Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/36—Adaptation or attenuation of cells
• • 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
  • A61K39/02—Bacterial antigens
  • A61K39/025—Enterobacteriales, e.g. Enterobacter
  • A61K39/0258—Escherichia
• • 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
  • A61K39/02—Bacterial antigens
  • A61K39/025—Enterobacteriales, e.g. Enterobacter
  • A61K39/0275—Salmonella
• • 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
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
• • 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
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • 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 present invention relates to attenuated bacterial mutants, in particular attenuated Salmonella enterica mutants, and to a live attenuated vaccine comprising same.
• Salmonellae are Gram-negative, facultative anaerobic, motile, non-lactose fermenting rods belonging to the family Enterobacteriaceae. Salmonellae are usually transmitted to humans by the consumption of contaminated foods and cause Salmonellosis. [0003] Salmonellae have been isolated from many animal species including, cows, chickens, turkeys, sheep, pigs, dogs, cats, horses, donkeys, seals, lizards and snakes. [0004] 95% of the important Salmonella pathogens belong to Salmonella enterica, with S. enterica serovar Typhimurium (S. Typhimurium) and S. enterica serovar Enteritidis (S. Enteritidis) as the most common serovars .
• S. enterica serovar Typhimurium S. Typhimurium
• S. enterica serovar Enteritidis S. Enteritidis
• Salmonella infections are a serious medical and veterinary problem world-wide and cause concern in the food industry. Contaminated food can not be readily identified. [0006] Control of Salmonellosis is important, to avoid potentially lethal human infections and considerable economic losses for the animal husbandry industry.
• Whole-cell killed vaccines and subunit vaccines are used in the prevention of Salmonella infections in animals and in humans with variable results. Inactivated vaccines in general provide poor protection against Salmonellosis .
• An object of the present invention is to provide attenuated Salmonella enterica. strains.
• Another object of the present invention is to provide a live attenuated vaccine against Salmonellosis and methods of prevention based thereon.
• Yet another object of the present invention is to provide live attenuated strains which are useful as live vector and as DNA-mediated vaccines expressing foreign antigens. Such strains are highly suitable for the development of vaccines including polyvalent vaccines.
• Still another object of this invention is to provide a method to achieve a S. enterica deletion mutant for use in a live attenuated vaccine.
• Yet a further object of this invention is to provide the same materials and methods for the preparation of attenuated strains of bacteria infecting veterinary specie, poultry more in particular.
• the general aim is to improve food safety and animal health.
• a first aspect of the invention relates to an attenuated Salmonella, enterica mutant strain which is incapable of forming de novo guanine nucleotides, and wherein said mutant contains a deletion mutation in the guaB gene .
• the principle as demonstrated here for S. enterica can be applied to any organism that can use the guanine nucleotide as an intermediary.
• An aspect of the invention therefore relates to an attenuated mutant strain of a bacterium infecting veterinary species that contains a deletion mutation in the guaB gene.
• the term "bacterium infecting veterinary species” in the context of the invention refers in particular to bacteria that are pathogenic to veterinary species, and which can be attenuated by a deletion mutation in the guaB gene.
• the bacterium infecting veterinary species may be a Gram- negative bacterium.
• Preferred are Gram-negative bacteria for poultry such as Salmonella, Pasteurella, Escherichia coli, etc. Most preferred are Salmonella enterica and (pathogenic) E. coli.
• pathogenic to is meant that the bacterium, if not attenuated, is capable of causing an infectious disease in the veterinary species .
• the mutants of the invention fail to express a functional GuaB gene product. In other words the guaB gene function is impaired, whereby an auxotrophic attenuated strain is obtained.
• the mutant strain of the invention carrying a deletion mutation in the guaB gene, can not grow on Minimal A medium, unless this medium is supplemented with 0.3mM guanine, xanthine, guanosine or xanthosine.
• the present invention in particular aims to provide attenuated S. Enteritidis and S. Typhimurium strains .
• the deletion mutation in the guaB gene is introduced into parent strain S. Enteritidis phage type 4 strain 76Sa88 or into parent strain S. Typhimurium 1491S96.
• One of the attenuated S. enterica strains obtained according to the invention is a S. Enteritidis strain SM69 having the deposit number LMG P-21641.
• Another example is the S. Typhimurium strain SM86 having the deposit number LMG P-21646.
• the attenuated mutant strains of the invention are highly suitable for use in a live attenuated vaccine including a polyvalent or multivalent vaccine.
• the attenuated mutant strain of the invention may encode and express a foreign antigen and may as such be used as a live vector and/or as a DNA-mediated vaccine.
• a second aspect of the invention concerns a pharmaceutical composition or a vaccine for immunizing a veterinary species against a bacterial infection (e.g. Salmonellosis caused by Salmonellae) comprising-.
• a bacterial infection e.g. Salmonellosis caused by Salmonellae
• compositions are those comprising a Salmonella enterica mutant strain according to the invention.
• the attenuated strain of the invention may transfer DNA encoding a foreign antigen in a eukaryotic cell.
• This foreign antigen may be encoded by and expressed from a plasmid comprised by the attenuated mutant strain of the invention.
• an immunizing dose varies according to the route of administration. Those skilled in the art may find that the effective dose for a vaccine administered parenterally may be smaller than a similar vaccine which is administered via drinking water, and the like.
• the attenuated strains of the invention, and pharmaceutical compositions or vaccines comprising same, are highly suitable for immunizing an animal or veterinary species against a bacterial infection (e.g. Salmonellosis) and possibly other diseases (in the case of a multivalent vaccine) .
• a further aspect of the invention therefore concerns a method of immunizing animals, preferably veterinary species, more preferably poultry such as chicken against an infection by a bacterium (e.g.
• said method comprising the step of: administering to the animal or veterinary species in need thereof an immunizing amount of an attenuated mutant strain of the invention and/or of a vaccine comprising same, whereby a protective immune response is then invoked in the animal or veterinary species .
• veterinary species to be immunized against Salmonellosis poultry, small or heavy livestock such as chicken, turkey, ducks, quails, guinea fowl, pigs, sheep, young calves, cattle etc.
• a last aspect of the invention relates to a mutant strain of the invention for use as a medicament
• Yet another aspect of the invention relates to the use of an attenuated mutant strain of the invention for the preparation of a medicament, such as a vaccine, for the prevention (and/or treatment) of a disease caused by a pathogen (the infecting bacterium) such as Salmonellosis.
• a pathogen the infecting bacterium
• Salmonellosis Salmonellosis
• guaB gene can lead to attenuated Salmonella enterica strains with significantly reduced virulence and capable of inducing an immune response in a livestock animal. Such deletion would attenuate any organism that can use the guanine nucleotide as an intermediary.
• gene refers to the coding sequence and its regulatory sequences such as promoter and termination signals.
• the deletion mutant according to the invention is one in which the purine metabolic pathway enzyme IMP dehydrogenase (encoded by guaB) is inactivated.
• Such inactivation may be obtained via a deletion by which the guaB gene function is impaired, leading to a null-function (no functional gene product formed) of the affected gene(s) .
• a person skilled in the art knows how to obtain such mutants and a simple test can tell whether the guaB gene function is impaired.
• the mutant strain which fails to express a functional guaB gene product cannot grow on Minimal A medium, unless this medium is supplemented with (e.g. 0.3mM) guanine, xanthine, guanosine or xanthosine.
• the invention aims to provide, amongst others, attenuated S. Enteritidis and S. Typhimurium strains since these are the most common S. enterica serovars .
• the present invention provides attenuated strains.
• the invention provides amongst others attenuated Salmonella enterica strains for use, inter alia, as live attenuated vaccines against Salmonellosis, as live vector and/or as DNA-mediated vaccines expressing foreign antigens.
• a "foreign antigen” means an antigen foreign to Salmonella.
• Live vector vaccines also called “carrier vaccines” and “live antigen delivery systems” comprise an exciting and versatile area of vaccinology (Levine et a.1, 1990, Microecol . Ther. 19:23-32).
• a live viral or bacterial vaccine is modified so that it expresses protective foreign antigens of another microorganism, and delivers those antigens to the immune system, thereby stimulating a protective immune response.
• Live bacterial vectors that are being promulgated include, among others, attenuated Salmonella.
• An object of the invention is to provide attenuated strains, like attenuated S. enterica strains for use in a live vaccine, possibly a polyvalent or multivalent live vaccine.
• One of the objects of the invention is therefore to provide a vaccine against e.g. Salmonellosis comprising :
• a pharmaceutically effective or an immunizing amount of a mutant of the invention e.g. a Salmonella enterica mutant
• a mutant of the invention which is incapable of forming de novo guanine nucleotides, wherein said mutant contains a deletion mutation in the guaB gene
• Another object of the invention is to provide a live vector vaccine comprising:
• a pharmaceutically effective or an immunizing amount of a mutant of the invention e.g. a Salmonella enterica mutant
• a mutant of the invention e.g. a Salmonella enterica mutant
• said mutant contains a mutation in the guaB gene, and wherein said mutant encodes and expresses a foreign antigen
• Still another object of the invention is to provide a DNA-mediated vaccine comprising:
• a pharmaceutically effective amount or an immunizing amount of a mutant of the invention e.g. a Salmonella enterica mutant
• a mutant of the invention e.g. a Salmonella enterica mutant
• said mutant contains a mutation in the guaB gene
• said mutant contains a plasmid which encodes and expresses in a eukaryotic cell, a foreign antigen
• DNA- mediated vaccines a pharmaceutically acceptable carrier or diluent .
• S. enterica using a prokaryotic promoter in a live vector vaccine
• S.g. S. enterica using a prokaryotic promoter in a live vector vaccine
• enterica (using a eukaryotic promoter in a DNA-mediated vaccine) may be based upon which vaccine construction for that particular antigen gives the best immune response in animal studies or in clinical trials, and/or, if the glycosylation of an antigen is essential for its protective immunogenicity, and/or, if the correct tertiary conformation of an antigen is achieved better with one form of expression than the other (US Patent 5,783,196) .
• the pharmaceutically effective amount or the immunizing amount of the mutants of the present invention to be administered will vary depending on the age, weight and sex of the subject.
• an “immunizing amount” as used herein is in fact meant an amount that is able to induce an immune response in the animal that receives the pharmaceutical composition/vaccine.
• the immune response invoked may be a humoral, mucosal, local and/or a cellular immune response.
• the particular pharmaceutically acceptable carrier or diluent employed is not critical to the present invention, and are conventional in the art. Examples of diluents include: buffer for buffering against gastric acid in the stomach, such as citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) alone, or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose, and optionally aspartame.
• the deletion mutants according to the invention have been created via standard homologous recombination techniques, whereby part of the guaB gene, for instance part of the guaB coding sequence, in a first step is replaced by a resistance gene and flanking FRT sites. [0059] Preferably, in a second step, said resistance gene is removed by recombination between the two FRT sites. One FRT site and the priming sites Pl and P2 remain by the molecular mechanism of the recombination removing the antibiotics resistance gene according to Datsenko and Wanner (2000) (see for instance Figure 4) .
• a particular example of the invention relates for instance to guaB deletion mutants of S. Enteritidis that comprise a mutated guaB gene or coding sequence comprising SEQ ID NO: 12.
• Figure 1 is a schematic representation of the biosynthetic pathway of guanosine monophosphate (adapted from Zalkin and Nygaard, 1996, in "Escherichia coli and Salmonella, Cellular and Molecular Biology, Second edition", 1996 F. C. Neidhardt ed. ASM Press, Washington D. C, Vol.l, Ch. 34:561-579).
• AICAR 5 ' -phosphoribosyl-4- carboxamide-5-aminoimidazole; ATP: adenosine triphosphate; G: guanine; GMP: guanosine monophosphate; GR: guanosine; Hx: hypoxanthine; HxR: hypoxanthine riboside (inosine) ;
• IMP Inosine monophosphate
• X Xanthine
• XMP Xanthosine monophosphate
• guaA GMP synthetase
• guaB IMP dehydrogenase
• guaC GMP reductase.
• Figure 2 represents contig 1294 of the S. Enteritidis genome (SEQ ID NO: 10) .
• the ATG initiation codon and TGA termination codon of the guaB gene are in bold.
• Figure 3 represents the sequence of the AguaB fragment of S. Enteritidis cloned in pUC18 (SEQ ID NO: 11) .
• the primers that were used are indicated by horizontal arrows.
• the fragment generated with primers GuaB6-GuaB7 was cloned in pUC18.
• the ATG initiation and TGA termination codon of the guaB gene and the CCCGGG Smal restriction site are indicated in bold.
• Figure 4 represents the nucleotide sequence of the S. Enteritidis PCR fragment, which includes the guaB deletion, obtained after sequencing, using primer GuaBlO (SEQ ID NO: 12) .
• the PCR fragment was amplified with primers GuaB6-GuaB7, using total genomic DNA of the mutant SM20.
• the remaining FRT site is indicated in bold italic and the Pl and P2 primers by arrows (Datsenko and Wanner, 2000, PNAS 97.-6640-6645) .
• the ATG initiation and TGA termination codon of the guaB gene are indicated in bold.
• Figure 5 represents the guaB gene of S.
• Figures 6-7 represent the deposit receipts of SM69 and SM86 respectively.
• Example 1 auxotrophic mutation affects the guaB gene
• An auxotrophic insertion mutant of a wild type S. Enteritidis was obtained via insertion mutagenesis. Only when supplemented with 0.3 mM guanine, xanthine, guanosine or xanthosine could the mutant strain grow on Minimal A medium.
• An insertion mutant can revert, thereby restoring the pathogenicity of the strain. This limits its applicability in a live attenuated vaccine. In that aspect deletion mutants are preferred. guaB deletion mutants of S. Enteritidis and S. Typhimurium were therefore created and tested. The guaB genes of both serovars are given in Figures 2 and 5.
• guaB deletion mutants were created according to the method for generating deletion mutations in the genome of Escherichia coli K12 (Datsenko and Wanner, 2000, PNAS 97 : 6640-5, incorporated by reference herein) .
• This method relies on homologous recombination, mediated by the bacteriophage ⁇ Red recombinase system, of a linear DNA fragment generated by PCR.
• the guaB sequence is hereby substituted by an antibiotic resistance gene. This resistance gene is flanked by FRT sites (FLP recognition target sites) and can be excised from the genome by site-specific recombination, mediated by the FLP recombinase.
• Overlap PCR ⁇ Ho et al . , 1989, Gene 77:51-59) was applied to construct a linear fragment. The principle relies on the use of two primer sets, one upstream pair
• Both sets contain primers (GuaB4, GuaB5) that are partially complementary and to which a Smal restriction site was added.
• PCR with the outward primers (GuaB6-GuaB7; GuaB6 : 5' GCAACAACTC CTGCTGGTTA 3' , SEQ ID NO 5; GuaB7 : 5' AGACCGAGGA TCACTTTATC 3' , SEQ ID NO 6) generated a fragment with a 6 basepair Smal site replacing an 861 basepair internal segment of the guaB coding sequence.
• This ⁇ guaB fragment was cloned in the vector pUCl ⁇ (see Figure 3) .
• the chloramphenicol resistance gene' ⁇ cat with its flanking FRT sequences was amplified using the primers Pl (5' GTGTAGGCTG GAGCTGCTTC 3', SEQ ID NO 8) and P2 (5' CATATGAATA TCCTCCTTAG 3', SEQ ID NO 9) (Datsenko and Wanner, 2000) and plasmid pKD3 DNA ⁇ Datsenko and Wanner, 2000) as a template.
• This PCR fragment was ligated in the Smal site of the cloned ⁇ guaB fragment.
• the desired fragment was generated using nested primers (GuaB6-GuaB7) .
• the resulting PCR fragment was electroporated into S. Enteritidis phage type 4 strain 76Sa88 (a clinical isolate from a turkey, obtained from the Veterinary and Agrochemical Research Centre, Groeselenberg 99, B-1180 Ukkel , Belgium) harboring the temperature sensitive replication plasmid pKD46, encoding the bacteriophage Lambda Red recombinase system.
• the chloramphenicol resistant transformants were tested on Minimal A medium and on Minimal A medium supplemented with 0.3 mM guanine.
• the ⁇ guaB: : catFRT mutants were confirmed by PCR using the following primer combinations: GuaB6-GuaB7, GuaB6-P2, GuaB7-Pl and P1-P2.
• the S. Enteritidis ⁇ guaB: : catFRT mutant (SM12) was transformed with the temperature sensitive replication plasmid pCP20 by electroporation.
• the plasmid pCP20 encodes the FLP recombinase, which recognizes the FRT- sites, to remove the cat gene.
• the resulting strain was named SM20.
• the PCR fragment in which the deletion is located was obtained using total genomic DNA of the mutant SM20 and the primer combination GuaB6-GuaB7 (see Figure 4) .
• the ⁇ guaB mutation was confirmed by sequencing of this fragment, using the primer GuaBlO (5' AGGAAGTTTG AGAGGATAA 3' , SEQ ID NO 7) .
• the ⁇ guaB: : catFRT mutation of the mutant SM12 was transduced by bacteriophage P22 HT i ⁇ t " ⁇ Davis, R.W. , Botstein D. and Roth, J. R. (1980) In Advanced Bacterial Genetics, A manual for genetic engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.) to wild type S. Enteritidis 76Sa88.
• the cat gene was removed using the plasmid pCP20.
• the resulting strain was called SM69
• the virulence of the mutant SM20 in mice was tested by oral infection of 6-8 week old female BALB/c mice in two independent experiments. These were performed as described above.
• the wild type strain S. Enteritidis 76Sa88 was tested in parallel as a positive control.
• the S. Enteritidis 76Sa88 ⁇ aroA mutant SM50 was included in the experiment as a vaccine control .
• This mutant carries a precise deletion of the complete aroA coding sequence and was constructed by the method of Datsenko and Wanner (2000) .
• mice BALB/c mice. These were performed as described above.
• Group 4 PBS-OG.
• the birds from groups 1 and 2 were placed in one isolator and those of groups 3 and 4 in another isolator.
• Chickens in groups 1 and 2 were inoculated with the SM69 master seed by the intratracheal (IT) route or oral gavage (OG) route, respectively, with an actual titer of 1.28 x 10 s cfu/0.2 ml per bird.
• Chickens in groups 3 and 4 were administered with 0.2 ml PBS (phosphate buffered saline) per bird by the intratracheal or oral gavage, respectively.
• I intratracheal
• OG oral gavage
• Chickens in groups 1 and 2 were inoculated with SM69 master seed by the intratracheal or oral gavage route, respectively, with the actual titer of 2.304 x 10 8 cfu/0.2 ml per bird.
• Chickens in group 3 were administered with Poulvac ® ST by the intratracheal route with 2.19 x 10 8 cfu/0.2 ml per bird.
• Chickens in group 4 were administered by the intratracheal route with 0.2 ml PBS per bird.
• Body weight was compared amongst groups in an analysis of variance (ANOVA) model with body weight as the dependent variable and treatment included as an independent variable. Group comparisons were made using Tukey' s test for multiple comparisons. The level of significance was set a p ⁇ 0.05. The study was considered valid because the control chickens (PBS control group) remained healthy and free of clinical signs of diseases or mortality throughout the study.
• ANOVA analysis of variance
• a deposit has been made according to the Budapest Treaty at the BCCM/LMG Culture Collection, Laboratorium voor Microbiologie, K. L. Ledeganckstraat 35, B-9000 Gent (Belgium) for the following micro-organisms: Salmonella Enteritidis SM69 under deposit number LMG P- 21641 (deposit date: 9 August, 2002) and S. Typhimurium SM86 under deposit number LMG P-21646 (deposit date: 28 August, 2002) .
• the deposits have been made in the name of Prof. J. -P.
• Table 2 Virulence test in BALB/c mice of the S. Enteritidis guaB deletion mutant SM20
• Table 7 Protection experiments with S . Typhimurium isogenic mutant strains in BALB/c mice . Oral inoculation with, approximately 10 8 cells of the S . Typhimurium strain 1491S96

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Veterinary Medicine (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Biotechnology (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Epidemiology (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Mycology (AREA)
• Immunology (AREA)
• Virology (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Biomedical Technology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Cell Biology (AREA)
• Communicable Diseases (AREA)
• Oncology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Applications Claiming Priority (1)

Publications (1)

Family

ID=37430822

Family Applications (1)

Country Status (7)

Families Citing this family (5)

Family Cites Families (1)

• 2006
  • 2006-03-20 MX MX2008012056A patent/MX2008012056A/es unknown
  • 2006-03-20 JP JP2009500671A patent/JP2009529895A/ja active Pending
  • 2006-03-20 EP EP06721540A patent/EP2004802A1/en not_active Withdrawn
  • 2006-03-20 WO PCT/BE2006/000019 patent/WO2007106956A1/en active Application Filing
  • 2006-03-20 CA CA002646421A patent/CA2646421A1/en not_active Abandoned
  • 2006-03-20 AU AU2006340699A patent/AU2006340699A1/en not_active Abandoned
  • 2006-03-20 BR BRPI0621489-4A patent/BRPI0621489A2/pt not_active IP Right Cessation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events