se présentant sous forme d'octapeptide, de nonapeptide ou de décapeptide.7. MHC ligand according to claim 6, characterized in that it is chosen from CTL epitopes in the form of octapeptide, nonapeptide or decapeptide.
8. Ligand du MHC selon l'une des revendications 2 à 4, caractérisé en ce qu'il est choisi parmi les ligands décrits dans les bases de données SYFPEITHI ou MHCPEP comportant un acide glutamique ou une glutamine à leur extrémité N- terminale. 8. MHC ligand according to one of claims 2 to 4, characterized in that it is chosen from the ligands described in the SYFPEITHI or MHCPEP databases comprising a glutamic acid or a glutamine at their N-terminal end.
9. Ligand du MHC selon l'une des revendications 2 ou 3, caractérisé en ce qu'il est choisi parmi les peptides SEQ ID N° 1 à SEQ ID N° 695. 9. MHC ligand according to one of claims 2 or 3, characterized in that it is chosen from peptides SEQ ID No. 1 to SEQ ID No. 695.
10. Ligand du CMH selon l'une des revendications 2 à 7, caractérisé en ce qu'il est choisi dans le groupe des peptides- correspondant à SEQ ID N° 81, SEQ ID N° 112, SEQ ID N° 2, SEQ ID N° 273, SEQ ID N° 110, SEQ ID N° 106, SEQ ID N° 10, SEQ ID N° 692, SEQ ID N° 257, SEQ ID N° 568, SEQ ID N° 464, SEQ ID N° 466, SEQ ID N° 567 et SEQ ID N° 695.10. MHC ligand according to one of claims 2 to 7, characterized in that it is chosen from the group of peptides- corresponding to SEQ ID No. 81, SEQ ID No. 112, SEQ ID N ° 2, SEQ ID N ° 273, SEQ ID N ° 110, SEQ ID N ° 106, SEQ ID N ° 10, SEQ ID N ° 692, SEQ ID N ° 257, SEQ ID N ° 568, SEQ ID N ° 464, SEQ ID N ° 466, SEQ ID N ° 567 and SEQ ID N ° 695.
11. Ligand du CMH selon l'une la revendication 10, caractérisé en ce qu'il s'agit du peptide correspondant à SEQ ID N° 81, sous forme de chlorhydrate ou de sulfate.11. MHC ligand according to one of claim 10, characterized in that it is the peptide corresponding to SEQ ID No. 81, in the form of hydrochloride or sulfate.
12. Composition pharmaceutique caractérisée en ce qu'elle comprend au moins une molécule d'intérêt pharmaceutique selon l'une des revendications 1 à 11.12. Pharmaceutical composition, characterized in that it comprises at least one molecule of pharmaceutical interest according to one of claims 1 to 11.
13. Vaccin caractérisé en ce qu'il comprend au moins un ligand du CMH selon l'une des revendications 2 à 11.13. Vaccine characterized in that it comprises at least one MHC ligand according to one of claims 2 to 11.
14. Vaccin selon la revendication 13, caractérisé en ce qu'il comprend en outre au moins un adjuvant.14. Vaccine according to claim 13, characterized in that it further comprises at least one adjuvant.
15. Vaccin selon la revendication 13 ou 14 caractérisé en ce que l'adjuvant est choisi parmi les sels d'Aluminium (Alum) ou de Calcium, les protéines OmpA d'entérobactérie, TT, DT, CRM197, PLGA, ISCOM, Montanide ISA 720, les15. Vaccine according to claim 13 or 14 characterized in that the adjuvant is chosen from Aluminum (Alum) or Calcium salts, OmpA enterobacterium proteins, TT, DT, CRM197, PLGA, ISCOM, Montanide ISA 720, the
-ammoniums quaternaires aliphatiques, le MPL-A, le Quil-A, les CpG; la Leif, la CT, la LT ou les versions détoxifiées de la CT ou la LT.aliphatic quaternary ammoniums, MPL-A, Quil-A, CpGs; Leif, CT, LT or detoxified versions of CT or LT.
16. Vaccin selon l'une des revendications 13 à 15 caractérisé en ce qu'il comprend en outre, un composé porteur mélangé ou couplé audit ligand. 16. Vaccine according to one of claims 13 to 15 characterized in that it further comprises, a carrier compound mixed or coupled to said ligand.
17. Vaccin selon la revendication 15 caractérisé en ce que ledit composé porteur est choisi parmi les anatoxines, dont le toxoïde diphtérique ou le toxoïde tétanique, les protéines dérivées du streptocoque, les protéines de membranes externes bactériennes de type Omp A, les complexes de protéines de membranes externes (OMPC), les vésicules de membranes externes (OMV) ou les HSP. 17. Vaccine according to claim 15 characterized in that said carrier compound is chosen from toxoids, including the diphtheria toxoid or tetanus toxoid, proteins derived from streptococcus, proteins from external bacterial membranes of Omp A type, protein complexes of outer membranes (OMPC), vesicles of outer membranes (OMV) or HSP.
18. Vaccin selon l'une des revendications 13 à 17 caractérisé en ce que ledit ligand associé éventuellement à un composé porteur est incorporé dans un vecteur choisi dans le groupe comprenant les liposomes, virosomes, nanosphères, microsphères, microcapsules ou biovecteurs.18. Vaccine according to one of claims 13 to 17 characterized in that said ligand optionally associated with a carrier compound is incorporated in a vector chosen from the group comprising liposomes, virosomes, nanospheres, microspheres, microcapsules or biovectors.
19. Vaccin anti-mélanome caractérisé en ce qu'il comprend au moins un peptide selon la revendication 11.19. Anti-melanoma vaccine characterized in that it comprises at least one peptide according to claim 11.
20. Vaccin anti-mélanome selon la revendication 19, caractérisé en ce qu'il comprend en outre une protéine OmpA d'entérobactérie. 20. Anti-melanoma vaccine according to claim 19, characterized in that it also comprises an enterobacterium OmpA protein.
21. Méthode de diagnostic in vitro de pathologies associées à la présence dans l'organisme d'un patient de ligand de CMH, et susceptibles d'être directement ou indirectement impliqués dans le processus de développement de ces pathologies chez l'homme ou l'animal, caractérisée en ce qu'elle comprend les étapes de : mise en contact d'un échantillon biologique provenant d'un patient, notamment du sang ou tout échantillon biologique susceptible de contenir des lymphocytes, avec un ligand du CMH selon l'invention, dans des conditions permettant la formation d'un complexe binaire entre ledit ligand du CMH et les molécules de CMH présentes dans ledit échantillon, et la réaction entre ledit complexe binaire et les récepteurs des cellules T susceptibles d'être présentes dans ledit échantillon biologique.21. Method for in vitro diagnosis of pathologies associated with the presence in the body of a patient of MHC ligand, and likely to be directly or indirectly involved in the process of development of these pathologies in humans or animal, characterized in that it comprises the steps of: bringing a biological sample from a patient, in particular blood or any biological sample capable of containing lymphocytes, into contact with a MHC ligand according to the invention, under conditions allowing the formation of a binary complex between said MHC ligand and the MHC molecules present in said sample, and the reaction between said binary complex and the T cell receptors likely to be present in said biological sample.
- détection in vitro du complexe ternaire CMH - ligand du CMH - récepteur T,- susceptible d ' être formé à 1 ' étape précédente.- in vitro detection of the MHC ternary complex - MHC ligand - T receptor, - capable of being formed in the previous step.
- 22. Nécessaire ou kit pour la mise en œuvre de méthodes de diagnostic in vitro selon la revendication 21, comprenant :- 22. Kit or kit for implementing in vitro diagnostic methods according to claim 21, comprising:
- un ligand du CMH selon l'une des revendications 2 à 11 ; éventuellement des réactifs pour permettre la formation d'une réaction immunologique entre ledit ligand, les molécules du CMH et les récepteurs des cellules T éventuellement présents dans l'échantillon biologique ; éventuellement des réactifs permettant de détecter le complexe ternaire selon l'invention, qui a été produit à l'issue de la réaction immunologique, lesdits réactifs contenant éventuellement un marqueur ou étant susceptibles d'être reconnus à leur tour par un réactif marqué. - a MHC ligand according to one of claims 2 to 11; optionally reagents to allow the formation of an immunological reaction between said ligand, MHC molecules and T cell receptors possibly present in the biological sample; optionally reagents for detecting the ternary complex according to the invention, which was produced at the end of the immunological reaction, said reagents optionally containing a marker or being capable of being recognized in turn by a labeled reagent.
23. Utilisation d'un ligand selon l'une des revendications 2 à 10, pour la préparation d'un vaccin destiné au traitement prophylactique ou thérapeutique des infections virales, bactériennes, parasitaires ou fongiques. 23. Use of a ligand according to one of claims 2 to 10, for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of viral, bacterial, parasitic or fungal infections.
24. Utilisation d'un ligand selon l'une des revendications 2 à 11, pour la préparation d'un vaccin destiné au traitement prophylactique ou thérapeutique des cancers et préférentiellement à inhiber la-croissance des tumeurs. 24. Use of a ligand according to one of claims 2 to 11, for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of cancers and preferably to inhibit the growth of tumors.
25. Utilisation d'un acide fort physiologiquement acceptable pour stabiliser et maintenir l'activité biologique d'une molécule à activité pharmaceutique comportant un acide glutamique ou une glutamine à son extrémité N-terminale.25. Use of a physiologically acceptable strong acid to stabilize and maintain the biological activity of a molecule with pharmaceutical activity comprising a glutamic acid or a glutamine at its N-terminal end.
26. Utilisation d'un acide fort pour diminuer et/ou supprimer la formation du dérivé pyroglutamique d'une molécule à activité pharmaceutique comportant un acide glutamique ou une glutamine à son extrémité N-terminale.26. Use of a strong acid to decrease and / or suppress the formation of the pyroglutamic derivative of a molecule with pharmaceutical activity comprising a glutamic acid or a glutamine at its N-terminal end.
27. Procédé de préparation d'une molécule d'intérêt pharmaceutique comportant un acide glutamique ou une glutamine à son extrémité N-terminale sous forme de sel d'addition d'acide fort physiologiquement acceptable selon l'une des revendications 1 à 11, caractérisé en ce qu'il comporte une étape de purification par RP-HPLC de ladite molécule à partir du sel de trifluoroacétate correspondant en utilisant un éluant à base dudit acide fort, suivie de façon optionnelle d'une é tape de lyophilisation de la solution ainsi obtenue.27. A method of preparing a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its N-terminal end in the form of a physiologically acceptable strong acid addition salt according to one of claims 1 to 11, characterized in that it comprises a step of purification by RP-HPLC of said molecule from the corresponding trifluoroacetate salt using an eluent based on said strong acid, optionally followed by a step of lyophilization of the solution thus obtained .
28. Procédé de préparation d'une molécule d'intérêt pharmaceutique comportant un acide glutamique ou une glutamine à son extrémité N-terminale sous forme de sel d'addition d'acide fort physiologiquement acceptable selon l'une des revendications 1 à 11, caractérisé en ce qu'il comporte une étape de dissolution d'un sel de trifluoroacétate de ladite molécule dans une solution en excès dudit acide fort, suivie de façon optionnelle d'une étape de lyophilisation de la solution ainsi obtenue.28. Method for preparing a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its N-terminal end in the form of a physiologically acceptable strong acid addition salt according to one of claims 1 to 11, characterized in that it comprises a step of dissolving a trifluoroacetate salt of said molecule in a solution in excess of said strong acid, optionally followed by a step of lyophilization of the solution thus obtained.
29. Procédé de préparation d'une molécule d'intérêt pharmaceutique comportant un acide glutamique ou une glutamine à son extrémité N-terminale sous forme de sel d'addition d'acide fort physiologiquement acceptable selon l'une des revendications 1 à 11, caractérisé en ce qu'il comporte une étape de chromatographie par échange ion à partir du sel de trifluoroacétate correspondant de ladite molécule d'intérêt pharmaceutique, après dissolution dudit sel dans une solution contenant ledit acide fort.29. Method for preparing a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its N-terminal end in the form of a physiologically acceptable strong acid addition salt according to one of claims 1 to 11, characterized in that it comprises a step of ion exchange chromatography from the corresponding trifluoroacetate salt of said molecule of pharmaceutical interest, after dissolution of said salt in a solution containing said strong acid.
30. Procédé pour la stabilisation d'une molécule d'intérêt pharmaceutique comportant un acide glutamique ou une glutamine à son extrémité N-terminale, caractérisé en ce que l'on fait réagir ladite molécule avec un acide fort dans des conditions permettant d'obtenir ladite molécule sous la forme d'un sel d'addition d'acide fort physiologiquement acceptable, en particulier selon un procédé selon l'une des revendications 27 à 29. 30. A method for stabilizing a molecule of pharmaceutical interest comprising a glutamic acid or a glutamine at its N-terminal end, characterized in that said molecule is reacted with a strong acid under conditions making it possible to obtain said molecule in the form of a physiologically acceptable strong acid addition salt, in particular according to a method according to one of claims 27 to 29.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0003711A FR2806727A1 (en) | 2000-03-23 | 2000-03-23 | MOLECULE OF PHARMACEUTICAL INTEREST COMPRISING IN THE N-TERMINAL END A GLUTAMIC ACID OR GLUTAMINE IN THE FORM OF A PHYSIOLOGICALLY ACCEPTABLE ACID ADDITION SALT |
FR0003711 | 2000-03-23 | ||
PCT/FR2001/000872 WO2001070772A2 (en) | 2000-03-23 | 2001-03-22 | Molecule of pharmaceutical interest comprising at its n-terminal a glutamic acid or a glutamine in the form of an addition salt to an acid |
Publications (1)
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EP1305332A2 true EP1305332A2 (en) | 2003-05-02 |
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ID=8848421
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EP01919544A Withdrawn EP1305332A2 (en) | 2000-03-23 | 2001-03-22 | Molecule of pharmaceutical interest comprising at its n-terminal a glutamic acid or a glutamine in the form of a physiologically acceptable strong acid |
Country Status (11)
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US (1) | US20030175285A1 (en) |
EP (1) | EP1305332A2 (en) |
JP (1) | JP2003528112A (en) |
CN (1) | CN1449407A (en) |
AU (1) | AU2001246623A1 (en) |
BR (1) | BR0109502A (en) |
CA (1) | CA2403803A1 (en) |
FR (1) | FR2806727A1 (en) |
MX (1) | MXPA02009359A (en) |
WO (1) | WO2001070772A2 (en) |
ZA (1) | ZA200207632B (en) |
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IL105554A (en) * | 1992-05-05 | 1999-08-17 | Univ Leiden | Peptides of human papilloma virus for use in human t cell response inducing compositions |
WO2002092120A1 (en) * | 2001-05-15 | 2002-11-21 | Ludwig Institute For Cancer Research | Structurally modified peptides and uses thereof |
WO2004104026A1 (en) * | 2003-05-21 | 2004-12-02 | Biotech Tools S.A. | Peptide complex |
DK1625148T3 (en) | 2003-05-21 | 2013-01-14 | Biotech Tools Sa | peptide Complex |
CA2539789A1 (en) * | 2003-09-22 | 2005-03-31 | Green Peptide Co., Ltd. | Peptide originating in hepatitis c virus |
GB0716992D0 (en) | 2007-08-31 | 2007-10-10 | Immune Targeting Systems Its L | Influenza antigen delivery vectors and constructs |
GB0408164D0 (en) | 2004-04-13 | 2004-05-19 | Immune Targeting Systems Ltd | Antigen delivery vectors and constructs |
JP2006248978A (en) | 2005-03-10 | 2006-09-21 | Mebiopharm Co Ltd | New liposome preparation |
BRPI0504117A (en) | 2005-09-05 | 2007-05-22 | Fundacao De Amparo A Pesquisa | epitopes, combination of epitopes, uses of epitopes or their combination, composition, uses of composition, anti-HIV-1 prophylactic vaccines, therapeutic vaccines, method for identifying epitopes and methods for treatment or prevention. |
GB0522748D0 (en) * | 2005-11-07 | 2005-12-14 | Univ Cambridge Tech | Collagen peptides, methods and uses |
CN101400363B (en) * | 2006-01-18 | 2012-08-29 | 昌达生物科技公司 | Pharmaceutical compositions with enhanced stability |
US8551493B2 (en) * | 2007-08-15 | 2013-10-08 | Circassia Limited | Peptide with reduced dimer formation |
CA2735278A1 (en) * | 2008-08-28 | 2010-03-04 | Aarhus Universitet | Hiv-1 envelope polypeptides for hiv vaccine |
JP5716018B2 (en) | 2009-09-16 | 2015-05-13 | 千寿製薬株式会社 | Lacritin partial peptide |
RS55670B1 (en) * | 2010-03-15 | 2017-06-30 | Academisch Ziekenhuis Leiden | Peptides, conjugates and method for increasing immunogenicity of a vaccine |
US20140227311A1 (en) * | 2011-08-23 | 2014-08-14 | Skau Aps | Method for Removing Immunosuppressive Properties of HIV Envelope Glycoproteins |
GB201410507D0 (en) * | 2014-06-12 | 2014-07-30 | Univ Bath | Drug delivery enhancement agents |
EP3279218A4 (en) * | 2015-03-30 | 2018-12-05 | Osaka University | Immunizing peptide, method for producing immunizing peptide, pharmaceutical composition for immune disorders containing same, and method for treating immune disorders |
WO2020090979A1 (en) * | 2018-10-31 | 2020-05-07 | 味の素株式会社 | Compound comprising substance with affinity for antibody, cleavage site and reactive group, or salt thereof |
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IT1226552B (en) * | 1988-07-29 | 1991-01-24 | Ellem Ind Farmaceutica | IMMUNOSTIMULANT PEPTIDES. |
US6696061B1 (en) * | 1992-08-11 | 2004-02-24 | President And Fellows Of Harvard College | Immunomodulatory peptides |
US5874560A (en) * | 1994-04-22 | 1999-02-23 | The United States Of America As Represented By The Department Of Health And Human Services | Melanoma antigens and their use in diagnostic and therapeutic methods |
EP1012173B1 (en) * | 1997-06-23 | 2007-10-31 | Ludwig Institute For Cancer Research | Isolated decapeptides which bind to hla molecules, and the use thereof |
EP1064022A4 (en) * | 1998-03-13 | 2004-09-29 | Epimmune Inc | Hla-binding peptides and their uses |
WO1999050637A2 (en) * | 1998-03-27 | 1999-10-07 | Ludwig Institute For Cancer Research | Isolated multimeric complexes useful in analysis of t cells, peptides useful in making the complexes, and uses thereof |
GB9808932D0 (en) * | 1998-04-27 | 1998-06-24 | Chiron Spa | Polyepitope carrier protein |
-
2000
- 2000-03-23 FR FR0003711A patent/FR2806727A1/en not_active Withdrawn
-
2001
- 2001-03-22 CA CA002403803A patent/CA2403803A1/en not_active Abandoned
- 2001-03-22 CN CN01808833A patent/CN1449407A/en active Pending
- 2001-03-22 WO PCT/FR2001/000872 patent/WO2001070772A2/en not_active Application Discontinuation
- 2001-03-22 EP EP01919544A patent/EP1305332A2/en not_active Withdrawn
- 2001-03-22 BR BR0109502-1A patent/BR0109502A/en not_active IP Right Cessation
- 2001-03-22 AU AU2001246623A patent/AU2001246623A1/en not_active Abandoned
- 2001-03-22 JP JP2001568973A patent/JP2003528112A/en not_active Withdrawn
- 2001-03-22 US US10/239,313 patent/US20030175285A1/en not_active Abandoned
- 2001-03-22 MX MXPA02009359A patent/MXPA02009359A/en unknown
-
2002
- 2002-09-23 ZA ZA200207632A patent/ZA200207632B/en unknown
Non-Patent Citations (1)
Title |
---|
ELLIOTT S.L. ET AL: "Peptide based cytotoxic T-cell vaccines; delivery of multiple epitopes, help, memory and problems", VACCINE, vol. 17, no. 15-16, 9 April 1999 (1999-04-09), pages 2009 - 2019 * |
Also Published As
Publication number | Publication date |
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WO2001070772A3 (en) | 2003-02-13 |
BR0109502A (en) | 2004-01-13 |
CA2403803A1 (en) | 2001-09-27 |
JP2003528112A (en) | 2003-09-24 |
FR2806727A1 (en) | 2001-09-28 |
CN1449407A (en) | 2003-10-15 |
US20030175285A1 (en) | 2003-09-18 |
WO2001070772A2 (en) | 2001-09-27 |
AU2001246623A1 (en) | 2001-10-03 |
ZA200207632B (en) | 2003-10-27 |
MXPA02009359A (en) | 2003-02-12 |
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