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
  • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
• • 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
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the subject of the present invention is polypeptides derived from the Tbp2 subunit of the transferrin receptor of Neisseria meningitidis, their use for therapeutic purposes, in particular vaccine, as well as the DNA fragments coding for these polypeptides.
• meningitis is either of viral or bacterial origin.
• the bacteria mainly responsible are: N. meningitidis and Haemophilus influenzae respectively involved in around 40 and 50% of cases of bacterial meningitis.
• the N meningitidis species is subdivided into serogroups according to the nature of the capsular polysaccharides Although there are a dozen serogroups, 90% of meningitis cases are attributable to 3 serogroups: A, B and C.
• N. meningitidis group B is not or little immunogenic in humans, whether it is in conjugated form or not. Thus, it appears highly desirable to seek a vaccine against meningitis induced by N. meningitidis, in particular serogroup B, other than a polysaccharide-based vaccine.
• iron can only be taken from human iron transport proteins such as transferrin and lactoferrin since the amount of iron in free form is negligible in humans (of the order of 10 - 18 ), in any case insufficient to allow bacterial growth.
• N. meningitidis has a receptor for human transferrin and a receptor for human lactoferrin which allow it to fix these iron chelating proteins and subsequently capture the iron necessary for its growth.
• the transferrin receptor of the N. meningitidis B16B6 strain was purified by Schryvers et al (WO 90/12591) from a membrane extract.
• This protein as purified appears to consist essentially of 2 types of polypeptides: a polypeptide of a high apparent molecular weight of 100 kD and a polypeptide of a lower apparent molecular weight of approximately 70 kD, as revealed after electrophoresis on gel of polyacrylamide in the presence of SDS.
• the purification product used in particular by Schryvers is by definition arbitrary and for the purposes of this patent application, called a transferrin receptor and the polypeptides constituting it, subunits.
• the high molecular weight and lower molecular weight subunits are respectively called Tbpl and Tbp2.
• the recognition of the transferrin receptor subunits is revealed by addition of a rabbit anti-immunoglobulin antibody coupled to peroxidase, then by addition of the substrate for this enzyme.
• Tables I and II below indicate the profile of certain representative strains as it appears on 7.5% polyacrylamide gel after electrophoresis in the presence of SDS; the bands are characterized by their apparent molecular weights expressed in kilodaltons (kD):
• NB In brackets, the serogroup, the serotype, the subtype and the immunotype are indicated in order.
• the first type corresponds to strains which have a receptor whose 2 subunits under the experimental conditions used are recognized by the anti-receptor antiserum IM2394 while only the high molecular weight subunit is recognized by the anti-receptor IM2169 antiserum.
• the second type corresponds to strains which have a receptor whose 2 subunits under the experimental conditions used are recognized by the anti-receptor antiserum IM2169 while only the high molecular weight subunit is recognized by the anti-receptor IM2394 antiserum.
• strains of type IM2169 are, for example, strains S3032 (12, P 1.12.16), 6940 (19, P 1.6), M978 (8, P 1.1, 7), 2223 (B : nd), 1610 (B: nd), C708 (A: 4, P 1.7), M981 (B: 4), also called 891, and 2996 (B: 2b, P 1.2).
• the IM2154 strain (serogroup C) is cited by way of example as being of the IM2394 type.
• IM2394 were disclosed in EPA patent application 586,266 (published March 9, 1994) along with the corresponding DNA fragments. These sequences are listed in SEQ ID NO 1 to 4 of the present application. In SEQ ID NOs 5 to 10, the sequences of the Tbp2 subunits of the strains of type IM2169 are presented, namely the strains M978, 6940 and S3032.
• sequence of the Tbp2 IM2154 subunit differs by two amino acids from the sequence of the Tbp2 IM2394 subunit, at positions 306 and 510.
• Tbp2 subunit whatever the original strain, has in terms of structures, three main domains associated for at least one of them with particular properties.
• the domains of Tbp2 IM2169 and Tbp2 IM2394 have been fixed as shown in the table below, indicating the position of the amino acids, limits included in the different domains, and by reference to the numbering appearing in SEQ ID NO 1 and 3.
• first domain (1 - 346)
• second domain (347 - 557)
• third domain 558 - 705
• the N-terminal domain or first domain and / or the hinge domain or second domain could be necessary and sufficient, in order to induce a vaccine effect in humans; consequently, it would not be essential to use a Tbp2 in a complete form.
• the first domain contains almost the entire transferrin binding site, is therefore very likely to be exposed to the outside and therefore constitutes an element of choice for vaccine purposes.
• sequence which derives from another sequence is obviously understood a sequence resulting by intellectual process from this other sequence.
• a polypeptide according to the invention has an amino acid sequence which derives from a Tbp2 subunit of the IM2169 or IM2394 type: (i) in particular by total or partial deletion of at least one domain of said domain Tbp2 subunit selected from the second and third domains; preferably by total or partial deletion of the third domain or of the second and third domains; (ii) in particular by total deletion of the first and third domains, or (iii) in particular by complete deletion of the third domain and by partial deletion of the first domain, optionally by partial deletion of the second domain.
• a polypeptide according to the invention has a partial, almost total or total deletion of the third domain, preferably total.
• the first as well as the second domain can be maintained in their entirety, partially or totally deleted; this independently of each other.
• the following combinations are possible (knowing that the first, second and third domains in their entirety are respectively represented by 1, 2 and 3, and that O and ⁇ mean respectively, partially and totally deleted):
• polypeptide according to the invention derived from a Tbp2 subunit of type IM2169 by partial deletion of the second domain, which comprises in their entirety or almost entirely the first and third domains; or the combination 1, O2, 3. (By "domain maintained in almost its entirety” is meant here and in the remainder of the text, a domain modified in a very small number of positions, approximately 5 maximum.)
• a polypeptide according to l The invention can also respond to the combination O1, O2, 3, the partial deletion of the first domain advantageously relating to the region homologous to that of Tbp2 IM2169 ranging from the amino acid in position 1 to the amino acid approximately in position 40.
• this partial deletion advantageously relates to one or more regions of the second domain which is (are) the (the) homolog (s) of the regions of the IM2169 sequence ranging from: (i) from amino acid at position 362 to amino acid at position 379;
• the partial deletion relates simultaneously to the four regions (i) to (iv) described above.
• a polypeptide according to the invention derives in particular by complete deletion of the third domain and almost integral deletion of the second domain from a Tbp2 subunit of type IM2169 and comprises the entire first domain or also derives by deletion from the part N-terminal of the first domain
• the almost integral deletion of the second domain extends over the region which: in the case of a polypeptide derived from a Tbp2 subunit of type IM2169, is the homolog of the region of the second domain of the Tbp2 IM2169 subunit ranging from the amino acid in one of positions 346 to 361 to the amino acid in position 543; in the case of a polypeptide derived from a Tbp2 subunit of type IM2394, is the homolog of the region of the second domain of the Tbp2 subunit IM2394 ranging from the amino acid in one of positions 326 to 341 with amino acid in position 442.
• this partial deletion advantageously relates to all or part of the region:
• sequence of type IM2169 or IM2394 from which is derived that of a polypeptide according to the invention has a degree of homology with the respective reference sequence, IM2169 or IM2394, advantageously at least 70-75%, of preferably at least 80%, more particularly preferably at least 90%.
• a polypeptide according to the invention has a sequence derived from that of the Tbp2 subunit IM2169 or IM2394.
• the degree of homology can be easily calculated by aligning the sequences so as to obtain the maximum degree of homology; to do this, it may be necessary to artificially introduce vacant locations, as illustrated in Figures 1 to 4 and 8 to 10. Once the optimal alignment is achieved, the degree of homology is established by accounting all the positions in which the amino acids of the two sequences are found identically, relative to the total number of positions.
• a polypeptide according to the invention is cited, the sequence of which has at least 70-75%, advantageously at least 80%, preferably at least 90%), very preferably 100% of homology with:
• a polypeptide according to the invention the sequence of which is substantially as shown in ID SEQ NO 1, 5, 7, 9, 36 or 38, from the amino acid in position 1 to the amino acid in position 350, 351, 354, 358, 322 or 346 respectively;
• a polypeptide according to the invention has an amino acid sequence which comprises at least 10, advantageously at least 20, preferably at least 50, very preferably at least 100 amino acids.
• a polypeptide according to the invention can also additionally comprise an amino acid sequence which does not have any homology with the sequences of the Tbp2 subunits of the strains IM2169 and IM2394; sequences which are shown in ID SEQ NO 1 and 3 from the amino acid in position 1 to the amino acid in position C-terminal.
• an additional sequence can be that of any other polypeptide to the exclusion of Tbp2.
• an additional sequence may be that of a signal peptide located in the N-terminal position of a polypeptide according to the invention.
• Examples of signal sequence are shown in ID SEQ NOs 1 to 4.
• a suitable heterologous signal sequence may be a signal sequence of a gene coding for a lipoprotein.
• a subject of the invention is also: (i) an isolated DNA fragment coding for a polypeptide according to the invention
• isolated DNA fragment it is meant that a DNA fragment according to the invention is not integrated into a DNA fragment coding for a complete Tbp2 subunit.
• the DNA fragment according to the invention may or may not be associated with a DNA block coding for a heterologous signal peptide or not, with the polypeptide coded by said DNA fragment, depending on whether the whether or not secretion of the polypeptide is sought. Preferably, this secretion will be sought.
• signal region or a promoter already exist in fairly large numbers and are known to those skilled in the art. His general skills will allow him to choose a particular signal region or promoter which will be adapted to the host cell in which he envisages expression.
• the host cell can be a mammalian cell, a bacterium or a yeast; the latter two being preferred. Again, the choice of a particular line is within the reach of the skilled person.
• the invention also relates to a monoclonal antibody:
• sequences of the third domain of the Tbp2 IM2394 subunit which are two by two homologous to those of the first domain are respectively in position 443 - 447, 472 - 485, 537 - 548 and 568 - 573;
• a monoclonal according to the invention is: (i) capable of recognizing the region present in the first domain of a Tbp2 subunit of type IM2169 or IM2394, the sequence of which is homologous to the sequence EGGFYGPKGEEL present in the first domain of the Tbp2 subunit of the strain IM2394; and optionally,
• a preferred monoclonal is: (i) capable of recognizing the GFYGPK epitope, present in the first domain of a Tbp2 subunit of the strain IM2394;
• the invention also relates to a pharmaceutical composition comprising, as active principle, at least one polypeptide according to the invention.
• a pharmaceutical composition according to the invention is in particular useful for inducing an immune response in humans against N. meningitidis, inter alia a vaccine effect so as to protect humans against N. meningitidis infections, in prevention or in therapy.
• a composition according to the invention advantageously comprises, as active principle, at least two polypeptides according to the invention; or at least a first polypeptide whose sequence derives from that of a Tbp2 subunit of type IM2169 and at least a second polypeptide whose sequence derives from that of a Tbp2 subunit of type IM2394.
• a composition according to the invention can also contain at least one polypeptide whose sequence derives from that of a Tbp2 subunit of type IM2169 and at least one Tbp2 subunit of type IM2394.
• polypeptide of type IM2394 element of the pharmaceutical composition
• it is very preferable that it comprises all or part of the sequence which is homologous to that of the first domain of the Tbp2 IM2394 subunit from which it is derived.
• the part of the sequence which should preferably be maintained is the homolog of the region of the Tbp2 IM2394 subunit ranging from the amino acid at position 267 to the amino acid at position 325.
• the sequence of such a polypeptide can be derived from that of a Tbp2 subunit of type IM2394 in particular by total or partial deletion of the region of the second or third domain of the Tbp2 subunit of type IM2394.
• polypeptides of type IM2394 are more particularly preferred:
• polypeptide of type IM2394 associates with the polypeptide of type IM2394, a polypeptide which comprises all or part of the sequence which is homologous to that of the first domain of the Tbp2 subunit IM2169 from which it is derived.
• the part of the sequence which should preferably be maintained is the homolog of the region of the Tbp2 IM2169 subunit ranging from the amino acid at position 282 to the amino acid at position 345.
• the sequence of such a polypeptide can derive from that of a Tbp2 subunit of type IM2169 in particular by total or partial deletion of the region of the second or third domain of the Tbp2 subunit of type IM2169.
• polypeptides of type IM2169 are more particularly preferred:
• the partial deletion of the second domain can very advantageously relate to one or more regions of the second domain which is (are) the (Ies) homolog (s) of the regions of the IM2169 sequence ranging from:
• the partial deletion relates simultaneously to the four regions (i) to (iv) described above.
• the pharmaceutical composition with two types of elements can advantageously contain several polypeptides ( ⁇ 1, 2, ⁇ 3) of type IM2169; for example two or more of the polypeptides selected from ( ⁇ 1, 2, ⁇ 3) IM2169, M978, 6940 and S3032.
• a pharmaceutical composition according to the invention can be manufactured in a conventional manner.
• the polypeptide (s) according to the invention are combined with an adjuvant, a diluent or a support which is acceptable from a pharmaceutical point of view.
• a composition according to the invention can be administered by any conventional route in use in the field of vaccines, in particular by the subcutaneous route, by the intramuscular route or by the intravenous route, for example in the form of a suspension. injectable.
• the administration can take place in single dose or repeated one or more times after a certain interval of interval.
• the appropriate dosage will vary depending on various parameters, for example, the individual being treated or the mode of administration. In order to determine the subject of the present invention, it is specified that the strains of N.
• meningitidis IM2394 and IM2169 are publicly available from the National Collection of Culture of Microorganisms (CNCM), Institut Pasteur, 25 rue du Dr Roux 75015 Paris under the respective registration numbers LNP N 1511 and LNP N 1520. The invention is described in more detail in the examples below and with reference to
• Figures 1 to 3, 8 and 9 respectively show the alignments of the sequences Tbp2, M978, 6940, S3032, BZ83 and BZ163 with the sequence Tbp2 IM2169, at maximum homology.
• the respective degrees of homology are 78.9, 81.2, 79.6, 71.3 and 81.8%.
• Figure 4 shows the maximum homology alignments of the sequences of the hinge domains (second domain) of Tbp2 IM2169 (1), 6940 (2), 2223 (3), C708
• Figures 5 to 7 respectively illustrate the construction of plasmids pTG5782, pTG5755 and pTG5783.
• Figure 10 shows the maximum homology alignments of the sequences of the hinge domains (second domain) of Tbp2 IM2169 (1), 2223 (2), 708 (3), M528 (4), 6940 (5), M978 (6 ), 1610 (7), S3032 (8), 867 (9), BZ83 (10) and BZ163 (11).
• IM2169 the sequences which can be deleted according to a preferred mode.
• C indicates the consensus sequence.
• EXAMPLE 1 Polypeptide T / 2169 (1, O2, ⁇ 3; 1-350) whose sequence as shown in ID SEQ NO 1 (IM2169), from amino acid in position 1 to amino acid in position 350.
• a fragment comprising the sequence coding for the RlpB secretion signal and the start of the sequence coding for mature Tbp2 to the internal H ⁇ ell site is amplified by PCR, using primers OTG4915 and OTG4651. .
• PCR fragment is then digested with BspHI and Haell and inserted simultaneously with the Haell-HindIII fragment of pTG4704 which contains the 3 ′ part of the region coding for Tbp2, in the plasmid pTG3704 described in EPA application 586 266, digested with Ncol and Hindlll, to generate the plasmid pTG5768.
• plasmid pTG3721 a fragment comprising the sequence coding for the N-terminal part of Tbp2 is amplified by PCR, using the primers OTG4928 and OTG501 1.
• This PCR fragment is digested with Sphl and HindIII, then cloned into the plasmid pTG4710 described in application EPA 586,266; the plasmid pTG5740 is thus generated.
• the Haell-Hindlll fragment of pTG5740 comprising the 3 ′ part of the sequence coding for the human transferrin binding domain (hTf) (3 ′ of the region coding for the first domain) is inserted into the plasmid pTG3704 digested with
• This vector includes the ⁇ raB promoter, the sequence coding for the RlpB secretion signal fused to the sequence coding for the N-terminal domain of Tbp2 (1 - 350).
• a strain of E. coli (Xac-I) is transformed by pTG5782.
• the transformants are cultured at 37 ° C. in M9 medium + 0.5% succinate + arginine 50 ⁇ g / ml + ampicillin 100 ⁇ g / ml.
• 0.2% arabinose (inducer) is added.
• cells are taken and extracts are prepared.
• Western blot analysis followed by revelation with hTF-peroxidase makes it possible to detect a majority band whose PM corresponds to that expected for this truncated form of Tbp2.
• T / 2169 is found to be capable of inducing bactericidal antibodies and therefore should be useful for vaccine purposes.
• This fragment is then digested with Mlul and Hindlll.
• the plasmid pTG4710 is digested with Mlul and Hindlll.
• the Mlul-Hindlll fragment comprising the 3 ′ part of the sequence coding for Tbp2 is replaced by the PCR fragment coding for the C-terminal part of the hTf binding domain.
• the plasmid pTG5707 is thus generated.
• the plasmid pTG5755 is thus generated.
• This vector includes the araB promoter, the sequence coding for the RlpB secretion signal fused to the sequence coding for the N-terminal domain of Tbp2 (1 - 340). 2B - Production and purification of T / 2394 (1-340)
• T / 2394 (1-340) is produced and purified as described in Example 1B.
• WO93 / 6861 published: 15. 04. 93
• Purified T / 2394 is found to be able to induce bactericidal antibodies and therefore should be useful for vaccine purposes.
• the DNA fragment coding for the Tbp2 subunit of the N. meningitidis IM2169 strain is amplified by PCR (Polymerase chain reaction) using specific primers complementary to the 5 'and 3' regions (respectively A5 ' and A3 ') on 10 ng of genomic DNA extracted from a culture of bacteria of the strain IM2169.
• a DNA fragment is thus obtained and after digestion with EcoRI, it counts 2150 nt. This EcoRI fragment is then ligated to the dephosphorylated EcoRI ends of the phagemid pBluescriptSK (-) (Stratagene) to give the recombinant phagemid pSK / 2169tbp2. 1.2. Implementation of deletions.
• the phage form of the recombinant phagemid pSK / 2169tbp2 is obtained after rescue by the "helper" phage VCS M13 according to the technique described by Stratagene, supplier of the basic vector, and used to infect the bacterial strain CJ236.
• the mutations dut and ung carried by the strain CJ236 result in the synthesis of DNA molecules which have incorporated the nucleotide precursor dUTP.
• the phages are harvested and the single-stranded DNA is extracted with a phenol / chloroform mixture. This DNA is hybridized, under conventional conditions, to the following oligonucleotides:
• the hybridization reaction is continued for 30 min, at a decreasing temperature from 70 ° C. to 30 ° C.
• the second complementary strand is then completed by complete synthesis in the presence of the four deoxynucleotides, of T4 DNA polymerase and of T4 DNA ligase, according to conventional conditions.
• the E. coli SURE strain (Stratagene) is transformed by the DNA thus obtained.
• the molecules carrying dUTP that is to say non-mutated, are destroyed.
• the phages obtained are analyzed by standard techniques for rapid preparation of plasmid DNA and digestion with the appropriate restriction enzymes. The presence of the mutation sought is then verified by nucleotide sequencing.
• the clone pSK2169 # 7, carrying the four mutations ⁇ 1203-1256, ⁇ 1371-1451, ⁇ 1512-1562, and ⁇ 1617-1679 is selected.
• the plasmid pTG5768 described above is digested with Hpal and Xcml.
• An Xcml-Xcml fragment from pTG5768 and the Hpal-Xcml fragment from the plasmid pSK / 2169ed # 7 are inserted simultaneously into this vector, to generate the plasmid pTG5783.
• This vector includes the araB promoter, the sequence coding for the RlpB secretion signal fused to the modified tbpl sequence (deletions dl to d4).
• primers specific for each of the second domains are created by introducing appropriate cleavage sites for future cloning in phase with signal sequence rlpB, under the control of the araB promoter. These primers are used in PCR to amplify the region coding for the second domain of each of the Tbp2. These regions are cloned as indicated above in a plasmid comprising the signal sequence rlpB, under the control of the araB promoter.
• EXAMPLE 9 Vaccine composition (T / 2169 - T / 2394) intended to prevent infections with N. meningitidis Sterile solutions of T / 2169 and T / 2394 as purified in Examples 1B and
• EXAMPLE 10 Vaccine composition (D4 / 2169 - Tbp2 / 2394) intended to prevent infections with N. meningitidis
• a sterile solution of D4 / 2169 as purified in Example 3C is thawed. The same is done with a sterile solution of Tbp2 / 2394 as prepared and purified in Example 3 of EPA 586 266.
• the solutions are mixed in a sterile manner. following:
• EXAMPLE 1 1: Obtaining an antibody capable of recognizing the GFYGPKGE epitope of the first domain of Tbp2 IM2394. 11A - Immunization of mice and production of hybridomas
• MRL / Lpr-Lpr mice known to produce more IgG2a, IgG2b and IgG3 than Balb / C mice (J. Immunol. Methods (1991) 144: 165) receive a first intraperitoneal injection of 50 ⁇ g of the membrane fraction IM2394 in the presence of Freund's complete adjuvant.
• the membrane fraction that is used is prepared as follows:
• the IM2394 strain preserved in lyophilized form is taken up and cultured on Mueller-Hinton agar overnight at 37 ° C. in an atmosphere containing 20% CO 2 .
• the tablecloth is taken up and used to seed an Erlenmeyer flask containing Mueller-Hinton broth supplemented with 30 ⁇ M EDDA (ethylene diamine di ortho-hydroxy acetic acid - Sigma). After 5 hours of incubation at 37 ° C with rotary shaking, the culture is centrifuged. The pellet is taken up in Tris-HCl pH 8 buffer and the suspension is lysed in an ultrasonic device operating at high pressure.
• the booster doses contain 25 ⁇ g of the protein Tbp2 as purified in Example 3 of EPA 586 266, in the form of an emulsion in the incomplete adjuvant of Freund.
• the mouse having developed the highest antibody titer (control of the immune sera by ELISA) is selected for the production of specific monoclonal antibodies.
• the latter receives a final booster injection (78 days after the initial injection) by inoculating 25 ⁇ g of the protein Tbp2 as purified in Example 3 of EPA 586 266 both intravenously and intraperitoneally.
• the animal's spleen is removed and the splenocytes are fused with the murine myeloma cells P3 x 63 Ag 8653 in a ratio of one myeloma cell to 4 spleen cells.
• the fusion protocol used is derived from that initially described by G. Köhler and C.
• the cells are placed in sterile microwells (Nunc) covered with a feeder "feeder" at a rate of 100,000 cells per well in a volume of 200 ⁇ l of selective medium [DMEM medium containing 20% FCS and a hypoxanthine - azaserine - thymidine mixture at 2% (V / V) (Gibco. Ref 043-01060H)] .
• selective medium [DMEM medium containing 20% FCS and a hypoxanthine - azaserine - thymidine mixture at 2% (V / V) (Gibco. Ref 043-01060H)] .
• the selective medium is replaced 6 days later, by a non-selective medium [D.M.E.M medium containing 20% of FCS and a hypoxanthine - thymidine mixture at 2%
• the wells are covered with 100 ⁇ l of a mixed solution of antibodies conjugated to alkaline phosphatase (PA) specific for the isotypes IgG2 a , IgG 2b and lgG 3 murins so as not to select only hybridomas secreting specific and functional antibodies in the bactericidal test.
• the mixed solution of conjugated antibodies is prepared by diluting the following 3 goat immune systems: goat anti IgG 2a - PA (Caltag), goat anti IgG 2b -PA (Caltag), goat anti IgG 3 -PA (Caltag) 1500th in PBS-T-AB buffer.
• the enzymatic reaction is revealed by 100 ⁇ l of a solution of paranitrophenyl phosphate at 5 mg / ml in 0.1 M diethanolamine buffer, pH 9 , 8. The development of the reaction is stopped after 30 min. by adding 50 ⁇ l of 1N sodium hydroxide before analysis with a spectrophotometer at 405 nm.
• Clones positive after this first screening are analyzed for their ability to recognize the Tbp2 subunit by Western blot.
• the transferrin receptors IM2394 (0.863 mg / ml) and IM2169 (0.782 mg / ml) as prepared in examples 1 and 2 of WO93 / 6861, are diluted 1/10 in 1 M Tris buffer pH 6 , 8, then denatured by adding 10% ”(V / V) of a 25% SDS solution in TE buffer (100 mM Tris / HCl, 10 mM EDTA) pH
• Positive clones are characterized by their ability to reveal a band corresponding to a protein of approximately 69 kD (Tbp2 subunit) after electrotransfer of the transferrin receptor IM2394 on a nitrocellulose membrane.
• the clones are analyzed for their capacity to produce an immunoglobulin reacting with the peptide sequence
• GFYGPKGE in an ELISA system; the methodology is identical to that described above except for the sensitization of the plates which is carried out by adding to each well of 100 ⁇ l of a solution of peptide GFYGPKE at 2 ⁇ g / ml.
• the hybridomas that are tested one is selected which proves capable of reacting with the peptide; then it is stabilized by successive cloning (at least 2) at the rate of 5 cells / well during the first cloning, of one cell / well during the following. 11C -Production and purification of the monoclonal antibody
• the monoclonal antibody is produced in ascites from Nude Swiss Swiss mice.
• the nude mice receive a second intraperitoneal injection of 7 million cells originating from the hybridoma.
• the ascites liquids are taken sterile and then purified by affinity chromatography on a protein G column.
• the ascites diluted 1 / 5th in 0.1M phosphate buffer pH 7.4 and filtered on a 0.22 ⁇ millipore filter is passed through a column of protein G previously equilibrated in the same phosphate buffer, at a rate of 40 ml / hour.
• the antibodies fixed on the column are eluted using a 0.1 M glycine buffer, pH 2.7.
• the eluted fractions are immediately neutralized using a Tris buffer
• the eluate is then dialyzed overnight at + 4 ° C. in a 0.1M phosphate buffer, pH 7.4, aliquoted and stored frozen.
• the purity of the antibody is checked by electrophoresis on 7.5% polyacrylamide gel and by permeation chromatography on Superose 12. The purity rate is generally greater than 95%.
• GFYGKNAI GFYGKNAI
• This titer was determined as follows: From a solution of Mab 475 E 7 , dilutions of reason two are carried out and incubated in the presence of 50 ⁇ l of a meningococcus suspension at 1.10 4 CFU / ml and of 50 ⁇ l of rabbit complement [the bacterial suspension is obtained by culture of the strain N. meningitidis B16B6 at 37 ° C for 5 hours in Mueller-Hinton-Difco broth containing 30 ⁇ M EDDA (ethylene diamine di ortho hydroxyphenyl acetic acid - Sigma )].
• bactericidal titer is expressed as the inverse of the last dilution in the presence of which 50% or more lysis of the bacteria is observed relative to the control.
• a strain of E. coli B is transformed with the plasmid pTG5782 described in Example 1.
• the selected transformant is amplified to give lots of seed.
• the culture is amplified in the M9 medium + 0.5% succetate. The culture is carried out in a 20 L fermenter.
• arabinose expression inducer
• the cells are harvested, broken in a device operating at high pressure (Rannie) and the membrane fraction is harvested by centrifugation.
• the purification is carried out according to the supplier's recommendations.
• the fraction of purified IgG is lyophilized and the lyophilisate is taken up in a certain volume so that the final protein concentration of the solution is close to 25 mg / ml. 12C -Bactericidal test
• a purification by SDS- Page is carried out of an E fraction.
• coli B obtained after transformation with the plasmid pTG3704 (this vector is identical to the plasmid pTG5782 but does not comprise any sequence of Tbp2).
• the protein fraction obtained by preparative SDS-Page is used to immunize rabbits as described above, and the IgGs are purified from the collected serum.
• IgG T / 2169 and Control IgG serum fractions
• D4 / 2169 is produced and purified according to Example 12A. 13B -Production of D4 / 2169 specific immunoglobulins
• IgG D4 / 2169 immunoglobulin fractions
• Control IgG immunoglobulin fractions
• the bactericidal titers are expressed in reverse of the dilution for which 50% of lysis of the initial colonies is observed.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Oncology (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Communicable Diseases (AREA)
• Genetics & Genomics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=9463680

Family Applications (1)

Country Status (9)

Families Citing this family (19)

Family Cites Families (4)

• 1994
  • 1994-05-31 FR FR9406594A patent/FR2720408B1/fr not_active Expired - Fee Related
• 1995
  • 1995-05-30 HU HU9600210A patent/HU220116B/hu not_active IP Right Cessation
  • 1995-05-30 JP JP8500434A patent/JPH09501059A/ja active Pending
  • 1995-05-30 WO PCT/FR1995/000701 patent/WO1995033049A2/fr not_active Application Discontinuation
  • 1995-05-30 AU AU26757/95A patent/AU706090B2/en not_active Ceased
  • 1995-05-30 EP EP95921860A patent/EP0720653A1/fr not_active Withdrawn
  • 1995-05-30 CA CA002167936A patent/CA2167936A1/fr not_active Abandoned
• 1996
  • 1996-01-26 NO NO960332A patent/NO960332L/no not_active Application Discontinuation
  • 1996-01-30 FI FI960428A patent/FI960428A/fi unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events