WO2003010313A1 - Novel diphtheria anatoxins and their use as carrier - Google Patents
Novel diphtheria anatoxins and their use as carrier Download PDFInfo
- Publication number
- WO2003010313A1 WO2003010313A1 PCT/FR2002/002598 FR0202598W WO03010313A1 WO 2003010313 A1 WO2003010313 A1 WO 2003010313A1 FR 0202598 W FR0202598 W FR 0202598W WO 03010313 A1 WO03010313 A1 WO 03010313A1
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- WIPO (PCT)
- Prior art keywords
- peptide
- antigen
- diphtheria
- sequence
- hapten
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- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- 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
- A61P33/00—Antiparasitic agents
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to new derivatives of diphtheria toxoids and a pharmaceutical composition comprising in a pharmaceutically acceptable medium such derivatives, as well as the use of these derivatives for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of viral infections, bacterial, parasitic or fungal or for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of cancers.
- Vaccination is an effective way to prevent or reduce viral or bacterial infections.
- the success of vaccination campaigns in these fields has made it possible to extend the concept of vaccine previously used in the field of infectious diseases to the fields of cancer and autoimmune diseases.
- tetanus and diphtheria toxoids are also used as carriers.
- tetanus toxoid Rosauly et al., Infect. Immun., 67: 5547-5551, 1999
- tetanus toxoid is a protein used in vaccines for human use.
- CRM 197 As an example of CRM, mention may be made of CRM 197 (Giannini G. et al., Nucleic Acids Res., 12: 4063, 1984) which is remarkable in that it only exhibits a modification of a single amino acid. (substitution of the residue Gly (position aa 52) with Glu) and is not distinguished from an antigenic point of view from the native toxin. This unique mutation completely abolished the toxic nature of the protein.
- CRM 45 (Giannini G. et al, Nucleic Acids Res., 12: 4063, 1984) which results from the deletion of a C-terminal part of the B subunit of DT, around 17 Kda , part responsible for fixation on the cell receptor.
- the CRM 45 protein no longer has the ability to bind to the surface of the cell. It has been found by the inventors that the production of peptides derived from toxoids poses multiple problems, in particular when the carrier is coupled to a peptide antigen. It was thus noted that it could form during the production of toxoid derivatives, multimers of said derivatives.
- the initial configurations in particular with regard to the formation of disulfide bridges, can change.
- the initial configuration in particular at the level of the formation of the disulfide bridges of the toxoid derivative or of the peptide of interest can change.
- unwanted pairings namely new disulfide bridges, can occur.
- the object of the present invention is to obtain new peptides derived from toxoid which respond to the problems mentioned above, which are easy to produce industrially and which make it possible to obtain an immune response against any antigen which is coupled or fused, the least possible risk of immunological side effects and presenting a negative HSI (Immediate Hypersensitivity).
- the present invention thus relates to new derivatives of diphtheria toxoids, the latter being easier to produce industrially while making it possible to increase the immunogenicity of a covalently associated antigen, either chemically conjugated or fused.
- diphtheria toxoids is meant in particular any peptide of amino acid sequence included in the amino acid sequence of diphtheria toxoid which, when associated with an antigen or hapten specific for an infectious agent or of a tumor cell, is capable of generating or increasing an immune response directed against said infectious agent or said tumor cell.
- the term “diphtheria toxoids” is also intended to denote any genetically modified protein similar from an antigenic point of view to the native toxin but which does not exhibit any toxicity.
- the term “diphtheria toxoids” also includes CRMs such as CRM 197 and CRM 45, and in particular those described in Uchida et al., J. Biol.
- the invention also relates to a peptide having, after optimal alignment, at least 80%, preferably at least 85%, 90%, 95% and 99% homology with a peptide derived from diphtheria toxoid according to the invention and comprising the deletion said at least cysteine residue of the peptide derived from diphtheria toxoid.
- the peptide derived from diphtheria toxoid can be obtained recombinantly.
- the diphtheria toxoid is chosen from the atoxic derivative of the diphtheria toxin obtained after heating and treatment with formalin, the CRM 197 of sequence SEQ ID No. 10 and the CRM 45 of sequence SEQ ID No. 1 l.
- Cysteines are preferentially deleted in the N-terminal or C-terminal regions, in particular in six of the fusion proteins between a toxoid derivative and a peptide or protein of interest so as to avoid unwanted pairings, such as new disulfide bridges.
- all the cysteines are deleted.
- the peptide derived from diphtheria toxoid comprises or has for sequence: a) an amino acid sequence chosen from the amino acid sequences SEQ ID N ° 1, SEQ ID N ° 2 and SEQ ID N ° 3; b) the amino acid sequence of a sequence having after optimal alignment a homology of at least 80%, preferably 85%, 90%, 95% and 99% with the reference amino acid sequence SEQ ID N ° 1, SEQ ID N ° 2 or SEQ ID N ° 3.
- the peptide of sequence SEQ ID No. 1, called DTa corresponds to CRM 197 and also has a C-terminal deletion after the residue Ala position 185, just before the residue Cys 186.
- the peptide of sequence SEQ ID No. 2, called DTb contains a deletion of the part responsible for binding of DT to the receptor of the cell, namely the N-terminal part of 8 aa (Cys in position 8), and in C-terminal after AA K (456).
- DTaDTb The peptide of sequence SEQ ID No. 3, called DTaDTb is a conjugate of DTa and DTb.
- nucleic acid or amino acid sequence having a homology of at least 80% after optimal alignment with a determined nucleic acid or amino acid sequence is meant a sequence which after optimal alignment with said determined sequence includes a percentage identity of at least 80% with said determined sequence.
- percentage of identity between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length.
- Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window” to identify and compare the regions. sequence similarity locale.
- the optimal alignment of the sequences for comparison can be achieved, besides manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App.
- the percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window. and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.
- BLAST 2 sequences available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used being those given by default (in particular for the parameters “open gap penaltie”: 5, and “extension gap penaltie”: 2; the chosen matrix being for example the “BLOSUM 62” matrix proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.
- sequences of, or coding for, peptides capable of inducing an immune response directed specifically against the antigen or hapten associated with it are preferred, such as the induction of an immune response measured using standard techniques described in the examples below.
- the present invention also relates to a nucleic acid coding for a derivative of diphtheria toxoids according to the invention, and preferably for a derivative of diphtheria toxoids of SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.
- L A further subject of the invention is a pharmaceutical composition characterized in that it comprises, in a pharmaceutically acceptable medium, at least one peptide derived from diphtheria toxoids according to the invention or a nucleic acid coding for said peptide.
- the present invention also relates to a pharmaceutical composition characterized in that it comprises in a medium which is pharmaceutically acceptable to at least one transformed host cell capable of expressing said peptide derived from diphtheria toxoids according to the invention.
- a subject of the invention is also the composition according to the invention, characterized in that said pharmaceutical composition further comprises an antigen, immunogen or hapten.
- immunogen antigen or hapten specific for an infectious agent or a tumor cell
- an infectious agent such as a virus, a bacterium, a yeast, a fungus or a parasite
- a tumor cell or one of their structural analogues, which alone or in combination with an adjuvant or carrier is capable of inducing a specific immune response of said infectious agent or of said tumor cell.
- immunogen, antigen or hapten is also intended to denote in the present description a compound having a structural analogy with said antigen or hapten capable of inducing an immunological response directed against said antigen or hapten in an organism previously immunized with said analogous compound.
- Said antigen or hapten can in particular be chosen from proteins, glycopeptides, lipopeptides, polysaccharides, oligosaccharides, nucleic acids and lipids.
- said antigen, immunogen or hapten is derived from a virus, a bacterium, a parasite or a fungus.
- said antigen, immunogen or hapten comprises at least one peptide derived from a microorganism responsible for airway pathologies chosen from RSV, the para influenza virus (PFV), the infuenza virus , hantaviruses, streptococci, pneumococci, haemophilus influenzae type b, rhinoviruses, coronoviruses and meningococci.
- PFV para influenza virus
- hantaviruses hantaviruses
- streptococci pneumococci
- haemophilus influenzae type b haemophilus influenzae type b
- rhinoviruses coronoviruses and meningococci.
- said antigen, immunogen or hapten comprises at least one fragment of the protein G of the respiratory syncytial virus.
- said antigen, immunogen or hapten is described in patent applications WO 87/04185 relating to structural proteins of RSV, WO 89/02935 which describes the entire F protein of RSV, optionally modified in monomeric or deglycosylated form, WO 95/27787 which relates to peptides derived from the G protein of RSV and more particularly the peptide called G2Na (fragment aa 130-230 of protein G of human RSV type A identified by sequence SEQ ID No.
- said antigen corresponds to an immunogenic peptide derived from RSV protein G of subgroup A or B comprising at least:
- a first peptide derived from the G protein of the RSV of the subgroup A or B comprising at least in position 173, 176, 182 and 186 a cysteine, and the C-terminal end of which comprises at most the amino acid in position 192; and a second peptide derived from an RSV protein from subgroup A or B, said second peptide being located downstream of said first peptide, so that the immunogenic peptide produced has a disulfide bridge connecting residues 173 and 186 and a second disulfide bridge connecting residues 176 and 182.
- peptide comprising at least in position 173, 176, 182 and 186 a cysteine, and the C-terminal end of which comprises at most the amino acid in position 192
- peptide having at least 4 cysteines in the same configuration as native G protein.
- said first peptide has a sequence chosen from the sequence of protein G of RSV of subgroup A or B 130-190, 130-192, 140-190, 140-192, 145 -190, 145-192, 148-190, 148-192, 130-188, 140-188, 145-188 or 148-188, and preferably the sequence 140-190.
- said second peptide has a sequence chosen from the sequence 144-158, 144-159 of protein G of RSV.
- the antigen called G20a corresponding to the sequence 140-190 of the GRS protein of RSV, in group A, coupled to the sequence 144-158 of protein G of RSV, in group A.
- the antigen, immunogen or hapten can also be associated or specific for a tumor cell.
- the present invention also relates to a composition according to the invention, characterized in that said antigen, immunogen or hapten is coupled or mixed with the peptide derived from diphtheria toxoids according to the invention.
- the diphtheria toxoid derivative according to the invention can be coupled by covalent bond, in particular by chemical coupling, with the immunogen, antigen or hapten specific for an infectious agent or a tumor cell.
- one or more linking elements are introduced into the diphtheria toxoid derivative according to the invention and / or into said antigen or hapten to facilitate chemical coupling, preferably, said element bonding agent is an amino acid.
- binding elements in particular amino acids to facilitate the coupling reactions between the peptide derived from diphtheria toxoids according to the invention and said immunogen, antigen or hapten.
- the covalent coupling between the peptide derived from diphtheria toxoids according to the invention and said immunogen, antigen or hapten according to the invention can be carried out at the N- or C-terminal end of said peptide.
- the bifunctional reagents allowing this coupling will be determined according to the end of said peptide chosen to effect the coupling and the nature of said immunogen, antigen or hapten to be coupled.
- the coupling between said immunogen, antigen or hapten and said peptide derived from diphtheria toxoids according to the invention is carried out by genetic recombination, when said immunogen, antigen or hapten is of peptide nature.
- the conjugates resulting from a coupling between the immunogen, antigen or hapten and said peptide derived from diphtheria toxoids according to the invention can be prepared by genetic recombination.
- the chimeric or hybrid protein (the conjugate) can be produced by recombinant DNA techniques by insertion or addition to the DNA sequence coding for said peptide derived from diphtheria toxoids according to the invention, of a sequence coding for said protein-based immunogen, antigen or hapten.
- the methods for synthesizing hybrid molecules include the methods used in genetic engineering to construct hybrid polynucleotides encoding the desired polypeptide sequences.
- the invention also includes such a conjugate between a diphtheria toxoid derivative according to the invention and an immunogen, antigen or hapten specific for an infectious agent or a tumor cell.
- conjugates mention may be made of peptides of sequences SEQ
- the present invention also relates to a pharmaceutical composition which comprises a nucleic construct coding for said conjugate, or which comprises a vector containing a nucleic construct coding for said conjugate or a cell transformed host containing said nucleic construct capable of expressing said conjugate.
- compositions according to the invention may also contain an adjuvant.
- an adjuvant can in particular be chosen from MPL-A (“MonoPhosphoryl Lipid A”), MF-59, Quil-A (adjuvant derived from saponin), ISCOM (“ImmunoStimulating
- COMplex Dimethyl Dioctadecyl Ammonium in the form of bromide (DDAB) or chloride (DDAC), CpG (oligodeoxynucleotides containing a specific motif centered on a dinucleotide CpG), Leif (protein antigen derived from Leishmania capable of stimulating cells PBMC and antigen presenting, and produce a cytokine reaction type Th-1), CT (Cholera Toxin), LT (“heat Labil Toxin”) and detoxified versions of CT or LT.
- DDAB bromide
- DDAC chloride
- CpG oligodeoxynucleotides containing a specific motif centered on a dinucleotide CpG
- Leif protein antigen derived from Leishmania capable of stimulating cells PBMC and antigen presenting, and produce a cytokine reaction type Th-1
- CT Choera Toxin
- LT heat Labil Toxin
- detoxified versions of CT or LT
- the pharmaceutically acceptable medium is the medium in which the compounds of the invention are administered, preferably a medium injectable into humans. It can consist of water, an aqueous saline solution or an aqueous solution based on dextrose and or glycerol.
- the invention also comprises a composition according to the invention, characterized in that said pharmaceutical composition is conveyed in a form making it possible to improve its stability and / or its immunogenicity; thus, it can be conveyed in the form of liposomes, virosomes, nanospheres, microspheres or microcapsules.
- a peptide derived from diphtheria toxoids according to the invention as defined above as a carrier in the manufacture of a vaccine constitutes another object of the invention.
- the invention also relates to the use of a peptide derived from diphtheria toxoids according to the invention as defined above for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of viral, bacterial, parasitic or fungal infections or for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of cancers.
- the invention also relates to the use of a peptide derived from diphtheria toxoids according to the invention as defined above for the preparation of a pharmaceutical composition intended to generate or increase an immune response against an infectious agent or a tumor cell.
- a pharmaceutical composition intended to generate or increase an immune response against an infectious agent or a tumor cell.
- Figures 1A and 1B Anti-G2Na and VRS-A titers after one or two immunizations in cotton rats immunized with different immunogens.
- Example 1 Cloning of diphtheria toxoid derivatives
- the gene coding for DTa (aal - aal85 of CRM 197), obtained by PCR directly from the genome of Corynebacterium Diphtheria (strain CRM 197), was cloned in an expression vector whose promoter is based on the Tryptophan operon (Trp).
- Trp Tryptophan operon
- pTEXDTa the DNA of the insert of which has been verified by DNA sequencing.
- the vector was transformed into an Escherishia coli Kl 2 bacterium called ICONE®.
- the same cloning procedure was performed for the cloning of DTb (aa202 - aa456 of CRM 197).
- the fusion protein DTaDTb results from the fusion of the genes coding for DTa and DTb.
- the G20aDTa protein results from the fusion of the genes coding for G20a and DTa.
- the following example illustrates the production and purification of G20aDTa.
- Example 2 Expression of the diphtheria toxoid derivative in Escherichia coli In a 30 L fermenter (CHEMAP CMF400) containing 18 L of minimum culture medium (g / 1) (KH 2 PO 4 , 6 / K 2 HPO 4 , 4 / Na 3 citrate 2H 2 O, 9 / Yeast extract 1 / (NH4) 2 SO 4 , 5 / CaCl 2 , 0.3 / MgSO 4 , 7H 2 O, 2 / Glycerol 100), Trace elements (1 ml / 1) and Struktol antifoam (0.4 ml / 1) supplemented with a solution of Tetracycline and Tryptophan at a final concentration of 0.008 g / 1 and 0.3 g / 1 respectively, 1,400 ml of the same are inoculated medium derived from a recombinant E.
- minimum culture medium g / 1
- a solution of Tetracycline and Tryptophan at a final
- coli preculture having the expression vector of G20aDTa in a 2 liter fermenter.
- the following physicochemical parameters are kept constant: Temperature at 37 ° C, pH at 7 regulated with NH OH, Agitation 500 - 1000 rpm to maintain the level of dissolved O 2 at 30%.
- the optical density (OD 620 nm) of the culture medium reaches the value of 50, it is possible to induce the expression of the recombinant protein by adding 2 ml / liter of culture of a solution of indolacrylic acid (IAA) at 12.5 g / 1. A few hours later, the fermentation is stopped by cooling to 4 ° C.
- IAA indolacrylic acid
- the biomass (approximately 500 g of dry cells) is taken up in 10 l of TST buffer (25 mM Tris HC1 pH 8, MgCl 2 6H 2 O 5 mM, 2 mM EDTA).
- TST buffer 25 mM Tris HC1 pH 8, MgCl 2 6H 2 O 5 mM, 2 mM EDTA.
- the bacterial suspension is ground with Manton-Gaulin (3 cycles at 560 bar). Since the recombinant protein G20aDTa is predominantly soluble, the purification can be carried out directly from the ground suspension. It is possible, for example, to capture DTa by ion exchange chromatography in an expanded bed (Streamline, Pharmacia). Two or three additional chromatography steps (ion exchange and exclusion) are necessary in order to remove DNA and protein contaminants from the host cell.
- the purified proteins are analyzed on SDS-PAGE gel under reduced conditions, on the MINI PROTEAN II SYSTEM device (BioRads). They can be viewed with Coomassie brilliant blue R250.
- Example 4 Coupling of the G20a Peptide A. Chemical Synthesis and Analytical Characterization of the G20a Peptide
- Peptide G20a is a fragment of protein G of VRS-A (140-190) - (144-158) of 69 amino acids. It contains 4 cysteines capable of forming 2 disulfide bridges.
- the sequence of the G20a peptide is as follows (SEQ ID No. 12): MEFQ 14 oTQPSKPTTKQRQNKPPNKPNNDFHFEVFNFVPC 173 SIC 176 SNNPTC ⁇ 82 WA IC ⁇ 86 KRIP 19 oS ⁇ 4 KPTTKQRQNKPPNK 158
- the G20a peptide is obtained by automatic synthesis in solid phase in Fmoc / tBu chemistry on a 0.25 mmol scale from a hydroxymethylphenoxymethyl resin (HMP) preloaded with a Lys (Boc) (0.70 mmol / g) and Fmoc-amino acids protected at the side chains by the following groups: trityl (Trt) for Asn, Gln and His; tert-butyl ether (tBu) for Ser and Thr; tert-butyl ester (OtBu) for Asp and Glu; tert-butyloxycarbonyl (Boc) for Lys and Trp and 2,2,5,7,8- pentamethylchroman-6 sulfonyl (Pmc) for Arg.
- HMP hydroxymethylphenoxymethyl resin
- Boc Lys
- the cysteines used had the following orthogonal protective groups: Trt for Cys 176 and 182 on the one hand and acetamidomethyl (Acm) for Cys 173 and 186.
- Trt for Cys 176 and 182 on the one hand
- acetamidomethyl (Acm) for Cys 173 and 186.
- 1000 mg of the 2500 mg of peptide-resin have been cleaved with a TFA / EDT / thioanisol / phenol / TIS / H 2 O mixture: 20 ml / 0.25 ml / 1 ml / 1.5 g / 0.22 ml / 1 ml.
- the mixture is filtered to remove the resin and the crude peptide is precipitated by the addition of cold diethyl ether.
- the precipitate is dissolved in a H 2 O / CH 3 CN / TFA mixture: 80/20 / 0.1: v / v / v and then lyophilized.
- the crude peptide is purified by RP-HPLC ("Reverse Phase High Performance Liquid Chromatography") using a water / acetonitrile gradient and analyzed by RP-HPLC (RP-HPLC purity>75%; yield: 38%) and ES-MS (calculated mass: 8,186.42 Da / measured mass: 8,186.40).
- the lyophilized peptide is dissolved (1 mg / ml) in a DMSO-H O mixture at 20% (v / v) and stirred at room temperature for 4 days (Tam et al., J. Am. Chem. Soc, 113, 6657, 1991).
- the peptide is purified by RP-HPLC under the same conditions as the reduced peptide. The fractions corresponding to the main peak are collected and lyophilized.
- the peptide is dissolved (1 mg / ml) in an acetic acid mixture / 80% water (v / v) and 10% 1 N HCl are added, the solution is saturated with nitrogen, then 10 equivalents of iodine dissolved in a mixture of acetic acid / 80% water (v / v) are added quickly and the medium is stirred for 5 hours at room temperature The excess iodine is reduced by the dropwise addition of an aqueous solution of ascorbic acid until the characteristic color of the iodine disappears.
- the crude oxidized peptide is purified by RP-HPLC, lyophilized and analyzed by RP-HPLC and ES-MS. Formation of disulphide bridges in 1 step.
- the pairing of the disulfide bridges is studied by LC-MS ("Liquid Chromatography Mass Spectrometry") and by microsequencing of the fragments obtained following a cleavage of the peptide with thermolysin.
- the fragments obtained and their interpretation are described in Table 1 below.
- the protocol used makes it possible to obtain only the native form G20a (1-4 / 2-3).
- the rats are challenged with 10 5 pfu VRS-A / 50 ⁇ l intranasally. 5 days after challenge, they are euthanized and the lungs recovered to assess the residual viral load.
- the anti-G2Na and VRS-A antibody titers are analyzed.
- the carrier effect is clearly demonstrated: the G20a antigen does not make it possible to obtain an immune response as strong as that obtained using the DTa or DTaDTb carriers.
- the G20 antigen does not make it possible to obtain an immune response as strong as that obtained using the DTa or DTaDTb carriers.
- the death of the guinea pigs is then evaluated.
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Abstract
Description
NOUVEAUX DERIVES D'ANATOXINES DIPHTERIQUES ET LEUR UTILISATION COMME PORTEURNOVEL DIPHTERIC ANATOXIN DERIVATIVES AND THEIR USE AS CARRIERS
L'invention concerne de nouveaux dérivés d'anatoxines diphtériques et une composition pharmaceutique comprenant dans un milieu pharmaceutiquement acceptable de tels dérivés, tout comme l'utilisation de ces dérivés pour la préparation d'un vaccin destiné au traitement prophylactique ou thérapeutique des infections virales, bactériennes, parasitaires ou fongiques ou pour la préparation d'un vaccin destiné au traitement prophylactique ou thérapeutique des cancers. La vaccination est un moyen efficace de prévenir ou de réduire les infections virales ou bactériennes. Le succès des campagnes de vaccination dans ces domaines a permis d'étendre le concept de vaccin jusqu'alors utilisé dans le domaine de l'infectiologie aux domaines du cancer et des maladies auto-immunes.The invention relates to new derivatives of diphtheria toxoids and a pharmaceutical composition comprising in a pharmaceutically acceptable medium such derivatives, as well as the use of these derivatives for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of viral infections, bacterial, parasitic or fungal or for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of cancers. Vaccination is an effective way to prevent or reduce viral or bacterial infections. The success of vaccination campaigns in these fields has made it possible to extend the concept of vaccine previously used in the field of infectious diseases to the fields of cancer and autoimmune diseases.
La mise au point de vaccins parfaitement définis et dépourvus d'effets secondaires marqués, nécessite l'emploi d'antigènes vaccinants de faible masse moléculaire, tels que des peptides ou des oligosaccharides. Ces antigènes de faible masse, mais aussi certains antigènes de masse moléculaire supérieure tels que les polysaccharides de la paroi bactérienne, ne peuvent induire seuls une réponse immunitaire durable et intense. Il est indispensable de lier ces antigènes, par voie chimique ou par génie génétique, à des protéines porteuses.The development of perfectly defined vaccines with no marked side effects requires the use of low molecular weight vaccinating antigens, such as peptides or oligosaccharides. These low mass antigens, but also certain higher molecular mass antigens such as the polysaccharides of the bacterial wall, cannot alone induce a lasting and intense immune response. It is essential to bind these antigens, chemically or by genetic engineering, to carrier proteins.
Comme protéines porteuses, on peut notamment citer les extraits de protéines membranaires bactériennes telles que les OMPC de Neisseria meningitidis (Vella et al.,As carrier proteins, mention may in particular be made of extracts of bacterial membrane proteins such as the OMPCs of Neisseria meningitidis (Vella et al.,
Infect. Immun., 60:4977-4983, 1992), TraT d'Escherichia coli (Croft et al., J. Immunol.,Infect. Immun., 60: 4977-4983, 1992), TraT of Escherichia coli (Croft et al., J. Immunol.,
146:793-798, 1991) ou PorB de Neisseria meningitidis (Fusco et al., J. Infect. Dis., 175:364-372, 1997).146: 793-798, 1991) or PorB of Neisseria meningitidis (Fusco et al., J. Infect. Dis., 175: 364-372, 1997).
Sont également utilisés comme porteurs, les anatoxines tétanique et diphtérique ; l'anatoxine tétanique (Rauly et al., Infect. Immun., 67:5547-5551, 1999), quant à elle, est une protéine utilisée dans les vaccins à usage humain.Also used as carriers are tetanus and diphtheria toxoids; tetanus toxoid (Rauly et al., Infect. Immun., 67: 5547-5551, 1999), for its part, is a protein used in vaccines for human use.
L'anatoxine au sens strict du terme est un dérivé atoxique de la toxine diphtérique obtenue après chauffage et traitement au formol. L'anatoxine est utilisée pour la vaccination antidiphtérique. Les CRM (Cross Reacting Materials) sont des protéines modifiées obtenues par mutagénèse avec du nitrosoguanidine sur le corynephage β DNA contenant le gène tox de DT (toxine diphthérique) (Uchida et al., J. Biol. Chem., 248:3838, 3845, 3851,The toxoid in the strict sense of the term is an atoxic derivative of the diphtheria toxin obtained after heating and treatment with formalin. The toxoid is used for diphtheria vaccination. CRMs (Cross Reacting Materials) are modified proteins obtained by mutagenesis with nitrosoguanidine on the corynephage β DNA containing the tox gene of DT (diphtheria toxin) (Uchida et al., J. Biol. Chem., 248: 3838, 3845 , 3851,
1973). Ces protéines sont similaires d'un point de vue antigénique à la toxine native mais ne présentent pas de toxicité.1973). These proteins are antigenically similar to the native toxin but do not exhibit toxicity.
Comme exemple de CRM, on peut citer le CRM 197 (Giannini G. et al., Nucleic Acids Res., 12:4063, 1984) qui est remarquable du fait qu'il ne présente qu'une modification d'un seul acide aminé (substitution du résidu Gly (position aa 52) avec Glu) et ne se distingue pas d'un point de vue antigénique de la toxine native. Cette unique mutation a totalement aboli le caractère toxique de la protéine. On peut également citer le CRM 45 (Giannini G.et al, Nucleic Acids Res., 12:4063, 1984) qui résulte de la délétion d'une partie C-terminale de la sous-unité B de DT, environ de 17 Kda, partie responsable de la fixation sur le récepteur de la cellule. Par conséquent, la protéine CRM 45 n'a plus la capacité de se lier à la surface de la cellule. II a été constaté par les inventeurs que la production de peptides dérivés d'anatoxines posaient de multiples problèmes, notamment lorsque le porteur est couplé à un antigène peptidique. Il a ainsi été constaté qu'il pouvait se former lors de la production des dérivés d'anatoxines, des multimères desdits dérivés. Les configurations initiales, notamment au niveau de la formation des ponts disulfures, peuvent se modifier. De même, lors de la production de protéine de fusion entre un dérivé d'anatoxine et un peptide ou protéine d'intérêt, la configuration initiale, notamment au niveau de la formation des ponts disulfures du dérivé d'anatoxine ou du peptide d'intérêt peut se modifier. De plus dans le cas de protéine de fusion entre un dérivé d'anatoxine et un peptide ou protéine d'intérêt des appariements non désirés, à savoir de nouveaux ponts disulfures, peuvent se produire.As an example of CRM, mention may be made of CRM 197 (Giannini G. et al., Nucleic Acids Res., 12: 4063, 1984) which is remarkable in that it only exhibits a modification of a single amino acid. (substitution of the residue Gly (position aa 52) with Glu) and is not distinguished from an antigenic point of view from the native toxin. This unique mutation completely abolished the toxic nature of the protein. We can also cite the CRM 45 (Giannini G. et al, Nucleic Acids Res., 12: 4063, 1984) which results from the deletion of a C-terminal part of the B subunit of DT, around 17 Kda , part responsible for fixation on the cell receptor. As a result, the CRM 45 protein no longer has the ability to bind to the surface of the cell. It has been found by the inventors that the production of peptides derived from toxoids poses multiple problems, in particular when the carrier is coupled to a peptide antigen. It was thus noted that it could form during the production of toxoid derivatives, multimers of said derivatives. The initial configurations, in particular with regard to the formation of disulfide bridges, can change. Likewise, during the production of fusion protein between a toxoid derivative and a peptide or protein of interest, the initial configuration, in particular at the level of the formation of the disulfide bridges of the toxoid derivative or of the peptide of interest can change. In addition, in the case of fusion protein between a toxoid derivative and a peptide or protein of interest, unwanted pairings, namely new disulfide bridges, can occur.
Ainsi, les rendements obtenus ne pouvaient être considérés comme satisfaisants. L'homme de l'art recherche donc des dérivés d'anatoxine plus facile à synthétiser. Ainsi, l'objet de la présente invention est d'obtenir de nouveaux peptides dérivés d'anatoxine répondant aux problèmes ci-dessus mentionnés, faciles à produire industriellement et permettant d'obtenir une réponse immune contre tout antigène qui lui est couplé ou fusionné, le moins de risque possible d'effets secondaires immunologiques et présentant une HSI (Hypersensibilité Immédiate) négative.Thus, the yields obtained could not be considered satisfactory. Those skilled in the art are therefore looking for toxoid derivatives which are easier to synthesize. Thus, the object of the present invention is to obtain new peptides derived from toxoid which respond to the problems mentioned above, which are easy to produce industrially and which make it possible to obtain an immune response against any antigen which is coupled or fused, the least possible risk of immunological side effects and presenting a negative HSI (Immediate Hypersensitivity).
De manière surprenante, il a été mis en évidence que certains dérivés d'anatoxines diphtériques répondent à ces besoins. La présente invention a ainsi pour objet de nouveaux dérivés d'anatoxines diphtériques, ces derniers étant plus faciles à produire industriellement tout en permettant d'augmenter l'immunogénicité d'un antigène associé de manière covalente, soit conjugué de manière chimique soit fusionné.Surprisingly, it has been demonstrated that certain derivatives of diphtheria toxoids meet these needs. The present invention thus relates to new derivatives of diphtheria toxoids, the latter being easier to produce industrially while making it possible to increase the immunogenicity of a covalently associated antigen, either chemically conjugated or fused.
Par "anatoxines diphtériques", on entend désigner en particulier tout peptide de séquence d'acides aminés compris dans la séquence d'acides aminés de l'anatoxine diphtérique qui, lorsqu'il est associé à un antigène ou haptène spécifique d'un agent infectieux ou d'une cellule tumorale, est capable de générer ou accroître une réponse immunitaire dirigée contre ledit agent infectieux ou ladite cellule tumorale. Par "anatoxines diphtériques", on entend également désigner toute protéine génétiquement modifiée similaire d'un point de vue antigénique à la toxine native mais qui ne présente pas de toxicité. Ainsi, au sens de la présente invention, le terme "anatoxines diphtériques" comprend également les CRM tels que le CRM 197 et le CRM 45, et notamment ceux décrits dans Uchida et al., J. Biol. Chem., 248:3838, 3845, 3851, 1973 et Giannini G. et al., Nucleic Acids Res., 12:4063, 1984. L'invention concerne ainsi un peptide dérivé d'anatoxine diphtérique de séquence comprenant au moins un résidu cysteine, caractérisé en ce que ledit peptide présente une séquence identique à la séquence de ladite anatoxine diphtérique et comprenant en outre une délétion d'au moins un résidu cysteine.By “diphtheria toxoids” is meant in particular any peptide of amino acid sequence included in the amino acid sequence of diphtheria toxoid which, when associated with an antigen or hapten specific for an infectious agent or of a tumor cell, is capable of generating or increasing an immune response directed against said infectious agent or said tumor cell. The term “diphtheria toxoids” is also intended to denote any genetically modified protein similar from an antigenic point of view to the native toxin but which does not exhibit any toxicity. Thus, within the meaning of the present invention, the term "diphtheria toxoids" also includes CRMs such as CRM 197 and CRM 45, and in particular those described in Uchida et al., J. Biol. Chem., 248: 3838, 3845, 3851, 1973 and Giannini G. et al., Nucleic Acids Res., 12: 4063, 1984. The invention thus relates to a peptide derived from diphtheria toxoid of sequence comprising at least one residue cysteine, characterized in that said peptide has a sequence identical to the sequence of said diphtheria toxoid and further comprising a deletion of at least one cysteine residue.
De tels nouveaux dérivés selon l'invention sont d'autant plus surprenants sachant que l'art antérieur enseignait à l'homme du métier de rajouter des résidus cystéines. Ainsi, le brevet WO 87/02987 préconise de rajouter un codon cysteine en région C- terminale de l'ADN codant pour la toxine diphtérique.Such new derivatives according to the invention are all the more surprising given that the prior art taught those skilled in the art to add cysteine residues. Thus, patent WO 87/02987 recommends adding a cysteine codon in the C-terminal region of the DNA coding for the diphtheria toxin.
L'invention concerne également un peptide présentant après alignement optimal au moins 80 %, de préférence au moins 85 %, 90 %, 95 % et 99 % d'homologie avec un peptide dérivé d'anatoxine diphtérique selon l'invention et comprenant la délétion dudit au moins résidu cysteine du peptide dérivé d'anatoxine diphtérique. Le peptide dérivé d'anatoxine diphtérique peut être obtenu par voie recombinante.The invention also relates to a peptide having, after optimal alignment, at least 80%, preferably at least 85%, 90%, 95% and 99% homology with a peptide derived from diphtheria toxoid according to the invention and comprising the deletion said at least cysteine residue of the peptide derived from diphtheria toxoid. The peptide derived from diphtheria toxoid can be obtained recombinantly.
Les méthodes de préparation de protéines recombinantes sont aujourd'hui bien connues de l'homme de l'art et ne seront pas développées dans la présente description, on pourra néanmoins se référer à la méthode décrite dans les exemples. Parmi les cellules utilisables pour la production de ces protéines recombinantes, il faut citer bien entendu les cellules bactériennes (Olins P.O. et Lee S.C., Curr. Op. Biotechnology, 4:520-525, 1993), mais également les cellules de levure (Buckholz R.G., Curr. Op. Biotechnology, 4:538-542, 1993), de même que les cellules animales, en particulier les cultures de cellules de mammifère (Edwards C.P. et Aruffo A., Curr. Op. Biotechnology, 4:558-563, 1993) mais également les cellules d'insectes dans lesquelles on peut utiliser des procédés mettant en oeuvre par exemple des baculovirus (Luckow VA., Curr. Op. Biotechnology, 4:564-572, 1993).The methods for preparing recombinant proteins are today well known to those skilled in the art and will not be developed in the present description, however, reference may be made to the method described in the examples. Among the cells which can be used for the production of these recombinant proteins, mention must of course be made of bacterial cells (Olins PO and Lee SC, Curr. Op. Biotechnology, 4: 520-525, 1993), but also yeast cells (Buckholz RG, Curr. Op. Biotechnology, 4: 538-542, 1993), as well as animal cells, in particular mammalian cell cultures (Edwards CP and Aruffo A., Curr. Op. Biotechnology, 4: 558- 563, 1993) but also insect cells in which methods using, for example, baculoviruses can be used (Luckow VA., Curr. Op. Biotechnology, 4: 564-572, 1993).
De manière tout à fait préférée, l'anatoxine diphtérique est choisie parmi le dérivé atoxique de la toxine diphtérique obtenue après chauffage et traitement au formol, le CRM 197 de séquence SEQ ID N° 10 et le CRM 45 de séquence SEQ ID N° l l.Most preferably, the diphtheria toxoid is chosen from the atoxic derivative of the diphtheria toxin obtained after heating and treatment with formalin, the CRM 197 of sequence SEQ ID No. 10 and the CRM 45 of sequence SEQ ID No. 1 l.
Les cystéines sont préférentiellement délétées dans les régions N-terminales ou C-terminales, notamment dans six des protéines de fusion entre un dérivé d'anatoxine et un peptide ou protéine d'intérêt de manière à éviter des appariements non désirés, tels que de nouveaux ponts disulfures.Cysteines are preferentially deleted in the N-terminal or C-terminal regions, in particular in six of the fusion proteins between a toxoid derivative and a peptide or protein of interest so as to avoid unwanted pairings, such as new disulfide bridges.
Dans un mode de réalisation préféré de l'invention, toutes les cystéines sont délétées.In a preferred embodiment of the invention, all the cysteines are deleted.
Dans un mode de réalisation particulièrement préféré de l'invention, le peptide dérivé d'anatoxine diphtérique comprend ou a pour séquence : a) une séquence d'acides aminés choisie parmi les séquences d'acides aminés SEQ ID N° 1, SEQ ID N° 2 et SEQ ID N° 3 ; b) la séquence d'acides aminés d'une séquence présentant après alignement optimal une homologie d'au moins 80 %, de préférence 85 %, 90 %, 95 % et 99 % avec la séquence d'acides aminés de référence SEQ ID N° 1 , SEQ ID N° 2 ou SEQ ID N° 3. Le peptide de séquence SEQ ID N° 1, dénommé DTa, correspond au CRM 197 et possède en outre une délétion en C-terminal après le résidu Ala position 185, juste avant le résidu Cys 186.In a particularly preferred embodiment of the invention, the peptide derived from diphtheria toxoid comprises or has for sequence: a) an amino acid sequence chosen from the amino acid sequences SEQ ID N ° 1, SEQ ID N ° 2 and SEQ ID N ° 3; b) the amino acid sequence of a sequence having after optimal alignment a homology of at least 80%, preferably 85%, 90%, 95% and 99% with the reference amino acid sequence SEQ ID N ° 1, SEQ ID N ° 2 or SEQ ID N ° 3. The peptide of sequence SEQ ID No. 1, called DTa, corresponds to CRM 197 and also has a C-terminal deletion after the residue Ala position 185, just before the residue Cys 186.
Le peptide de séquence SEQ ID N° 2, dénommé DTb, comporte une délétion de la partie responsable du binding de DT au récepteur de la cellule, à savoir de la partie N- terminale de 8 aa (Cys en position 8), et en C-terminale après l'aa K(456).The peptide of sequence SEQ ID No. 2, called DTb, contains a deletion of the part responsible for binding of DT to the receptor of the cell, namely the N-terminal part of 8 aa (Cys in position 8), and in C-terminal after AA K (456).
Le peptide de séquence SEQ ID N° 3, dénommé DTaDTb est un conjugué de DTa et DTb.The peptide of sequence SEQ ID No. 3, called DTaDTb is a conjugate of DTa and DTb.
Par séquence d'acide nucléique ou d'acides aminés présentant une homologie d'au moins 80 % après alignement optimal avec une séquence d'acide nucléique ou d'acides aminés déterminée, on entend désigner une séquence qui après alignement optimal avec ladite séquence déterminée comprend un pourcentage d'identité d'au moins 80 % avec ladite séquence déterminée.By nucleic acid or amino acid sequence having a homology of at least 80% after optimal alignment with a determined nucleic acid or amino acid sequence, is meant a sequence which after optimal alignment with said determined sequence includes a percentage identity of at least 80% with said determined sequence.
Par «pourcentage d'identité» entre deux séquences d'acide nucléique ou d'acides aminés au sens de la présente invention, on entend désigner un pourcentage de nucléotides ou de résidus d'acides aminés identiques entre les deux séquences à comparer, obtenu après le meilleur alignement, ce pourcentage étant purement statistique et les différences entre les deux séquences étant réparties au hasard et sur toute leur longueur. Les comparaisons de séquences entre deux séquences d'acide nucléique ou d'acides aminés sont traditionnellement réalisées en comparant ces séquences après les avoir alignées de manière optimale, ladite comparaison étant réalisée par segment ou par «fenêtre de comparaison» pour identifier et comparer les régions locales de similarité de séquence. L'alignement optimal des séquences pour la comparaison peut être réalisé, outre manuellement, au moyen de l'algorithme d'homologie locale de Smith et Waterman (1981) [Ad. App. Math., 2:482], au moyen de l'algorithme d'homologie locale de Neddleman et Wunsch (1970) [J. Mol. Biol., 48:443], au moyen de la méthode de recherche de similarité de Pearson et Lipman (1988) [Proc. Natl. Acad. Sci., USA, 85:2444], au moyen de logiciels informatiques utilisant ces algorithmes (GAP, BESTFIT, FASTA et TFASTA dans le Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, ou encore par les logiciels de comparaison BLAST N ou BLAST P). Le pourcentage d'identité entre deux séquences d'acide nucléique ou d'acides aminés est déterminé en comparant ces deux séquences alignées de manière optimale par fenêtre de comparaison dans laquelle la région de la séquence d'acide nucléique ou d'acides aminés à comparer peut comprendre des additions ou des délétions par rapport à la séquence de référence pour un alignement optimal entre ces deux séquences. Le pourcentage d'identité est calculé en déterminant le nombre de positions identiques pour lesquelles le nucléotide ou le résidu d'acide aminé est identique entre les deux séquences, en divisant ce nombre de positions identiques par le nombre total de positions dans la fenêtre de comparaison et en multipliant le résultat obtenu par 100 pour obtenir le pourcentage d'identité entre ces deux séquences.By “percentage of identity” between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window" to identify and compare the regions. sequence similarity locale. The optimal alignment of the sequences for comparison can be achieved, besides manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math., 2: 482], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol., 48: 443], using the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci., USA, 85: 2444], using computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by BLAST N or BLAST P comparison software). The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences per comparison window in which the region of the nucleic acid or amino acid sequence to be compared. may include additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window. and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.
Par exemple, on pourra utiliser le programme BLAST, «BLAST 2 séquences», disponible sur le site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, les paramètres utilisés étant ceux donnés par défaut (en particulier pour les paramètres «open gap penaltie» : 5, et «extension gap penaltie» : 2 ; la matrice choisie étant par exemple la matrice «BLOSUM 62» proposée par le programme), le pourcentage d'identité entre les deux séquences à comparer étant calculé directement par le programme.For example, we could use the BLAST program, "BLAST 2 sequences", available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used being those given by default (in particular for the parameters “open gap penaltie”: 5, and “extension gap penaltie”: 2; the chosen matrix being for example the “BLOSUM 62” matrix proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.
Parmi lesdites séquences présentant une homologie d'au moins 80 % avec la séquence de référence, on préfère les séquences de, ou codant pour des, peptides capables d'induire une réponse immunitaire dirigée spécifiquement contre l'antigène ou haptène qui lui est associée, telle que l'induction d'une réponse immunitaire mesurée au moyen des techniques standard décrites dans les exemples ci-après.Among said sequences having at least 80% homology with the reference sequence, the sequences of, or coding for, peptides capable of inducing an immune response directed specifically against the antigen or hapten associated with it are preferred, such as the induction of an immune response measured using standard techniques described in the examples below.
La présente invention concerne encore un acide nucléique codant pour un dérivé d'anatoxines diphtériques selon l'invention, et préférentiellement pour un dérivé d'anatoxines diphtériques de SEQ ID N° 1, SEQ ID N° 2 ou SEQ ID N° 3. L'invention a encore pour objet une composition pharmaceutique caractérisée en ce qu'elle comprend dans un milieu pharmaceutiquement acceptable au moins un peptide dérivé d'anatoxines diphtériques selon l'invention ou un acide nucléique codant pour ledit peptide.The present invention also relates to a nucleic acid coding for a derivative of diphtheria toxoids according to the invention, and preferably for a derivative of diphtheria toxoids of SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. L A further subject of the invention is a pharmaceutical composition characterized in that it comprises, in a pharmaceutically acceptable medium, at least one peptide derived from diphtheria toxoids according to the invention or a nucleic acid coding for said peptide.
La présente invention concerne aussi une composition pharmaceutique caractérisée en ce qu'elle comprend dans un milieu pharmaceutiquement acceptable au moins une cellule hôte transformée capable d'exprimer ledit peptide dérivé d'anatoxines diphtériques selon l'invention.The present invention also relates to a pharmaceutical composition characterized in that it comprises in a medium which is pharmaceutically acceptable to at least one transformed host cell capable of expressing said peptide derived from diphtheria toxoids according to the invention.
L'invention a également pour objet la composition selon l'invention, caractérisée en ce que ladite composition pharmaceutique comprend en outre, un antigène, immunogène ou haptène.A subject of the invention is also the composition according to the invention, characterized in that said pharmaceutical composition further comprises an antigen, immunogen or hapten.
Par « immunogène, antigène ou haptène spécifique d'un agent infectieux ou d'une cellule tumorale », on entend désigner en particulier tout composé exprimé par un agent infectieux, tel qu'un virus, une bactérie, une levure, un champignon ou un parasite, par une cellule tumorale, ou un de leurs analogues structuraux, qui seul ou en association avec un adjuvant ou porteur est capable d'induire une réponse immunitaire spécifique dudit agent infectieux ou de ladite cellule tumorale.By “immunogen, antigen or hapten specific for an infectious agent or a tumor cell” is intended to denote in particular any compound expressed by an infectious agent, such as a virus, a bacterium, a yeast, a fungus or a parasite, by a tumor cell, or one of their structural analogues, which alone or in combination with an adjuvant or carrier is capable of inducing a specific immune response of said infectious agent or of said tumor cell.
On entend également désigner par "immunogène, antigène ou haptène" dans la présente description un composé présentant une analogie structurale avec ledit antigène ou haptène capable d'induire une réponse immunologique dirigée contre ledit antigène ou haptène dans un organisme préalablement immunisé avec ledit composé analogue.The term “immunogen, antigen or hapten” is also intended to denote in the present description a compound having a structural analogy with said antigen or hapten capable of inducing an immunological response directed against said antigen or hapten in an organism previously immunized with said analogous compound.
Ledit antigène ou haptène peut notamment être choisi parmi les protéines, les glycopeptides, les lipopeptides, les polysaccharides, les oligosaccharides, les acides nucléiques et les lipides.Said antigen or hapten can in particular be chosen from proteins, glycopeptides, lipopeptides, polysaccharides, oligosaccharides, nucleic acids and lipids.
Dans une forme de réalisation de l'invention, ledit antigène, immunogène ou haptène dérive d'un virus, d'une bactérie, d'un parasite ou d'un champignon.In one embodiment of the invention, said antigen, immunogen or hapten is derived from a virus, a bacterium, a parasite or a fungus.
Dans un mode de réalisation préféré de l'invention, ledit antigène, immunogène ou haptène comprend au moins un peptide dérivé de micro-organisme responsable de pathologies des voies aériennes choisi parmi le VRS, le para influenza virus (PFV), l'infiuenza virus, les hantavirus, les streptocoques, les pneumocoques, haemophilus influenza type b, les rhinovirus, les coronovirus et les méningocoques.In a preferred embodiment of the invention, said antigen, immunogen or hapten comprises at least one peptide derived from a microorganism responsible for airway pathologies chosen from RSV, the para influenza virus (PFV), the infuenza virus , hantaviruses, streptococci, pneumococci, haemophilus influenzae type b, rhinoviruses, coronoviruses and meningococci.
Dans un mode de réalisation encore plus préféré de l'invention ledit antigène, immunogène ou haptène comprend au moins un fragment de la protéine G du virus respiratoire syncytial.In an even more preferred embodiment of the invention, said antigen, immunogen or hapten comprises at least one fragment of the protein G of the respiratory syncytial virus.
Dans un autre mode de réalisation préféré de l'invention ledit antigène, immunogène ou haptène est décrit dans les demandes de brevets WO 87/04185 relative à des protéines structurales du VRS, WO 89/02935 qui décrit la protéine F entière du VRS, éventuellement modifiée sous forme monomérique ou déglycosylée, WO 95/27787 qui concerne des peptides issus de la protéine G du VRS et plus particulièrement le peptide dénommé G2Na (fragment aa 130-230 de la protéine G du VRS humain de type A identifié par la séquence SEQ ID N° 1 du document WO 95/27787 appelé encore "G2A"), WO 97/46581 qui divulgue des peptides structurellement homologues à la séquence 149-197 de la protéine G et dans laquelle aucun oligosaccharide n'est lié à une serine, thréonine ou asparagine ou dans la demande WO 99/03987 qui décrit des épitopes spécifiques de la protéine G du VRS.In another preferred embodiment of the invention, said antigen, immunogen or hapten is described in patent applications WO 87/04185 relating to structural proteins of RSV, WO 89/02935 which describes the entire F protein of RSV, optionally modified in monomeric or deglycosylated form, WO 95/27787 which relates to peptides derived from the G protein of RSV and more particularly the peptide called G2Na (fragment aa 130-230 of protein G of human RSV type A identified by sequence SEQ ID No. 1 of document WO 95/27787 also called "G2A"), WO 97/46581 which discloses peptides structurally homologous to the sequence 149-197 of protein G and in which no oligosaccharide is linked to a serine, threonine or asparagine or in application WO 99/03987 which describes specific epitopes of the G protein of RSV.
Dans un autre mode de réalisation préféré de l'invention ledit antigène correspond à un peptide immunogène dérivé de la protéine G du VRS du sous-groupe A ou B comprenant au moins :In another preferred embodiment of the invention, said antigen corresponds to an immunogenic peptide derived from RSV protein G of subgroup A or B comprising at least:
- un premier peptide dérivé de la protéine G du VRS du sous-groupe A ou B comprenant au moins en position 173, 176, 182 et 186 une cysteine, et dont l'extrémité C-terminale comprend au plus l'acide aminé en position 192 ; et - un deuxième peptide dérivé d'une protéine du VRS du sous-groupe A ou B, ledit deuxième peptide étant situé en aval dudit premier peptide, de manière à ce que le peptide immunogène produit présente un pont disulfure reliant les résidus 173 et 186 et un deuxième pont disulfure reliant les résidus 176 et 182.a first peptide derived from the G protein of the RSV of the subgroup A or B comprising at least in position 173, 176, 182 and 186 a cysteine, and the C-terminal end of which comprises at most the amino acid in position 192; and a second peptide derived from an RSV protein from subgroup A or B, said second peptide being located downstream of said first peptide, so that the immunogenic peptide produced has a disulfide bridge connecting residues 173 and 186 and a second disulfide bridge connecting residues 176 and 182.
Par peptide "comprenant au moins en position 173, 176, 182 et 186 une cysteine, et dont l'extrémité C-terminale comprend au plus l'acide aminé en position 192", on entend désigner tout peptide présentant au moins 4 cystéines dans la même configuration que la protéine G native. Les numéros de position font référence à la protéine native et ne signifient pas que le premier peptide selon l'invention comprend forcément tous les 192 premiers acides aminés de la protéine G native, mais que ce peptide est un peptide de séquence n-m, avec n = 1-172 et m = 187-192.By peptide "comprising at least in position 173, 176, 182 and 186 a cysteine, and the C-terminal end of which comprises at most the amino acid in position 192", is intended to denote any peptide having at least 4 cysteines in the same configuration as native G protein. The position numbers refer to the native protein and do not mean that the first peptide according to the invention necessarily includes all the first 192 amino acids of the native protein G, but that this peptide is a peptide of sequence nm, with n = 1-172 and m = 187-192.
Dans un mode de réalisation préféré de l'invention, ledit premier peptide présente une séquence choisie parmi la séquence de la protéine G du VRS de sous groupe A ou B 130-190, 130-192, 140-190, 140-192, 145-190, 145-192, 148-190, 148-192, 130-188, 140-188, 145-188 ou 148-188, et préférentiellement la séquence 140-190. Dans un autre mode de réalisation préféré de l'invention, ledit deuxième peptide présente une séquence choisie parmi la séquence 144-158, 144-159 de la protéine G du VRS. On préfère dans le cadre de la présente invention tout particulièrement l'antigène dénommé G20a correspondant à la séquence 140-190 de la protéine G du VRS, sous groupe A, couplée à la séquence 144-158 de la protéine G du VRS, sous groupe A.In a preferred embodiment of the invention, said first peptide has a sequence chosen from the sequence of protein G of RSV of subgroup A or B 130-190, 130-192, 140-190, 140-192, 145 -190, 145-192, 148-190, 148-192, 130-188, 140-188, 145-188 or 148-188, and preferably the sequence 140-190. In another preferred embodiment of the invention, said second peptide has a sequence chosen from the sequence 144-158, 144-159 of protein G of RSV. We prefer in the framework of the present invention, in particular the antigen called G20a corresponding to the sequence 140-190 of the GRS protein of RSV, in group A, coupled to the sequence 144-158 of protein G of RSV, in group A.
L'antigène, immunogène ou haptène peut également être associé ou spécifique d'une cellule tumorale.The antigen, immunogen or hapten can also be associated or specific for a tumor cell.
Parmi les cancers dont les tumeurs expriment un antigène tumoral associé pouvant être prévenus ou traités par les utilisations selon la présente invention, on peut citer en particulier, mais sans s'y limiter :Among the cancers whose tumors express an associated tumor antigen which can be prevented or treated by the uses according to the present invention, there may be mentioned in particular, but not limited to:
• le cancer du sein, du poumon, du côlon, et le carcinome gastrique (Kawashima et al., Cancer Res., 59:431-5, 1999) ;• breast, lung, colon cancer and gastric carcinoma (Kawashima et al., Cancer Res., 59: 431-5, 1999);
• le mésothéliome, l'ostéosarcome, les cancers du cerveau (Xie et al., J. Natl. Cancer. Inst., 91:169-75, 1999) ;• mesothelioma, osteosarcoma, brain cancer (Xie et al., J. Natl. Cancer. Inst., 91: 169-75, 1999);
• le mélanome (Zheuten et al., Bratilsl. Lek. Listy, 99:426-34, 1998) ;• melanoma (Zheuten et al., Bratilsl. Lek. Listy, 99: 426-34, 1998);
• l'adénome cystique du pancréas (Hammel et al., Eur. J. gastroenterol. Hepatol., 10:345-8, 1998) ;• cystic adenoma of the pancreas (Hammel et al., Eur. J. gastroenterol. Hepatol., 10: 345-8, 1998);
• le cancer colorectal (Ogura et al., Anticancer Res., 18:3669-75, 1998) ;• colorectal cancer (Ogura et al., Anticancer Res., 18: 3669-75, 1998);
• le carcinome des cellules rénales (Jantzer et al., Cancer Res., 58:3078-86, 1998) ; et• renal cell carcinoma (Jantzer et al., Cancer Res., 58: 3078-86, 1998); and
• le cancer de l'ovaire et du cervix (Sonoda et al., Cancer, 77:1501-9, 1996).• ovarian and cervical cancer (Sonoda et al., Cancer, 77: 1501-9, 1996).
La présente invention a aussi pour objet une composition selon l'invention, caractérisée en ce que ledit antigène, immunogène ou haptène est couplé ou mélangé au peptide dérivé d'anatoxines diphtériques selon l'invention.The present invention also relates to a composition according to the invention, characterized in that said antigen, immunogen or hapten is coupled or mixed with the peptide derived from diphtheria toxoids according to the invention.
Ainsi, le dérivé d'anatoxine diphtérique selon l'invention peut être couplé par liaison covalente, notamment par couplage chimique, avec l'immunogène, antigène ou haptène spécifique d'un agent infectieux ou d'une cellule tumorale. Dans un mode de réalisation particulier de l'invention, il est introduit un ou plusieurs éléments de liaison dans le dérivé d'anatoxines diphtériques selon l'invention et/ou dans ledit antigène ou haptène pour faciliter le couplage chimique, de préférence, ledit élément de liaison introduit est un acide aminé.Thus, the diphtheria toxoid derivative according to the invention can be coupled by covalent bond, in particular by chemical coupling, with the immunogen, antigen or hapten specific for an infectious agent or a tumor cell. In a particular embodiment of the invention, one or more linking elements are introduced into the diphtheria toxoid derivative according to the invention and / or into said antigen or hapten to facilitate chemical coupling, preferably, said element bonding agent is an amino acid.
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 peptide dérivé d'anatoxines diphtériques selon l'invention et ledit immunogène, antigène ou haptène. Le couplage covalent entre le peptide dérivé d'anatoxines diphtériques selon l'invention et ledit immunogène, antigène ou haptène selon l'invention peut être réalisé à l'extrémité N- ou C-terminale dudit peptide. Les réactifs bifonctionnels permettant ce couplage seront déterminés en fonction de l'extrémité dudit peptide choisie pour effectuer le couplage et de la nature dudit immunogène, antigène ou haptène à coupler. Dans un autre mode de réalisation particulièrement préféré, le couplage entre ledit immunogène, antigène ou haptène et ledit peptide dérivé d'anatoxines diphtériques selon l'invention est réalisé par recombinaison génétique, lorsque ledit immunogène, antigène ou haptène est de nature peptidique. Les conjugués issus d'un couplage entre l'immunogène, antigène ou haptène et ledit peptide dérivé d'anatoxines diphtériques selon l'invention, peuvent être préparés par recombinaison génétique. La protéine chimérique ou hybride (le conjugué) peut être produite par des techniques d'ADN recombinant par insertion ou addition à la séquence d'ADN codant pour ledit peptide dérivé d'anatoxines diphtériques selon l'invention, d'une séquence codant pour ledit immunogène, antigène ou haptène de nature protéique.According to the invention, it is possible to introduce one or more binding elements, in particular amino acids to facilitate the coupling reactions between the peptide derived from diphtheria toxoids according to the invention and said immunogen, antigen or hapten. The covalent coupling between the peptide derived from diphtheria toxoids according to the invention and said immunogen, antigen or hapten according to the invention can be carried out at the N- or C-terminal end of said peptide. The bifunctional reagents allowing this coupling will be determined according to the end of said peptide chosen to effect the coupling and the nature of said immunogen, antigen or hapten to be coupled. In another particularly preferred embodiment, the coupling between said immunogen, antigen or hapten and said peptide derived from diphtheria toxoids according to the invention is carried out by genetic recombination, when said immunogen, antigen or hapten is of peptide nature. The conjugates resulting from a coupling between the immunogen, antigen or hapten and said peptide derived from diphtheria toxoids according to the invention, can be prepared by genetic recombination. The chimeric or hybrid protein (the conjugate) can be produced by recombinant DNA techniques by insertion or addition to the DNA sequence coding for said peptide derived from diphtheria toxoids according to the invention, of a sequence coding for said protein-based immunogen, antigen or hapten.
Les procédés de synthèse des molécules hybrides englobent les méthodes utilisées en génie génétique pour construire des polynucléotides hybrides codant pour les séquences polypeptidiques recherchées. On pourra, par exemple, se référer avantageusement à la technique d'obtention de gènes codant pour des protéines de fusion décrite par D.V. Goeddel (Gène expression technology, Methods in Enzymology, vol. 185, 3-187, 1990).The methods for synthesizing hybrid molecules include the methods used in genetic engineering to construct hybrid polynucleotides encoding the desired polypeptide sequences. One can, for example, advantageously refer to the technique for obtaining genes coding for fusion proteins described by D.V. Goeddel (Gene expression technology, Methods in Enzymology, vol. 185, 3-187, 1990).
L'invention comprend également un tel conjugué entre un dérivé d'anatoxine diphtérique selon l'invention et un immunogène, antigène ou haptène spécifique d'un agent infectieux ou d'une cellule tumorale. Comme exemple de tels conjugués, on peut citer les peptides de séquences SEQThe invention also includes such a conjugate between a diphtheria toxoid derivative according to the invention and an immunogen, antigen or hapten specific for an infectious agent or a tumor cell. As an example of such conjugates, mention may be made of peptides of sequences SEQ
ID N° 4 à SEQ ID N° 9 correspondant respectivement aux conjugués G2Na et DTa, G2Na et DTb, et G2Na et DTaDTb et aux conjugués G20a et DTa, G20a et DTb, et G20a et DTaDTb.ID No. 4 to SEQ ID No. 9 corresponding respectively to the conjugates G2Na and DTa, G2Na and DTb, and G2Na and DTaDTb and to the conjugates G20a and DTa, G20a and DTb, and G20a and DTaDTb.
La présente invention concerne aussi une composition pharmaceutique qui comprend une construction nucléique codant pour ledit conjugué, ou qui comprend un vecteur contenant une construction nucléique codant pour ledit conjugué ou une cellule hôte transformée contenant ladite construction nucléique capable d'exprimer ledit conjugué.The present invention also relates to a pharmaceutical composition which comprises a nucleic construct coding for said conjugate, or which comprises a vector containing a nucleic construct coding for said conjugate or a cell transformed host containing said nucleic construct capable of expressing said conjugate.
Les compositions selon l'invention peuvent contenir en outre un adjuvant. Ce dernier peut notamment être choisi parmi le MPL-A (« MonoPhosphoryl Lipid A »), le MF-59, le Quil-A (adjuvant dérivé de saponine), l'ISCOM (« ImmunoStimulatingThe compositions according to the invention may also contain an adjuvant. The latter can in particular be chosen from MPL-A (“MonoPhosphoryl Lipid A”), MF-59, Quil-A (adjuvant derived from saponin), ISCOM (“ImmunoStimulating
COMplex »), le Diméthyl Dioctadécyl Ammonium sous forme de bromure (DDAB) ou de chlorure (DDAC), les CpG (oligodésoxynucléotides contenant un motif spécifique centré sur un dinucléotide CpG), la Leif (antigène protéique dérivé de Leishmania capable de stimuler les cellules PBMC et présentatrices d'antigène, et de produire une réaction cytokine de type Th-1), la CT (Toxine Cholérique), la LT (« heat Labil Toxin ») et les versions détoxifiées de la CT ou la LT.COMplex ”), Dimethyl Dioctadecyl Ammonium in the form of bromide (DDAB) or chloride (DDAC), CpG (oligodeoxynucleotides containing a specific motif centered on a dinucleotide CpG), Leif (protein antigen derived from Leishmania capable of stimulating cells PBMC and antigen presenting, and produce a cytokine reaction type Th-1), CT (Cholera Toxin), LT ("heat Labil Toxin") and detoxified versions of CT or LT.
Au sens de la présente invention, le milieu pharmaceutiquement acceptable est le milieu dans lequel les composés de l'invention sont administrés, préférentiellement un milieu injectable chez l'homme. Il peut être constitué d'eau, d'une solution aqueuse saline ou d'une solution aqueuse à base de dextrose et ou de glycérol.Within the meaning of the present invention, the pharmaceutically acceptable medium is the medium in which the compounds of the invention are administered, preferably a medium injectable into humans. It can consist of water, an aqueous saline solution or an aqueous solution based on dextrose and or glycerol.
L'invention comprend également une composition selon l'invention, caractérisée en ce que ladite composition pharmaceutique est véhiculée sous une forme permettant d'améliorer sa stabilité et/ou son immunogénicité ; ainsi, elle peut être véhiculée sous forme de liposomes, virosomes, nanosphères, microsphères ou microcapsules. L'utilisation d'un peptide dérivé d'anatoxines diphtériques selon l'invention tel que défini ci-dessus comme porteur dans la fabrication d'un vaccin constitue un autre objet de l'invention.The invention also comprises a composition according to the invention, characterized in that said pharmaceutical composition is conveyed in a form making it possible to improve its stability and / or its immunogenicity; thus, it can be conveyed in the form of liposomes, virosomes, nanospheres, microspheres or microcapsules. The use of a peptide derived from diphtheria toxoids according to the invention as defined above as a carrier in the manufacture of a vaccine constitutes another object of the invention.
L'invention concerne encore l'utilisation d'un peptide dérivé d'anatoxines diphtériques selon l'invention tel que défini ci-dessus pour la préparation d'un vaccin destiné au traitement prophylactique ou thérapeutique des infections virales, bactériennes, parasitaires ou fongiques ou pour la préparation d'un vaccin destiné au traitement prophylactique ou thérapeutique des cancers.The invention also relates to the use of a peptide derived from diphtheria toxoids according to the invention as defined above for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of viral, bacterial, parasitic or fungal infections or for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of cancers.
L'invention a encore pour objet l'utilisation d'un peptide dérivé d'anatoxines diphtériques selon l'invention tel que défini ci-dessus pour la préparation d'une composition pharmaceutique destinée à générer ou accroître une réponse immunitaire contre un agent infectieux ou une cellule tumorale. Les légendes des figures et exemples qui suivent sont destinés à illustrer l'invention sans aucunement en limiter la portée. Légendes des figures :The invention also relates to the use of a peptide derived from diphtheria toxoids according to the invention as defined above for the preparation of a pharmaceutical composition intended to generate or increase an immune response against an infectious agent or a tumor cell. The legends of the figures and examples which follow are intended to illustrate the invention without in any way limiting its scope. Legends of the figures:
Figures 1A et 1B : Titres anti-G2Na et VRS-A après une ou deux immunisations chez des rats de cotton immunisés avec différents immunogènes.Figures 1A and 1B: Anti-G2Na and VRS-A titers after one or two immunizations in cotton rats immunized with different immunogens.
Figure 2 : Protection contre un challenge avec VRS-A chez le rat de cotton immunisé par différentes molécules.Figure 2: Protection against a challenge with VRS-A in cotton rats immunized with different molecules.
Exemple 1 : Clonage de dérivés d'anatoxines diphtériques A titre d'exemple, le gène codant pour DTa (aal - aal85 de CRM 197), obtenu par PCR directement à partir du génome de Corynebacterium Diphtheria (souche CRM 197), a été clone dans un vecteur d'expression dont le promoteur est basé sur l'opéron Tryptophan (Trp). Il en résulte le vecteur nommé pTEXDTa dont l'ADN de l'insert a été vérifié par séquençage ADN. Le vecteur a été transformé dans une bactérie Escherishia coli Kl 2 nommée ICONE®. La même procédure de clonage a été effectuée pour le clonage de DTb (aa202 - aa456 de CRM 197). La protéine de fusion DTaDTb résulte de la fusion des gènes codant pour DTa et DTb. De même, la protéine G20aDTa résulte de la fusion des gènes codant pour G20a et DTa. L'exemple suivante illustre la production et la purification de G20aDTa. Exemple 2 : Expression du dérivé d'anatoxines diphtériques chez Escherichia coli Dans un fermenteur de 30 1 (CHEMAP CMF400) contenant 18 1 de milieu minimum de culture (g/1) (KH2PO4, 6 / K2HPO4, 4 / Na3 citrate 2H2O, 9 / Extrait de levure 1 / (NH4)2SO4, 5 / CaCl2, 0,3 / MgSO4,7H2O, 2 / Glycérol 100), les Oligoéléments (1 ml/1) et de l' antimousse Struktol (0,4 ml/1) supplémentés par une solution de Tétracycline et du Tryptophane à concentration finale respectivement de 0,008 g/1 et de 0,3 g/1, on inocule 1 400 ml du même milieu issu d'une préculture de E. coli recombinante ayant le vecteur d'expression de G20aDTa dans un fermenteur de 2 litres. En culture type batch, les paramètres physico-chimiques suivants sont maintenus constants : Température à 37°C, pH à 7 régulé avec NH OH, Agitation 500 - 1000 tr/mn pour maintenir le taux d'O2 dissous à 30 %. Lorsque la densité optique (DO 620 nm) du milieu de culture atteint la valeur de 50, il est possible d'induire l'expression de la protéine recombinante en ajoutant 2 ml/litre de culture d'une solution d'acide 3 indolacrylique (IAA) à 12,5 g/1. Quelques heures après, la fermentation est arrêtée par refroidissement à 4°C après épuisement de substrat carboné (mesuré par un dosage enzymatique du glycérol dans le milieu de culture). La biomasse bactérienne est obtenue par centrifugation continue du milieu (14000 tr/mn, débit 100 1 h). Le rendement en biomasse est d'environ 40 g de cellules sèches/1. Exemple 3 : Extraction et purification du dérivé G20aDTaExample 1: Cloning of diphtheria toxoid derivatives For example, the gene coding for DTa (aal - aal85 of CRM 197), obtained by PCR directly from the genome of Corynebacterium Diphtheria (strain CRM 197), was cloned in an expression vector whose promoter is based on the Tryptophan operon (Trp). This results in the vector named pTEXDTa, the DNA of the insert of which has been verified by DNA sequencing. The vector was transformed into an Escherishia coli Kl 2 bacterium called ICONE®. The same cloning procedure was performed for the cloning of DTb (aa202 - aa456 of CRM 197). The fusion protein DTaDTb results from the fusion of the genes coding for DTa and DTb. Similarly, the G20aDTa protein results from the fusion of the genes coding for G20a and DTa. The following example illustrates the production and purification of G20aDTa. Example 2: Expression of the diphtheria toxoid derivative in Escherichia coli In a 30 L fermenter (CHEMAP CMF400) containing 18 L of minimum culture medium (g / 1) (KH 2 PO 4 , 6 / K 2 HPO 4 , 4 / Na 3 citrate 2H 2 O, 9 / Yeast extract 1 / (NH4) 2 SO 4 , 5 / CaCl 2 , 0.3 / MgSO 4 , 7H 2 O, 2 / Glycerol 100), Trace elements (1 ml / 1) and Struktol antifoam (0.4 ml / 1) supplemented with a solution of Tetracycline and Tryptophan at a final concentration of 0.008 g / 1 and 0.3 g / 1 respectively, 1,400 ml of the same are inoculated medium derived from a recombinant E. coli preculture having the expression vector of G20aDTa in a 2 liter fermenter. In batch-type culture, the following physicochemical parameters are kept constant: Temperature at 37 ° C, pH at 7 regulated with NH OH, Agitation 500 - 1000 rpm to maintain the level of dissolved O 2 at 30%. When the optical density (OD 620 nm) of the culture medium reaches the value of 50, it is possible to induce the expression of the recombinant protein by adding 2 ml / liter of culture of a solution of indolacrylic acid (IAA) at 12.5 g / 1. A few hours later, the fermentation is stopped by cooling to 4 ° C. after exhaustion of carbonaceous substrate (measured by an enzymatic assay of glycerol in the culture medium). The bacterial biomass is obtained by continuous centrifugation of the medium (14,000 rpm, flow rate 100 1 h). The biomass yield is approximately 40 g of dry cells / 1. EXAMPLE 3 Extraction and Purification of the G20aDTa Derivative
La biomasse (environ 500 g de cellules sèches) est reprise dans 10 1 de tampon TST (Tris HC1 25 mM pH 8, MgCl2 6H2O 5 mM, EDTA 2mM). La suspension bactérienne est broyée au Manton-Gaulin (3 cycles à 560 bar). La protéine recombinante G20aDTa étant majoritairement soluble, la purification peut être réalisée directement à partir de la suspension broyée. On peut réaliser par exemple la capture de DTa par chromatographie d'échange d'ions en lit expansé (Streamline, Pharmacia). Deux ou trois étapes de chromatographie supplémentaires (échange d'ions et exclusion) sont nécessaires afin d'éliminer les contaminants ADN et protéiques de la cellule hôte. Les protéines purifiées sont analysées sur gel SDS-PAGE dans des conditions réduites, sur l'appareil MINI PROTEAN II SYSTEM (BioRads). Elles peuvent être visualisées avec du Coomassie brilliant blue R250. Exemple 4 : Couplage du peptide G20a A. Synthèse chimique et caractérisation analytique du peptide G20aThe biomass (approximately 500 g of dry cells) is taken up in 10 l of TST buffer (25 mM Tris HC1 pH 8, MgCl 2 6H 2 O 5 mM, 2 mM EDTA). The bacterial suspension is ground with Manton-Gaulin (3 cycles at 560 bar). Since the recombinant protein G20aDTa is predominantly soluble, the purification can be carried out directly from the ground suspension. It is possible, for example, to capture DTa by ion exchange chromatography in an expanded bed (Streamline, Pharmacia). Two or three additional chromatography steps (ion exchange and exclusion) are necessary in order to remove DNA and protein contaminants from the host cell. The purified proteins are analyzed on SDS-PAGE gel under reduced conditions, on the MINI PROTEAN II SYSTEM device (BioRads). They can be viewed with Coomassie brilliant blue R250. Example 4 Coupling of the G20a Peptide A. Chemical Synthesis and Analytical Characterization of the G20a Peptide
Le peptide G20a est un fragment de la protéine G du VRS-A ( 140-190)-( 144- 158) de 69 acides aminés. Il comporte 4 cystéines capables de former 2 ponts disulfures. La séquence du peptide G20a est la suivante (SEQ ID N° 12) : MEFQ14oTQPSKPTTKQRQNKPPNKPNNDFHFEVFNFVPC173SIC176SNNPTCι82WA ICι86KRIP19oSι4 KPTTKQRQNKPPNK158 Peptide G20a is a fragment of protein G of VRS-A (140-190) - (144-158) of 69 amino acids. It contains 4 cysteines capable of forming 2 disulfide bridges. The sequence of the G20a peptide is as follows (SEQ ID No. 12): MEFQ 14 oTQPSKPTTKQRQNKPPNKPNNDFHFEVFNFVPC 173 SIC 176 SNNPTCι 82 WA ICι 86 KRIP 19 oSι 4 KPTTKQRQNKPPNK 158
Le peptide G20a est obtenu par synthèse automatique en phase solide en chimie Fmoc/tBu à l'échelle de 0,25 mmole à partir d'une résine hydroxyméthyl- phénoxyméthyle (HMP) préchargée avec une Lys (Boc) (0,70 mmole/g) et des Fmoc- acides aminés protégés au niveau des chaînes latérales par les groupes suivants : trityl (Trt) pour Asn, Gin et His ; tert-butyl éther (tBu) pour Ser et Thr ; tert-butyl ester (OtBu) pour Asp et Glu ; tert-butyloxycarbonyl (Boc) pour Lys et Trp et 2,2,5,7,8- pentaméthylchromane-6 sulfonyl (Pmc) pour Arg. Les cystéines utilisées possédaient les groupes protecteurs orthogonaux suivants : Trt pour les Cys 176 et 182 d'une part et acétamidométhyl (Acm) pour les Cys 173 et 186. A la fin de la synthèse, 1000 mg des 2500 mg de peptide-résine ont été clivés par un mélange TFA/ EDT / thioanisol / phénol / TIS/ H2O : 20 ml / 0,25 ml / 1 ml / 1,5 g / 0,22 ml / 1 ml. Après 3 heures de réaction sous agitation à température ambiante, le mélange est filtré pour éliminer la résine et le peptide brut est précipité grâce à l'addition de diéthyl éther froid. Le précipité est solubilisé dans un mélange H2O / CH3CN / TFA : 80 / 20 / 0,1 : v / v / v puis lyophilisé. Avant oxydation, le peptide brut est purifié par RP-HPLC ("Reverse Phase High Performance Liquid Chromatography") à l'aide d'un gradient eau / acétonitrile et analysé par RP-HPLC (pureté RP-HPLC > 75 % ; rendement : 38 %) et ES-MS (masse calculée : 8186,42 Da/masse mesurée : 8186,40).The G20a peptide is obtained by automatic synthesis in solid phase in Fmoc / tBu chemistry on a 0.25 mmol scale from a hydroxymethylphenoxymethyl resin (HMP) preloaded with a Lys (Boc) (0.70 mmol / g) and Fmoc-amino acids protected at the side chains by the following groups: trityl (Trt) for Asn, Gln and His; tert-butyl ether (tBu) for Ser and Thr; tert-butyl ester (OtBu) for Asp and Glu; tert-butyloxycarbonyl (Boc) for Lys and Trp and 2,2,5,7,8- pentamethylchroman-6 sulfonyl (Pmc) for Arg. The cysteines used had the following orthogonal protective groups: Trt for Cys 176 and 182 on the one hand and acetamidomethyl (Acm) for Cys 173 and 186. At the end of the synthesis, 1000 mg of the 2500 mg of peptide-resin have been cleaved with a TFA / EDT / thioanisol / phenol / TIS / H 2 O mixture: 20 ml / 0.25 ml / 1 ml / 1.5 g / 0.22 ml / 1 ml. After 3 hours of reaction with stirring at room temperature, the mixture is filtered to remove the resin and the crude peptide is precipitated by the addition of cold diethyl ether. The precipitate is dissolved in a H 2 O / CH 3 CN / TFA mixture: 80/20 / 0.1: v / v / v and then lyophilized. Before oxidation, the crude peptide is purified by RP-HPLC ("Reverse Phase High Performance Liquid Chromatography") using a water / acetonitrile gradient and analyzed by RP-HPLC (RP-HPLC purity>75%; yield: 38%) and ES-MS (calculated mass: 8,186.42 Da / measured mass: 8,186.40).
Formation des ponts disulfures en 2 étapes. Pour former le pont entre les Cys 176 et 182, non protégées, le peptide lyophilisé est solubilisé (1 mg/ml) dans un mélange DMSO-H O à 20 % (v/v) et agité à température ambiante pendant 4 jours (Tam et al., J. Am. Chem. Soc, 113, 6657, 1991). En fin de réaction pour éliminer le DMSO, le peptide est purifié par RP-HPLC dans les mêmes conditions que le peptide réduit. Les fractions correspondant au pic principal sont collectées et lyophilisées. Un aliquot est soumis à une analyse par ES-MS ("ElectroSpray Mass Spectrometry" pour vérifier que le premier pont disulfure a bien été formé. Le second pont, entre les Cys(Acm) 173 et 186 est obtenu par oxydation à (Buku et al., Int. J. Peptide. Res., 33:86, 1989 et Annis et al., Meth. Enzymol., 289:198, 1997). Le peptide est solubilisé (1 mg/ml) dans un mélange acide acétique / eau à 80 % (v/v) et 10 % d'HCl 1 N sont ajoutés. La solution est saturée par de l'azote. Puis 10 équivalents d'iode solubilisé dans un mélange d'acide acétique / eau à 80 % (v/v) sont ajoutés rapidement et le milieu est agité pendant 5 heures à température ambiante. L'excès d'iode est réduit par l'addition goutte à goutte d'une solution aqueuse d'acide ascorbique jusqu'à ce que la couleur caractéristique de l'iode disparaisse. Le peptide oxydé brut est purifié par RP-HPLC, lyophilisé et analysé par RP-HPLC et ES-MS. Formation des ponts disulfures en 1 étape. Un protocole d'obtention en une étape a également par oxydation directe à l'iode sur le peptide réduit permettant également d'obtenir le peptide d'intérêt. Le rendement passe alors de 22 à 44 % (pureté RP-HPLC > 90 % ; masse calculée : 8140,22 Da / masse mesurée : 8040,30 Da).Formation of disulfide bridges in 2 stages. To form the bridge between Cys 176 and 182, unprotected, the lyophilized peptide is dissolved (1 mg / ml) in a DMSO-H O mixture at 20% (v / v) and stirred at room temperature for 4 days (Tam et al., J. Am. Chem. Soc, 113, 6657, 1991). At the end of the reaction to remove the DMSO, the peptide is purified by RP-HPLC under the same conditions as the reduced peptide. The fractions corresponding to the main peak are collected and lyophilized. An aliquot is subjected to analysis by ES-MS ("ElectroSpray Mass Spectrometry" to verify that the first disulfide bridge has indeed been formed. The second bridge, between Cys (Acm) 173 and 186 is obtained by oxidation at (Buku and al., Int. J. Peptide. Res., 33:86, 1989 and Annis et al., Meth. Enzymol., 289: 198, 1997). The peptide is dissolved (1 mg / ml) in an acetic acid mixture / 80% water (v / v) and 10% 1 N HCl are added, the solution is saturated with nitrogen, then 10 equivalents of iodine dissolved in a mixture of acetic acid / 80% water (v / v) are added quickly and the medium is stirred for 5 hours at room temperature The excess iodine is reduced by the dropwise addition of an aqueous solution of ascorbic acid until the characteristic color of the iodine disappears. The crude oxidized peptide is purified by RP-HPLC, lyophilized and analyzed by RP-HPLC and ES-MS. Formation of disulphide bridges in 1 step. in one step also by direct oxidation with iodine on the reduced peptide allowing also to obtain the peptide of interest. The yield then goes from 22 to 44% (RP-HPLC purity>90%; calculated mass: 8140.22 Da / measured mass: 8040.30 Da).
L'appariement des ponts disulfures est étudié par LC-MS ("Liquid Chromatography Mass Spectrometry") et par microséquençage des fragments obtenus suite à une coupure du peptide à la thermolysine. Les fragments obtenus et leur interprétation sont décrits dans le tableau 1 ci-dessous. Le protocole utilisé permet d'obtenir uniquement la forme native G20a (1-4/2-3).The pairing of the disulfide bridges is studied by LC-MS ("Liquid Chromatography Mass Spectrometry") and by microsequencing of the fragments obtained following a cleavage of the peptide with thermolysin. The fragments obtained and their interpretation are described in Table 1 below. The protocol used makes it possible to obtain only the native form G20a (1-4 / 2-3).
Tableau 1 : Carte peptidique (thermolysine) du peptide G20aTable 1: Peptide map (thermolysin) of the G20a peptide
1010
B. Couplage du peptide G20a à l'aide d'un réactif homobifonctionnel (glutaraldéhyde)B. Coupling of the G20a peptide using a homobifunctional reagent (glutaraldehyde)
10 mg de DTa sont dialyses contre un tampon phosphate 0,1 M pH 7. La concentration est ajustée à 2 mg/ml à l'aide d'un tampon carbonate 0,1 M pH 9. 200 mg10 mg of DTa are dialyzed against a 0.1 M phosphate buffer pH 7. The concentration is adjusted to 2 mg / ml using a 0.1 M carbonate buffer pH 9. 200 mg
15 de SDS d'une solution à 4 % sont ajoutés.15 SDS of a 4% solution are added.
55,5 μl de glutaraldéhyde à 2,5 % sont ajoutés à 2,5 ml d'une solution de peptide G20a à 1 mg/ml dans un tampon carbonate 0,1 M pH 9 à un pH se situant entre 9 et 10. Le milieu réactiormel est agité à 4°C pendant 24 h puis ramené à température ambiante. 25 μl de lysine sont ajoutés pour bloquer la réaction. La solution est dialysée contre du 0 tampon phosphate 0.1 M pH 7 pendant 24 h à 4°C sous agitation. Le dialysat est récupéré et le SDS éliminé par précipitation à l'aide d'une solution de KCl 0,02 M à 6 1655.5 μl of 2.5% glutaraldehyde are added to 2.5 ml of a solution of peptide G20a at 1 mg / ml in a 0.1 M carbonate buffer pH 9 at a pH between 9 and 10. The reaction medium is stirred at 4 ° C for 24 h and then brought to room temperature. 25 μl of lysine are added to block the reaction. The solution is dialyzed against 0.1 M phosphate buffer pH 7 for 24 h at 4 ° C with stirring. The dialysate is recovered and the SDS eliminated by precipitation using a 0.02 M KCl solution at 6 16
récupéré et le SDS éliminé par précipitation à l'aide d'une solution de KCl 0,02 M à 6 reprises. Le dernier surnageant qui contient le conjugué Dta-G20a glutaraldéhyde est conservé sous forme congelé à 4°C après une filtration stérilisante (0,22 μm). Les conjugués sont caractérisés pas dosage de protéines et par électrophorèse de type SDS- PAGE ("SDS-PolyAcrylamide Gel Electrophoresis). Les conditions de couplage utilisées ont été étudiées sur l'hexadécapeptide modèle G4a (fragment aa 171-187 de la protéine G du VRS humain de type A, correspondant à la séquence SEQ ID N° 15 du document WO 95/27787) qui possède la même séquence en acides aminés et le même appariement 1-4/2-3 des 4 cystéines. Ces conditions ne modifient pas l'appariement natif du peptide modèle (Beck et al., J. Peptide Res., 55:24, 2000). Exemple 5 : Immunogénicité et protection du conjuguérecovered and the SDS eliminated by precipitation using a 0.02 M KCl solution 6 times. The last supernatant which contains the conjugate Dta-G20a glutaraldehyde is stored in frozen form at 4 ° C after sterilizing filtration (0.22 μm). The conjugates are characterized by protein assay and by SDS-PAGE electrophoresis ("SDS-PolyAcrylamide Gel Electrophoresis). The coupling conditions used were studied on the hexadecapeptide model G4a (fragment AA 171-187 of protein G of the Human RSV type A, corresponding to the sequence SEQ ID No. 15 of document WO 95/27787) which has the same amino acid sequence and the same 1-4 / 2-3 pairing of the 4 cysteines. These conditions do not modify the native pairing of the model peptide (Beck et al., J. Peptide Res., 55:24, 2000) Example 5: Immunogenicity and protection of the conjugate
A. Titres anti-G2Na et VRS-A après une ou deux immunisations chez des rats de cotton immunisés avec différents immunogènesA. Anti-G2Na and VRS-A Titers After One or Two Immunizations in Cotton Rats Immunized with Different Immunogens
Les rats des cottonniers (6/groupe) ont été immunisés en intramusculaire (100 μl) avec 6 μg d'équivalent G2Na, quel que soit l' immunogène testé, mélangé à 20 % (v/v) Al(OH)3 à J0 et J21 et J40.Cottonnier rats (6 / group) were immunized intramuscularly (100 μl) with 6 μg of G2Na equivalent, whatever the immunogen tested, mixed at 20% (v / v) Al (OH) 3 on D0 and J21 and J40.
10 jours après la dernière immunisation, les rats sont challenges avec 105 pfu VRS-A/50 μl en intranasal. 5 jours après challenge, ils sont euthanasiés et les poumons récupérés pour évaluer la charge virale résiduelle. Les titres anticorps anti-G2Na et VRS-A sont analysés.10 days after the last immunization, the rats are challenged with 10 5 pfu VRS-A / 50 μl intranasally. 5 days after challenge, they are euthanized and the lungs recovered to assess the residual viral load. The anti-G2Na and VRS-A antibody titers are analyzed.
Les résultats d' immunogénicité (voir figure 1) observés chez le rat des cottons indiquent que G20DTa et G20DTaDTb sont immunogéniques après une ou deux immunisations. L'immunigénicité obtenue est comparable à celle observée avecThe immunogenicity results (see FIG. 1) observed in the cottons rat indicate that G20DTa and G20DTaDTb are immunogenic after one or two immunizations. The immunogenicity obtained is comparable to that observed with
BBG2Na (peptide G2Na fusionné au fragment dénommé "BB", récepteur de la sérum albumine humaine de streptococcus tel que décrit dans le document WO 95/27787).BBG2Na (G2Na peptide fused to the fragment called "BB", receptor for human serum albumin from streptococcus as described in document WO 95/27787).
De plus, l'effet porteur est clairement démontré : l'antigène G20a ne permet pas d'obtenir une réponse immunitaire aussi forte que celle obtenue en utilisant les porteurs DTa ou DTaDTb.In addition, the carrier effect is clearly demonstrated: the G20a antigen does not make it possible to obtain an immune response as strong as that obtained using the DTa or DTaDTb carriers.
B. Protection contre un challenge avec VRS-A chez le rat des cottons immunisé par différentes molécules Après challenge des rats de cotton avec VRS-A, une protection des voies pulmonaires a été observée (voir figure 2) après immunisation des rats par G20DTa, G20DTaDTb comparable à celle observée avec BBG2Na.B. Protection against a challenge with VRS-A in rat cottons immunized with different molecules After challenge of the cotton rats with VRS-A, protection of the pulmonary tracts was observed (see FIG. 2) after immunization of the rats with G20DTa, G20DTaDTb comparable to that observed with BBG2Na.
A nouveau, l'effet porteur est clairement démontré : l'antigène G20 ne permet pas d'obtenir une réponse immunitaire aussi forte que celle obtenue en utilisant les porteurs DTa ou DTaDTb.Again, the carrier effect is clearly demonstrated: the G20 antigen does not make it possible to obtain an immune response as strong as that obtained using the DTa or DTaDTb carriers.
Ces résultats indiquent que G20DTa et G20DTaDTb sont immunogéniques et protecteurs chez le rat des cottons et que l'effet biologique de ces molécules est comparable à celle de BBG2Na. Exemple 6 : Détermination du HSIThese results indicate that G20DTa and G20DTaDTb are immunogenic and protective in the rat of cottons and that the biological effect of these molecules is comparable to that of BBG2Na. Example 6: Determination of the HSI
Les cobayes sont immunisés à JO et J8 avec le porteur DTaDTb adjuvante par de l'adjuphos 20 % (v/v) en i.m.. A J21, un rappel est effectué avec le porteur non adjuvante en i.v.The guinea pigs are immunized at OJ and D8 with the adjuvanting DTaDTb carrier by 20% (v / v) adjuphos at i.m. At D21, a recall is made with the non-adjuvanting carrier at i.v.
La mort des cobayes est alors évaluée.The death of the guinea pigs is then evaluated.
Un témoin positif d'expérience constitué d'ovalbumine à 200 μg est inclus pour chaque molécule testée.A positive experience control consisting of ovalbumin at 200 μg is included for each molecule tested.
Tableau 2 : Résultats obtenus après immunisationTable 2: Results obtained after immunization
Les résultats au tableau 2 indiquent que DTaDTb n'induit pas de HSI chez 6/6 animaux testés. The results in Table 2 indicate that DTaDTb does not induce HSI in 6/6 animals tested.
Claims
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| FR0109733A FR2827606A1 (en) | 2001-07-20 | 2001-07-20 | NOVEL DIPHTERIC ANATOXIN DERIVATIVES AND THEIR USE AS A BEARER |
| FR01/09733 | 2001-07-20 |
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| CN114057851A (en) * | 2020-08-07 | 2022-02-18 | 清华大学 | A kind of polypeptide, immunogenic conjugate and use for preventing novel coronavirus pneumonia COVID-19 |
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| FR2873378A1 (en) * | 2004-07-23 | 2006-01-27 | Pierre Fabre Medicament Sa | IMMUNOGENIC COMPLEXES, PROCESS FOR THEIR PREPARATION AND THEIR USE IN PHARMACEUTICAL COMPOSITIONS |
| IT1398927B1 (en) | 2009-06-25 | 2013-03-28 | Consorzio Interuniversitario Per Lo Sviluppo Dei Sistemi A Grande Interfase Csgi | BACTERIAL EXPRESSION OF AN ARTIFICIAL GENE FOR THE PRODUCTION OF CRM197 AND DERIVATIVES. |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1987002987A1 (en) * | 1985-11-13 | 1987-05-21 | Murphy John R | Cys codon-modified dna |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987002987A1 (en) * | 1985-11-13 | 1987-05-21 | Murphy John R | Cys codon-modified dna |
Non-Patent Citations (7)
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| C E BELL ET AL.: "Oligomerization of a 45 KD fragment of diphtheria toxin at pH 5.0 to a molecule of 20-24 subunits", BIOCHEMISTRY., vol. 36, no. 49, 1997, AMERICAN CHEMICAL SOCIETY. EASTON, PA., US, pages 15201 - 15207, XP002224942, ISSN: 0006-2960 * |
| DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1988, N V JAKUBOVICH ET AL.: "Cloning the structural gene for diphtheria toxin from Corynebacterium diphtheriae and expression of its deleted derivatives in E. coli", XP002224943, Database accession no. BA86:37356 * |
| G GIANNINI ET AL.: "The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM 197", NUCLEIC ACIDS RESEARCH, vol. 12, no. 10, 1984, OXFORD GB, pages 4063 - 4069, XP002198694 * |
| J CHANG ET AL.: "Unique chemical reactivity of His-21 of CRM-97, a mutated diphtheria toxin", FEBS LETTERS, vol. 427, no. 3, 1998, AMSTERDAM NL, pages 362 - 366, XP004257895 * |
| M PIZZA ET AL.: "Mutants of pertussis toxin suitable for vaccine development", SCIENCE., vol. 246, 27 October 1989 (1989-10-27), AAAS. LANCASTER, PA., US, pages 497 - 500, XP001016264, ISSN: 0036-8075 * |
| MOL GENET MIKROBIOL VIRUSOL, no. 11, 1987, pages 13 - 18 * |
| P O FALNES & S OLSNES: "Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway", EMBO JOURNAL., vol. 17, no. 2, 1998, OXFORD UNIVERSITY PRESS, SURREY., GB, pages 615 - 625, XP002198693, ISSN: 0261-4189 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057851A (en) * | 2020-08-07 | 2022-02-18 | 清华大学 | A kind of polypeptide, immunogenic conjugate and use for preventing novel coronavirus pneumonia COVID-19 |
| CN114057851B (en) * | 2020-08-07 | 2023-11-21 | 清华大学 | Polypeptide and immunogenic conjugate for preventing novel coronavirus pneumonia COVID-19 and application thereof |
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