Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,...
Reexamination Certificate
2000-03-27
2003-12-23
Navarro, Mark (Department: 1645)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
C424S141100, C424S150100, C424S164100, C424S167100, C530S350000, C530S387100, C530S388100, C530S388200, C530S388400, C530S389500
Reexamination Certificate
active
06667035
ABSTRACT:
The object of the invention are amino acid sequences (peptides) produced from antibody producing cells, particularly monoclonal antibody producing hybridoma cells, that neutralize the effect of the
Clostridium difficile
enterotoxin and/or cytotoxin. In addition, there is a description of humanized, monoclonal antibodies for use against the
Clostridium difficile
toxins, as well as the hypervariable regions of these antibodies. Finally a process for the production and application of these amino acid sequences (peptides) and monoclonal antibodies is shown.
It is known that the introduction of macrolide antibiotics such as Clindamycin leads to severe bowel diseases, which manifest themselves as diarrhoea, proceeding further to occasionally fatal pseudomembranous colitis (PMC). This connection gave the disease the name “Clindamycin associated diarrhoea”. We now know that nearly all the antibiotics and cytostatic agents used in medicine can trigger the clinical symptoms of PMC.
PMC is characterised clinically by severe diarrhoea that may lead to death due to heavy electrolyte and liquid losses. Depending on the severity of the symptoms, abdominal pain, bloody diarrhoea, fever and leukocytosis occur. Treatment has hitherto consisted in stopping of the administration of the antibiotic causing the disease, by the administration of Vancomycin, as well as balancing liquid and electrolyte losses.
The aetiological agent of pseudomembranous colitis was for a long time unknown. Only in 1977 could hitherto unknown toxic activity be demonstrated in a stool specimen, inducing a cytotoxic effect on CHO cells (Chinese hamster ovary carcinoma cells). Through further investigations it could finally be proved that the pseudomembranous colitis was caused by
Clostridium difficile
and its toxins.
Clostridium difficile is
an obligate anaerobic, gram-positive rod bacteria that builds subterminal oval spores. It is characterised biochemically by being able to ferment monosaccharides such as glucose, N-acetylglucosamine and N-acetylneuraminic acid, but not mannose, xylose or arabinose.
Clostridium difficile
is also not able to split these monosaccharides off from the side chains of the gastrointestinal mucin as enzymes such as neuraminidase, beta-galactosidase or sialidase are missing. Due to these biochemical shortcomings
Clostridium difficile
cannot flourish in the stomach of healthy individuals. If the gut flora are disturbed through the administration of antibiotics or cytostatic agents,
Clostridium difficile
outgrows the gut flora, leading to PMC.
Clostridium difficile
produces two major factors of pathogenicity, the enterotoxin (toxin A) and the cytotoxin (toxin B). Their production, purification and properties, together with their use to produce monoclonal antibodies are described in detail in European patents 153 519 and 209 273, U.S. Pat. Nos. 4,879,218 and 5,098,826 as well as international patent application WO 91/18 293.
Antibodies clearly play an important part in protecting against the consequences of infection with
Clostridium difficile
. Antibody titres against toxin A and toxin B could be established in patients suffering from PMC. After antibiotic treatment and infection with toxinogenic
Clostridium difficile
strains, hamsters develop the symptoms of PMC and die of it. Prior immunisation of the animals with the above-mentioned toxins protects them from disease. The toxins can be neutralised by antibodies, which are directed against the C-terminal repetitive ligand domains, the central translocation domain or the N-terminal catalytic domain (see reference [1] of the bibliography regarding domain structure). The antibodies directed against the C-terminal repetitive ligand domains hinder the binding of the toxins to the cell receptors, the antibodies directed against the N-terminal catalytic domain block the glucosylation reaction imparted by the toxins, while the antibodies directed against the central translocation domain restrain the translocation of toxins into the cell.
Antibodies neutralising toxin A and/or toxin B thus offer a possible therapy and/or prophylaxis for
Clostridium difficile
diseases in which they do not eliminate the bacteria but they block the action of the toxins produced. In this way the development of the symptoms of the disease can be hindered by acting on the toxins responsible.
Taking toxin A as an example, the cell line DSM ACC 2322 produces an antibody TTC8, which not only binds to toxin A, but is also able to neutralise its biological action. The binding site of TTC8 in toxin A was identified within the repetitive ligand domain. By binding of the antibodies to the toxins, their interaction with the cell receptors is. hindered. The binding site of mAb TTC8 is within amino acids 2480-2539 of toxin A with the (probable) antigen sequence TINGKKYYF (SEQ ID No. 17). A mAb PCG-4 known from U.S. Pat. No. 4,879,218 binds to a defined protein fragment through the amino acids 2098 to 2141 of toxin A.
The mAb 2CV produced by the cells DSM ACC 2321 also binds to a sequence of the ligand domain, in this case of toxin B. The binding occurs in a protein fragment between aa 2233 and 2366 of toxin B. Both monoclonal antibody PCG-4 and monoclonal antibody TCC8 are mouse antibodies, which are not used in therapy on humans but would be suitable as a diagnostic aid.
The monoclonal antibodies generated in the laboratory of the inventor and their reactivities are indicated below:
Directed against
toxin A
a)
Toxin B
a)
Catalytic domain
b)
1212
2612, 2688, 3110
1502, 1115
Translocation domain
b)
2825, 2836,
2703, 2740,
5288
2747, 2754,
5288, 2784,
2788
ligand domain
b)
2620, TTC8
2912, 2914,
2916, 2926,
2562, 2CV
Notes:
a)
The antibodies recognise recombinant protein of the listed regions in the Western blot procedure.
b)
The amino acid position of the domains in toxins A and (B) are: catalytic domain: 1-879 (1-877); translocation domain: 880-1848 (878-1850); ligand domain: 1849-2681 (1851-2360).
All the hitherto generated and described toxin specific monoclonal antibodies were obtained from mice. Therapeutic or prophylactic use of these antibodies in species other than mice (humans or other animals) is not possible since the antibodies are recognised as from a different species, and induce an immune reaction through which they are inactivated. Their adaptation to other species, particularly to humans, has not hitherto been possible since their variable and hypervariable regions were not sequenced (i.e. were not defined).
The objective of the invention was to provide peptides which possess toxin neutralising properties but no longer contain a strongly immunogenic portion, so the peptide is suitable for use in human and veterinary medicine. The neutralisation of the toxin's effect is based on the binding of the antibodies through the attachment of the antibody variable regions to the respective toxin. This binding is mainly determined through the hypervariable regions (or CDR, complementarity determining regions). It was therefore necessary to identify the regions (variable and hypervariable (CDR)) responsible for binding and neutralising the toxins, and to adapt them in such a way that they no longer triggered an immune reaction. Such peptides can be used both for the therapy of already existing
Clostridium difficile
diseases and for protection against such diseases in human and veterinary medicine.
The hitherto existing scientific results can be summarised in that, on the one hand, the toxin A of
Clostridium difficile
is able to trigger the full symptoms of pseudomembranous colitis (PMC) and on the other hand that the monoclonal antibody TTC8 directed against this toxin can neutralise the effect of this toxin during in vivo experiments (in mice). The mAb TTC8 is an antibody of the class IgG 2b. The specific recognition between mAb TTC8 and toxin A of
Clostridium difficile
is mediated by the hypervariable regions of the heavy and light chains. This means that exactly defined sections of the antibody (in other words, peptides) are responsible
Moos Michael
Von Eichel-Streiber Christoph
Morrison & Foerster / LLP
Navarro Mark
Von Eichel-Streiber Christoph
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