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
  • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (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
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
  • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/13—Immunoglobulins specific features characterized by their source of isolation or production isolated from plants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

• the invention relates to amino acid sequences (peptides) developed from antibody-producing cells, especially from hybridoma cells which produce monoclonal antibodies, which neutralize the action of the enterotoxin and / or the cytotoxin of Clostridium difficile.
• amino acid sequences peptides developed from antibody-producing cells, especially from hybridoma cells which produce monoclonal antibodies, which neutralize the action of the enterotoxin and / or the cytotoxin of Clostridium difficile.
• humanized, monoclonal antibodies against the toxins of Clostridium difficile and the hypervariable regions of these antibodies are described.
• methods for obtaining and using these amino acid sequences (peptides) and monoclonal antibodies are also disclosed.
• PMC is characterized by severe diarrhea, which can lead to death, among other things, due to the severe loss of electrolytes and fluids.
• abdominal pain, bloody diarrhea, fever and leukocytosis occur. So far treatment by stopping the antibiotic that triggers the disease, by giving vancomycin and by compensating for the loss of fluids and electrolytes.
• Clostridium difficile is an obligate anaerobic, gram-positive rod bacterium that forms subterminal, oval spores. It is biochemically characterized in that it can ferment monosaccharides such as glucose, N-acetylglucosamine and N-acetylneuraminic acid, but not mannose, xylose or arabinose. Clostridium difficile is also unable to split these monosaccharides off the side chains of gastrointestinal mucin because it lacks enzymes such as neuraminidase, ⁇ -galactosidase or sialidase. Because of these biochemical shortcomings, Clostridium difficile cannot prevail in the intestines of healthy people. If the intestinal flora is disturbed by the administration of antibiotics or cytostatics, the intestinal flora overgrows due to Clostridium difficile and consequently PMC.
• the toxins can be neutralized by antibodies which are directed against the C-terminal repetitive ligand domain, the central translocation domain or the N-terminal catalytic domain (for the structure of the domain, see lit [1]).
• the former prevent the binding of the toxins to the cell receptors, the latter block the glucosylation reaction mediated by the toxins, while the antibodies directed against the central translocation domain inhibit the penetration of the toxins into the cell.
• Antibodies neutralizing toxin A and / or toxin B thus offer the possibility of therapy and / or prophylaxis against C. difficile diseases, whereby they do not eliminate the bacteria, but instead block the action of the toxins formed. In this way, the development of the symptoms of the disease, which is due to the toxins, can be prevented.
• the cell line DSM ACC 2322 produces an antibody TTC8, which not only binds to toxin A, but is also able to neutralize its biological effect.
• the binding site of TTC8 in toxin A was located within the repetitive ligand domain. Binding of the antibodies to the toxins prevents their interaction with the cell receptors.
• the binding site of the mAb TTC8 is located within amino acids 2480-2539 of toxin A, with the (most likely) antigenic sequence TINGKKYYF.
• a mAb PCG-4 known from US Pat. No. 4,879,218 binds to a protein fragment defined by amino acids 2098 to 2141 of toxin A.
• the mAb 2CV produced by the cell DSM ACC 2321 also binds to a sequence of the ligand domain, in this case the toxin B. The binding takes place in the protein fragment between AS 2233 and 2366 of toxin B.
• Both with the monoclonal antibody PCG- 4 and the monoclonal antibody TCC8 are mouse antibodies that are not used in human therapy, but would be suitable as diagnostic aids.
• Ligand domain b 2620, TTC8 2912, 2914,
• toxin-specific monoclonal antibodies generated and described so far that they were obtained from mice. Therapeutic or prophylactic use of such antibodies in species other than the mouse (humans or other animals) is not possible, because the antibodies are recognized as foreign, induce an immune response, which inactivates them. Their adaptation to other species, especially their humanization, has not been possible until now because their variable and hypervariable regions are not sequenced, i.e. were not defined.
• the aim of the invention was to make peptides available which have toxin-neutralizing properties but no longer have a strongly immunogenic fraction, so that the peptides are suitable for use in human and veterinary medicine.
• the neutralization of the effect of the toxins is based on the binding of the antibodies, mediated by the attachment of the variable regions of the antibodies to the respective toxin. This binding is mainly determined by the hypervariable regions (CDR: complementarity determining region).
• CDR hypervariable regions
• the task was therefore to identify the regions responsible for the binding and neutralization of the toxins (variable and hypervariable (CDR)) and to adapt them in such a way that they no longer trigger an immune response.
• Such Peptides can be used for the therapy of existing Clostridium difficile diseases as well as for protection against such diseases in the field of human and also veterinary medicine.
• the scientific results available so far can be summarized to the extent that on the one hand the toxin A from Clostridium difficile is able to trigger the full symptoms of pseudomembranous colitis (PMC) and on the other hand the monoclonal antibody TTC8 directed against this toxin has the effect in vivo (in the mouse) can neutralize this toxin.
• the mAb TTC8 is an antibody of the class IgG 2b.
• the specific recognition between the mAb TTC8 and the toxin A from Clostridium difficile is mediated by the hypervariable regions of the heavy and light chains. This means that precisely defined sections of the antibody (in other words peptides) are responsible for the antigen recognition.
• hypervariable regions make it possible to transfer these peptides (structures) to antibody sequences of other species, which then e.g. in humans, in the form of humanized monoclonal antibodies, are suitable for therapy and prophylaxis of C. difficile diseases and do not have the undesirable side effects of non-species antibodies, which are usually obtained from the mouse.
• the sequences SEQ ID No 1-12 and / or SEQ ID Nol3-16 were used to determine the hypervariable and variable peptide sequences which exert the neutralizing activity of the mAb TTC8.
• Knowledge of the variable regions of neutralizing antibodies allows peptides to be produced which, by binding to the enterotoxin and / or the cytotoxin of Clostridium difficile, abolish their biological effects.
• These peptide sequences like the sequences SEQ ID No. 13 and / or SEQ ID No. 16 of the sequence listing, can be used as such or incorporated in immunoglobulins for the therapy of Clostridium difficile diseases.
• CDRs complenentarity determining regions
• CDR-1 -Asn-Tyr-Trp-Met-Asn- (SEQ ID No. 2)
• CDR-2 -Arg-Ile-Tyr-Pro-Gly-Asp-Gly-Asp-Ala-His-Tyr-Asn-Gly-Lys-Phe-Lys-Gly- (SEQ ID No. 4)
• CDR-3 -Gly-Gly-Asn-Tyr-Asp-Asp-Arg-Val-Phe-Asp-Tyr- (SEQ ID No. 6)
• CDR-4 -Lys-Ala-Ser-Gln-Asn-Val-Gly-Thr-Asn-Val-Ala- (SEQ ID No. 8)
• CDR-6 -Gln-Gln-Tyr-Asn-Ser-Tyr-Pro-Leu-Thr- (SEQ ID No. 12)
• RNA of the hybridoma cells which form the mAb TTC8 was prepared by a method known per se.
• the mRNA was then separated from the total RNA in a second step. This was done with the help of magnetic polystyrene balls (beads) to which 01igo (dT) 25 chains are covalently bound.
• the purified mRNA was then used to synthesize cDNA and, using the polymerase chain reaction (PCR), the VH and VL genes of the mAb TTC8 were amplified, which for the light Code (VL) or heavy chain (VH) of the antibody TTC8. It was of crucial importance here that primers were selected which had a suitable restriction site in order to facilitate the subsequent cloning.
• VL light Code
• VH heavy chain
• VH and VL genes of the mAb TTC8 obtained in this way were then cloned in the vector pUC 19 and then sequenced.
• the nucleotide (SEQ ID No. 13 and 15) and the amino acid sequences derived from it were derived in accordance with SEQ ID No. 14 and SEQ ID No. 16 found.
• the hypervariable areas can be identified from this on the basis of a comparison with the germline gene V 102. It can be seen from the heavy chain gene (SEQ ID No. 13) that the CDR-1 of the heavy chain comprises 5 amino acids and begins in position 30 of the sequence. The heavy chain CDR-2 begins at position 49 and contains 17 amino acids. The hypervariable region CDR-3 starts at position 98 and contains 11 amino acids.
• the VH gene of the mAb TTC8 has 36 point mutations compared to the germline gene V 102. Three of the mutations lie in the binding area of the PCR primer and can therefore be caused by the sequence of the primer. Almost all mutations in the hypervariable regions lead to a change in the amino acid sequence, while almost half of all mutations in the framework regions are silent.
• CDR-4 begins in position 22 and comprises 11 amino acids.
• the CDR-5 has 7 amino acids and starts at position 48.
• the CDR-6 region starts at position 87 and has 9 amino acids.
• the VL gene of the mAb TTC8 has only 9 compared to the mAb A23 Mutations. 4 of them are in the binding area of the PCR primer. Of the remaining five differences in the nucleotide sequence, two mutations at the protein level are silent. Of the mutations that lead to a change in the amino acid sequence, one is in CDR-4, the other two in framework regions 2 and 3.
• the sequence of the hypervariable regions of the monoclonal antibody 2CV, which recognize the cytotoxin (toxin B) of Clostridium difficile, can be determined in a corresponding manner from the hybridoma cell line deposited at DSMZ under the number DSM ACC 2321.
• mice derived monoclonal antibody elicits an immune response in humans.
• an antibody can be adapted to the species to be treated (here humans), i.e. be humanized.
• species to be treated here humans
• the humanized monoclonal antibodies obtained by such a method are much more suitable for therapeutic use in humans than the antibodies obtained from the mouse.
• humanizing antibodies can also be applied to the mAb TTC8 and other toxin A and toxin B neutralizing monoclonal antibodies and result in a humanized monoclonal antibody that represents a chimera from the hypervariable regions (e.g. for TTC8: SEQ ID No. 1 - 12) of the neutralizing mAb TTC8 inserted into the Framework region of a human immunoglobulin.
• the latter can be of the IgG subtype (preferred for parenteral administration) or of the IgA subtype (preferred for oral administration).
• the hypervariable regions of other monoclonal antibodies directed against Clostridium difficile can also be cloned, sequenced and used to produce humanized antibodies.
• the invention also relates to humanized monoclonal antibodies in which the amino acid sequence is modified by allelic variations within the variable and / or constant regions of the light and / or heavy chains of human immunoglobulin, as long as the ability to bind to toxin A and / or to the toxin B from Clostridium difficile is retained.
• All biological “building blocks” required for the production of the monoclonal antibodies according to the invention are commercially available or generally available. Unless stated otherwise, they are used only as examples and are not critical to the practice of the invention, but can also be replaced by other suitable biological "building blocks”.
• Bacterial host cells are preferably used to amplify the DNA sequences mentioned according to the invention. Examples include E. coli or Bacillus spec.
• the humanized antibodies can be produced in eukaryotic cells such as yeast cells, fungi or, for example, CHO (Chinese hamster ovary cells). Production in plants can take place in monocotyledonous or dicotyledonous plants. The production of antibodies in plants is described below by way of example. The production in other organisms is carried out analogously.
• the finished transformation vector contains all DNA sequences to be transferred into the plants. These are transferred from the vector into the plant cell.
• the genes for the light and the heavy chain can also be incorporated separately into two plant transformation vectors and transferred separately into plant cells in order to combine them later into the finished antibody.
• the transformation of plants can be done by any suitable method, e.g. with the help of Agrobacterium tumefaciens or by direct gene transfer or with the help of the particle gun.
• the production of the antibodies can be directed to individual compartments of the cell.
• the sequence coding for the signal peptide is removed, the antibodies are expressed cytoplasmically.
• a DNA sequence is coupled to the 5 'end of the gene or the naturally existing one is left, which codes for a signal peptide for transport into the endoplasmic reticulum.
• DNA sequences which code for the peptide sequences responsible for them can be fused to or into the gene.
• the incorporation of the antibody genes in the transgenic plant is analyzed and detected by suitable restriction digestion of the isolated genomic plant DNA and subsequent Southern hybridization.
• the transcription of the genes can be detected in the Northern blot.
• the biosynthesized antibody is e.g. detected in plant extracts using a specific secondary antibody or a antibody directed against the constant region or against a specially introduced (so-called tag) sequence.
• Monocot plants such as e.g. Barley and wheat as well as dicotyledonous plants such as the potato, rapeseed, carrot or pea.
• the expression of the antibody can be expressed in various organs of the plants, e.g. in leaves, seeds, tubers or other tissues.
• the purification of the antibody expressed in the plant takes place e.g. with chromatographic methods, which are usually also used for the purification of antibodies from hybridoma cell lines.
• chromatographic methods which are usually also used for the purification of antibodies from hybridoma cell lines.
• recombinant antibodies that contain a tag sequence can also be produced using specially established affinity chromatography methods, e.g. metal chelate chromatography.
• the humanized monoclonal antibodies according to the invention can be administered to the human patient for the therapy and prophylaxis of diseases caused by Clostridium difficile by methods known per se.
• the antibodies or antibody fragments according to the invention are parenterally, but preferably by administered os.
• a special "galenical preparation" is the direct oral intake of plants containing the antibodies in the form of raw food.
• the suitable dosage of the antibodies according to the invention is to be adapted to the respective patient and depends, for example, on his body weight, his age and his condition. It is determined by the experienced doctor and is usually between 0.1 mg / kg and 70 mg / kg, which is administered once or several times a day over a period of several days.
• Example 1 Collection of the total RNA of the hybridoma cells which form the mAb TTC8 (DSM ACC 2322)
• the cells are initially disrupted by guanidinium thiocyanate buffer and then completely destroyed mechanically using an ultrathorax.
• Cell debris is centrifuged off and the solution is applied to a prepared CsCl cushion (5.7 M).
• the RNA pellets onto the bottom of the tube. This bottom is separated with a hot scalpel and the pellet is dissolved by adding H 2 0.
• the RNA is then further purified from the solution by precipitation. At the end it is taken up in 100 ⁇ l H 2 0.
• the concentration of the RNA prepared in this way is usually 1.5-3 ⁇ g / ⁇ l.
• RNA from the other RNA species was applied to 01igo (dT) 25 beads using the method described by Dynal ren hybridized.
• the binding capacity of the beads is 2 ⁇ g RNA per mg beads.
• the proportion of mRNA in the total RNA is about 1-5%, so that 1 mg beads were loaded with 80 ⁇ g total RNA.
• the cleaning was carried out according to the Dynal protocol with the following deviations.
• the bound mRNA was washed twice with a buffer of the composition 10MM Tris / HCl (pH 7.5), 0.15 mM LiCl, ImM EDTA, 1% SDS and then twice with a modified buffer of lower salt concentration [5mM Tris / HCL (pH 7.5), 75mM LiCl, 0.5mM EDTA]. All further steps corresponded to the information from Dynal. The addition of SDS and the additional washing step at low salt concentration (higher stringency) significantly improved the purity of the mRNA.
• CDNA was synthesized from the purified mRNA using MMLV reverse transcriptase (Moloney Murine Leukemia Virus).
• 01igo (dT) 12 was 18 _ 2x primer in excess used to keep high the proportion of the hybridized mRNA Oligo (dT) molecules.
• concentration of the nucleotide triphosphates was greater than ImM.
• a premature increased degradation of the RNA in the solution was counteracted by adding human and placental ribonuclease inhibitor.
• the ssDNA obtained in this way was used in the PCR reactions for the amplification of the variable regions of the mAb TTC8.
• Example 4 The polymerase chain reaction for amplifying the cDNA
• VH5Prim 5 '-AGGTCGACCTGCAG (C / G) AGTC (A / T) GG-3'
• VH3Prim 5 '-ACGGTGACAGTCGACCCTTGGCCCC-3
• VL5Prim 5 '-GACATTGAGCTCACCCAGTCTCCA-3'
• VL3Prim 5 '-GTTTGAGCTCCAGCTTGGTCCC-3'
• the optimal primer concentration for the extraction of the variable regions was 0.5 ⁇ M, the dNTPs were used in a final concentration of 400 ⁇ M.
• the PCR temperature was determined as the second essential parameter.
• the final reaction sequence for VH amplification was: denaturation 1 min at 92 ° C; Annealing 1.5 min 50 ° C; Elongation 3 min 72 ° C, at 30 cycles.
• the final reaction sequence for VL amplification was: denaturation 1 min at 92 ° C; Annealing 1.5 min 60 ° C; Elongation 3 min 72 ° C, at 30 cycles.
• PCR products were cut with Sall (VH) or Sacl (VL) and purified on an agarose gel.
• the cloning was carried out in pUC19 into the homologous interfaces.
• Constructs pVHT8 and pVLT8.10 were identified as target clones by control digestion. They contain the VH and VL segments in a positive orientation with respect to the pLac promoter of pUC19.
• the insert of the pVHT8 has a size of 341 bp, that of the clone pVLT ⁇ .10 one of 312 bp. Both clones each have an open reading frame that spans the entire insert. The sequences of the PCR primers and the interfaces were easy to identify.
• VH segment The assignment of the VH segment to the multi-gene family J558 results from a sequence comparison. The highest degree of homology was found in the germline sequence V102.
• the gene for the VL region of the mAb TTC8 has 94% homology to a sequence of an antibody which is considered to be the germline gene Vkl9d. Accordingly, the VL region of TTC8 can also be assigned to this gene family.
• Example 7 The preparation of a humanized monoclonal antibody
• the immunogenic regions of the mAb must be replaced by homologous human sequences. There are two main options:
• Regions of a human antibody are coupled to the sequences for the variable region of the nonhuman mAb.
• a complete chimeric antibody can be produced or only a part of the chimeric antibody, a so-called. Fab fragment [3]. The production of chimeric antibodies has the
• a human antibody is first identified by sequence comparison and 3D modeling, the structure of which roughly corresponds to that of the original antibody. Subsequently, by using five oligonucleotide primers that bind in the sequence of the variable region of the human antibody (VL or VH) and three oligonucleotides that contain the sequences of the CDR regions of the mouse antibody, with the help of each other constructing PCR amplifications complete, humanized gene for the variable portion of the antibody generated [4].
• VL or VH variable region of the human antibody
• a second modified approach is to assemble the gene from several synthetic, overlapping oligonucleotides that code for the target sequence [for strategy and implementation see 4].

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• 1997
  • 1997-09-10 DE DE19739685A patent/DE19739685A1/de not_active Ceased
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