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Title:  Helicobacter pylori adhesin binding group antigen
United States Patent: 
7,271,251
Issued: 
September 18, 2007

Inventors: 
Boren; Thomas (Umea, SE), Arnqvist; Anna (Umea, SE), Hammarstrom; Lennart (Huddinge, SE), Normark; Staffan (Stockholm, SE), Ilver; Dag (Umea, SE)
Appl. No.: 
10/761,201
Filed: 
January 22, 2004


 

Covidien Pharmaceuticals Outsourcing


Abstract

A novel Helicobacter pylori blood group antigen binding (BAB) adhesin protein was isolated and purified, whereby said protein or fractions thereof bind specifically to fucosylated blood group antigens. The protein sequence of said adhesin is disclosed in this application. Simultaneously the DNA sequences for two genes, babA and babB, producing highly similar proteins, are disclosed. Said adhesin and/or DNA is useful for diagnose and therapy and/or prophylaxis directed against H. pylori induced infections, e.g. gastritis and acid peptic disease, i.e. active vaccination.A new immunoglobulin composition, which exhibits specific activity to a Lewis.sup.b antigen binding Helicobacter pylori adhesin, or preferably, monoclonal and/or polyclonal antibodies to said adhesin offer a new and more efficient method of treatment and/or prevention of gastrointestinal diseases, caused by Helicobacter pylori or other Helicobacter species, i.e. passive vaccination.

SUMMARY OF THE INVENTION

The above problem of providing specific, cost-efficient and therapeutically superior immunoglobulin preparations for the treatment and prevention of H. pylori has now been solved through the composition and methods according to the attached patent claims. The present inventors have now surprisingly shown, that highly specific and therapeutically efficient polyclonal and/or monoclonal immunoglobulin preparations can be provided through the immunization of an animal with an adhesin protein, specific for H. pylori. The invention will now be described in closer detail with reference to the attached, non-limiting figures and examples.

One objective of the present invention was to further purify and characterize the H. pylori blood group antigen binding (BAB) adhesin to make possible the development of methods and materials for specific and selective diagnosing and treatment of H. pylori induced infections and related diseases and the development of said methods and materials. A further and equally important objective was to determine the DNA sequences of the genes involved in the expression of this protein. These objectives were fulfilled through the protein, the DNA and the methods and materials specified herein The DNA sequences are SEQ ID NOS: 1 and 2, disclosing the babA (SEQ ID NO:1) and babB (SEQ ID NO:2) sequences, respectively. The full protein sequences are disclosed in SEQ ID NOS: 3 and 4.

DESCRIPTION OF THE INVENTION

The blood group antigen binding adhesin, BabA, has now been biochemically characterized and purified by a novel technique, receptor Activity Directed Affinity Tagging (Retagging). Two genes, babA and babB were found to code for two different but very similar proteins. The present invention thus comprises a novel blood group antigen binding adhesin. The DNA sequences are disclosed in SEQ ID NO:1 (babA) and SEQ ID NO:2 (babB). The protein sequences are disclosed in SEQ ID NOS: 3 and 4. The invention also includes any pharmaceutical composition comprising said adhesin protein and/or fractions thereof. Examples of such pharmaceutical compositions are for example medicaments for the prevention or treatment of Helicobacter pylori induced gastritis, gastric and duodenal ulcers and gastric adenocarcinoma. Optionally said pharmaceutical composition additionally encompasses pharmaceutically acceptable excipients.

Further, the present invention comprises the BAB-adhesin gene or genes for expression of an adhesin protein according to the invention. Said invention also comprises a novel method for the isolation and purification of said adhesin. The disclosed genes are contemplated to function as a cassette system, the organism alternating between these to avoid immunity in the host. It is very likely, that homologies of the disclosed sequences exist and additionally supplement said cassette function in other strains of H. pylori. Also, genes corresponding to a homology of the first 40 amino acids or genes, corresponding to a homology of the last, about 300 amino acids, can function to this effect. It is further highly likely, that Helicobacter pylori is able to switch between several genes, similar to the disclosed genes, in a so-called cassette system.

The invention additionally comprises monospecific antisera produced using the novel adhesin protein and/or fractions thereof. Said monospecific antisera is preferably produced according to any suitable, conventional method for producing monospecific antisera in vitro or in vivo, e.g. by inoculating a suitable animal. Such methods are familiar to a person skilled in the art. Antibodies raised in a suitable animal or in the patient to be treated, can subsequently be administered locally, e.g. orally to the patient.

The invention further comprises the use of said monospecific antisera for the manufacturing of a test kit for quantitative or qualitative determinations of adhesin protein or fractions thereof in cells, tissues or body fluids.

The invention further comprises the use of said adhesin protein or corresponding DNA for use in therapy or immunisation and/or in the manufacture of compositions for said uses. The invention specifically encompasses the use of said DNA for immunisation therapy and for the manufacture for compositions for such therapy. Preferably, in an immunisation therapy where said composition is administered orally to a patient, the adhesin protein, fractions thereof or said DNA is administered in combination with a pharmaceutically suitable immunostimulating agent. Examples of such agents include, but are not limited to the following: cholera toxin and/or derivatives thereof, heat labile toxins, such as E. coli toxin and similar agents. The composition according to the present invention can further include conventional and pharmaceutically acceptable adjuvants, familiar to a person skilled in the art of immunisation therapy. Preferably, in an immunisation therapy using the inventive DNA or fractions thereof, said DNA is preferably administered intramuscularly, whereby said DNA is incorporated in suitable plasmide carriers. An additional gene or genes encoding a suitable immunostimulating agent can preferably be incorporated in the same plasmide.

Said immunisation therapies are not restricted to the above-described routes of administration, but can naturally be adapted to any one of the following routes of administration: oral, nasal, subcutaneous and intramuscular. Especially the oral and nasal methods of administration are promising, in particular for large-scale immunisations.

The present inventors have surprisingly shown, that highly specific and therapeutically efficient polyclonal and/or monoclonal immunoglobulin preparations can be provided through the immunisation of an animal with an adhesin protein or fractions thereof, specific for H. pylori. When considering immunisation against H. pylori, it is worth noting that the infection is known to be lifelong despite a vigorous immune response in the gastric mucosa. An increased local production of IgA in the mucosa is not necessarily enough and the administration of monospecific antibodies directed against a central virulens factor, such as the adhesin according to the present invention, may constitute a more effective approach.

The term "immunisation" refers here to a method for inducing a continous high level of antibody and/or cellular immunresponse. The term "animal" here preferentially denotes any member of the subphylum Vertebrata, a division that includes all animals, including mammals, which are characterized by a segemented bony or cartilaginous spinal column. All vertebrates have a functional immune system and respond to antigens by producing antibodies. The term "protein" is used here to denote a naturally occurring polypeptide and the term "polypeptide" is used here in its widest meaning, i.e. any amino acid polymer (dipeptide or longer) linked through peptide bonds. Accordingly the term "polypeptide" comprises proteins, oligopeptides, protein fragments, analogues, muteins, fusion proteins and the like. The term "antibody" as used in this context includes an antibody belonging to any of the immunological classes, such as immunoglobulins A, D, E, G or M. Of particular interest are nevertheless immunoglobulin A (IgA) since this is the principle immunoglobulin produced by the secretory system of warm-blooded animals. However, in cow colostrum, the main antibody class is IgG 1.

Boren et al. have recently isolated and characterized a Lewis.sup.b binding protein with a molecular weight of about 73500 Da (See the priority applications SE 9602287-6 and SE 9701014-4, which are referred to in their entirety). This adhesin protein is thought to be a conserved structure and specific for pathogenic strains of H. pylori. Said protein is specific for at least one of the H. pylori strains included in the following group: CCUG 17875, NCTC 11637, A5, P466, G109, G56, Ba 185, Ba 99, 931 and 932.

This adhesin protein or immunologically effective fractions thereof are characterized in that the following amino acid sequence (SEQ ID NO:5) is included:

EDDGFYTSVGYQIGEAAQMV or homologues thereof.

The following DNA sequence (SEQ ID NO:6) or homologues thereof is included in DNA for expression of said adhesin protein or fractions thereof:

5'-GAAGACGACGGCTTTTACACAAGCGTAGGCTATCAAATCGGT

GAAGCCGCTCAAATGGTA-3'

According to one embodiment of the invention, a pregnant mammal, preferably a cow or another suitable domestic animal, is immunised with said Lewis.sup.b binding adhesin protein or fractions thereof. The adhesin protein or fractions thereof is/are preferably injected intramuscularly or subcutaneously in the chosen animal, optionally together with suitable adjuvants. Examples of such adjuvants include, but are not limited to immunostimulating agents such as cholera toxin and/or derivatives thereof, heat labile toxins, such as E. coli toxin and similar, conventional agents, such as classical adjuvants including mineral and vegetable oils. Subsequent to the regimen of immunization, comprising a necessary amount of doses, including so called booster-doses, over a time span allowing for optimal immunoglobulin expression, milk or sera is collected from said animal. Preferably the cow colostrum, which is specially high in immunoglobulins, is collected. The specific immunoglobulin fraction according to the present invention is then separated and purified in a conventional manner, e g including separation of fats, protein precipitation and concentration by ultrafiltration.

According to another embodiment of the invention, a bird, preferably a chicken or another suitable domestic bird, is immunized with said Lewis.sup.b binding adhesin protein or fractions thereof. The adhesin protein or fractions thereof is preferably injected intramuscularly or subcutaneously in the chosen bird, optionally together with suitable adjuvants. Examples of such adjuvants include, but are not limited to immunostimulating agents such as cholera toxin and/or derivatives thereof, heat labile toxins, such as E. coli toxin and similar, conventional agents, such as classical adjuvants including mineral and vegetable oils. Subsequent to the regimen of immunization, comprising a necessary amount of doses, including so called booster-doses, over a time span allowing for optimal immunoglobulin expression, sera or eggs is/are collected from said animal. Preferably the egg yolk, which is specially high in immunoglobulins, is collected. The specific immunoglobulin fraction according to the present invention is then separated and purified in a conventional manner, e g including protein precipitation and ultrafiltration. Alternatively, the egg yolk being of high nutritional value in addition to containing a high titer of specific antibodies according to the present invention, can be administered as such.

According to a preferred embodiment of the present invention, monoclonal immunoglobulin is produced by establishing transgenic animals. Said transgenic animals can be chosen from the following group of species: mammals, e.g. cow, goat and rabbit, and birds: e.g. chicken, duck, turkey. The mammal most preferably used is cow and the most preferable bird is chicken. Further developments of transgenic animals such as mice and rats could also offer new possibilities. The choice of animal is naturally governed by availability and local adaptation.

According to one embodiment, a stock of transgenic animals according to the present invention, adapted to the local conditions, are kept locally, e.g. in villages in developing countries to function as local units for the production of immunoglobulins for oral administration. For example transgenic cows, goats or chicken are suitable for this purpose and preferably chicken are used. Consumption of the milk or preferably the eggs, produced by the transgenic animals, can help to eradicate presently very difficult infectious diseases, e.g. diseases caused by H. pylori.

According to yet another embodiment of the present invention, monoclonal antibodies can be produced using the hybridoma method. The hybridoma method is well known to a skilled worker in the field of biochemistry and it is described e. g. in Galfre, G. And Milstein, C., Preparation of monoclonal antibodies: strategies and procedures (Methods in Enzymology, 73:3 46, 1981). A suitable host animal is immunized with the Lewis.sup.b binding adhesin protein or fractions thereof. When the immunization is accomplished, the animal is sacrificed, spleen cells collected and fused with cells from a neoplastic cell line, preferably myeloma cells. By choosing the growth conditions, the successfully fused hybridoma cells can be selected. The monoclonal antibodies produced by the hybridoma cell line can then be administered orally in a regimen for treatment and/or prevention of H. pylori infections.

Preferably the polyclonal and/or monoclonal antibodies are purified prior to administration and, more preferably, admixed with pharmaceutically suitable carriers and/or adjuvants. Examples of suitable carriers are saline, pharmaceutically acceptable fats, oils, carbohydrates and proteins. The carrier or carriers is/are preferably chosen so that the solubility and absorption of the immunoglobulin in the mucus layer lining the stomach is enhanced. Using suitable adjuvants the stability, therapeutic efficiency and nutritional value of the composition can be improved. To improve stability under storage, the immunoglobulin composition can be lyophilized. Regardless of the exact preparation and formulation, it is of central importance to avoid denaturating the immunoglobulins.

The higher specificity, exhibited by the immunoglobulin preparation of polyclonal and/or monoclonal antibodies according to the invention, makes it possible use substantially lower doses compared to those presently used, thus lowering the cost and improving the availability of the treatment. The use of specific, monoclonal antibodies can make it possible to further lower the doses. The doses are in all cases a function of the antibody titer of the preparation. A high titer naturally allows the use of lower doses.

According to one embodiment of the invention, an immunoglobulin preparation is manufactured as follows: an animal is immunized with a Lewis.sup.b binding adhesin protein or fractions thereof, expressed by Helicobacter pylori, the immunoglobulin fraction is isolated from a excretion of said animal and subsequently purified. The purified immunoglobulin composition is admixed with suitable carriers and adjuvants to form a immunoglobulin preparation for the prevention or treatment of H. pylori infections. In cases where the antibody titer is sufficiently high and the other constituents of the immunoglobulin composition isolated from the animal are harmless, for example in the case of colostrum from immunized cows or egg yolk from immunized chicken, there is always the option of administering the colostrum or egg yolk to the patient without any further treatment of the colostrum or egg yolk.

The immunoglobulin composition according to the invention is preferably administered orally to the patient, in the smallest therapeutically or prophylactically effective dose. Presently conceived are doses in the interval of 0.1 to 1000 mg/day, preferably in the interval of 0.1 to 100 mg/day. The chosen doses naturally depend on the antibody titer of the preparation in question. The exact doses and the regimen of administration can be chosen by the physician responsible for the patient, infected by Helicobacter pylori. Routine experimentation and later, with increasing experience of this method, empirical information will suffice to establish the required amount. Multiple dosages may be used, as needed, to provide the desired level of therapeutic or profylactic effect. The immunoglobulin preparations according to the present invention can also, being free from adverse side effects and imposing practically no danger of overdosing, be taken prophylactically or therapeutically by a person without medical supervision.

A therapeutical effect can be attained, except with the specific antibody according to the present invention, also with at least two Fab-fragments of said antibody. Said embodiment is also encompassed by the scope of the present invention.

According to yet another embodiment, avirulent microorganisms, preferably bacteria, are used as expression systems for the specific antibody according to the present invention. An "avirulent microorganism" in this context is a microorganism which has the ability to colonize and replicate in an infected individual, but which does not cause disease symptoms associated with virulent strains of the same species of microorganism. The definition inherent in the GRAS (Generally Regarded As Safe) concept can be applied here. A GRAS-organism is suitable for use according to the present invention, provided that the organism externalises the antibody or can be modified to this effect The term "microorganism" as used herein includes bacteria, protozoa and unicellular fungi. Preferably, bacteria are used as expression systems, e.g. bacteria of the genus Lactobacillus, Streptococcus or Enterobacteriae. The above mentioned expression system can be utilised in vitro for the production of the specific antibody according to the present invention or, according to a further embodiment of the invention, the micro-organism constituting the expression system can be administered directly to the patient. The micro-organisms can be harvested and administered as such, but they are preferably mixed with a suitable carrier, mixed in a suitable foodstuff, lyophilised, encapsulated or treated in any other conventional way, used for the delivery of viable micro-organisms to the gastrointestinal tract.

According to yet another embodiment, avirulent microorganisms, preferably bacteria, are used as expression systems for the specific adhesin protein according to the present invention. An "avirulent microorganism" in this context is a microorganism which has the ability to colonize and replicate in an infected individual, but which does not cause disease symptoms associated with virulent strains of the same species of microorganism. The definition inherent in the GRAS (Generally Regarded As Safe) concept can be applied here. A GRAS-organism is suitable for use according to the present invention, provided that the organism externalises the adhesin protein or can be modified to this effect. The term "microorganism" as used herein includes bacteria, protozoa and unicellular fungi. Preferably, bacteria are used as expression systems, e.g. bacteria of the genus Lactobacillus, Streptococcus or Enterobacteriae. The above mentioned expression system can be utilised in vitro for the production of the specific adhesin according to the present invention or, according to a further embodiment of the invention, the micro-organism constituting the expression system can be administered directly to the patient. The micro-organisms can be harvested and administered as such, but they are preferably mixed with a suitable carrier, mixed in a suitable foodstuff, lyophilised, encapsulated or treated in any other conventional way, used for the delivery of viable micro-organisms to the gastrointestinal tract.

The exact doses and the regimen of administration of said micro-organisms can be chosen by the physician responsible for the patient, infected by Helicobacter pylori. Routine experimentation and later, with increasing experience of this method, empirical information will suffice to establish the required amount. Multiple dosages may be used, as needed, to provide the desired level of therapeutic or prophylactic effect. The avirluent micro-organism expressing the antibody or adhesin protein according to the present invention can also, being free from adverse side effects and imposing practically no danger of overdosing, be taken prophylactically or therapeutically by a person without medical supervision. A preferred carrier in this specific application is a foodstuff, e.g. a fermented product such as fermented cereal or dairy product.

The creation of previously mentioned expression systems and still earlier mentioned methods of creating hybridomas and transgenic animals can include steps involving recombinant DNA techniques. Recombinant DNA techniques are now sufficiently well known and widespread so as to be considered routine. In very general and broad terms, recombinant DNA techniques consist of transferring part of the genetic material of one organism into a second organism, so that the transferred genetic material becomes a permanent part of the genetic material of the organism to which it is transferred. Methods for achieving this are well known and the mere choice of specific methods for achieving the objectives, set out in the present description and claims, fall under the scope of the invention.

It is possible, that H. pylori alone or together with related slow-acting bacteria are involved in the genesis and aggravation of other chronic inflammatory diseases in the gastrointestinal tract. It is obvious for a skilled practitioner how to modify the present invention, within the scope of the claims, to gain utility in the treatment and/or prevention of such diseases. Examples of such diseases are ulcerative colitis, Crohn's disease, sarcoidosis, Wegener's granulomatosis and other vasculithic disorders, as well as various neoplasms, including carcinomas of the colon, pancreas and prostate.
 


Claim 1 of 7 Claims

1. A test kit comprising a monospecific antisera that recognizes a BabA antigen and comprises an immunoglobulin that binds said BabA antigen via a variable region, wherein said Bab A adhesin protein comprises SEQ ID NO:5 and is produced using an isolated and purified bacterial blood group antigen binding protein (BabA) from Helicobacter pylori species, wherein said BabA protein binds specifically to fucosylated Lewis.sup.b type I and H-1 blood group antigen-glycoconjugates and, wherein said BabA protein contains less than 20% bacterial protein impurities, has a molecular weight in the interval of 70 to 77 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and is not a HopA, HopB, HopC, HopD, or HopE protein.

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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