Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-06-07
2003-11-11
Spector, Lorraine (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S252300, C435S320100, C536S023500
Reexamination Certificate
active
06645740
ABSTRACT:
The present invention relates to the nucleotide sequence of the gene encoding the horse cytokine GM-CSF, to expression vectors containing it, and to its use as adjuvant in equine vaccination and as nonspecific immunity stimulant.
The documents cited here are incorporated by reference into the present application. (All documents cited herein, and all documents cited in documents cited herein are hereby incorporated herein by reference).
The first discovery of a granulocyte-macrophage colony-stimulating factor (GM-CSF) dates from 1977 (Burgess A.W. et al. J. Biol. Chem. 1977, 252, 1998-2003). It is the murine GM-CSF, purified from mouse lung culture supernatants.
The biological activities of GM-CSF have been demonstrated by the work carried out on the murine and human GM-CSFs (Clark S. C. et al. Science 1987, 230, 1229; Grant S. M. et al. Drugs 1992, 53, 516).
GM-CSF has many physiological roles (Dy M. in “Les cytokines” Cavaillon J.-M., 1996, ed. Masson, Paris, France, 43-56). In particular, GM-CSF stimulates the production, the development and the formation of colonies of granulocytes, macrophages, eosinophils and megakaryocytes. GM-CSF induces in particular a macrophagic cytotoxocity, stimulates antibody-dependent cytotoxic activity (ADCC) and the recruitment of leukocytes at the level of the sites of inflammation.
The GM-CSFs from various animal species have already been identified.
The sizes of the nucleotide sequences encoding the known GM-CSFs from various species vary from 381 to 432 nucleotides. The human and murine nucleotide sequences have a degree of homology of 69%. The degree of homology is 54% at the level of the amino acid sequence (Cantrell M. A. et al. Proc. Natl. Acad. Sci. USA 1985, 82, 6250-6254). However, this homology does not allow any cross-activity between the two human and murine species (Metcalf D. et al. Blood 1986, 67, 37-45).
The administration of heterologous GM-CSF, that is to say obtained from a species other than the one treated, does not make it possible to obtain an optimum adjuvant effect, in particular a stimulation of the activity of the haematopoietic cells and a substantial increase in the immune response.
Up until now, it has not been possible to identify the equine GM-CSF. Yet this cytokine is of great interest for therapeutic and vaccinal applications for use in horses.
The applicant has succeeded in isolating and sequencing the equine GM-CSF gene. This gene was isolated after polymerase chain reaction (PCR) with the aid of the oligonucleotides described in the examples.
The equine GM-CSF gene has a size of 432 nucleotides (SEQ ID No. 8 and
FIG. 1
) and encodes a protein of 144 amino acids (SEQ ID No. 9 and FIG.
1
). The protein encoded by this gene exhibits a homology of at least 75% with the GM-CSF polypeptide sequences of other animal species.
The subject of the present invention is therefore an isolated DNA fragment encoding equine GM-CSF, e.g. a fragment comprising SEQ ID No. 8. Its subject is also the DNA fragment having or consisting essentially of this sequence.
The subject of the present invention is also an isolated DNA fragment encoding the amino acid sequence SEQ ID No. 9.
The invention covers the equivalent nucleotide sequences of equine or synthetic origin, that is to say the nucleotide sequences encoding a protein of equivalent functionality and specificity in horses. The nucleotide sequences which differ by the degeneracy of the genetic code will of course be included. In particular, DNA sequences having an homology equal or greater than 90%, particularly than 92%, preferably than 95% with SEQ ID No. 8, are equivalent sequences.
An object is also DNA fragments comprising such a nucleotide sequence encoding the equine GM-CSF, e.g. according to SEQ ID No. 8 or a sequence encoding the amino acid sequence SEQ ID No. 9, this nucleotide sequence being associated, in the form of a fusion, with the nucleotide sequence encoding at least one immunogen or at least one immunogenically active fragment or at least one epitope of an immunogen. The DNA fragment then does not comprise a stop codon between the sequence encoding GM-CSF and the associated immunogen encoding sequence. For instance, referring to SEQ ID No. 8, the coding sequence inserted ends at nucleotide 432, and does not include the stop codon.
The subject of the present invention is also the isolated equine GM-CSF protein or polypeptide, e.g. that encoded by the nucleotide sequence SEQ ID No. 8 or by the equivalent of the latter as defined above.
The subject of the present invention is also the equine GM-CSF protein having the amino acid sequence SEQ ID No. 9.
The equine GM-CSF protein has a size of 144 amino acids. However, the present invention also comprises the proteins, protein fragments and polypeptides of equine origin or which are synthetic, having a size greater or equal than or less than these 144 amino acids, as well as the recombinant proteins (having one or more substitutions, deletions or additions) and the fusion proteins, as long as their biological activity (for the part which is common to GM-CSF) is substantially equivalent to that of the natural equine GM-CSF protein in vivo in horses and their species-specificity is not modified. Are encompassed as equivalents any of the amino acid sequences encoded by any of the equivalent nucleotide sequences as defined above.
The subject of the present invention is also a pure preparation of equine GM-CSF protein.
The subject of the present invention is also the expression vectors containing, as insert, any of the above defined DNA fragments or nucleotide sequences, in particular the equine GM-CSF gene (SEQ ID No. 8) or an equivalent thereof as defined above, as well as any of the nucleotide sequences encoding any of the above defined amino acid sequences. Also, the vector may further comprise a nucleotide sequence encoding at least one immunogen or at least one immunogenically active fragment or at least one epitope of an immunogen, which can be or not associated under the form of a fusion as described above.
The nucleotide sequence may be inserted into conventional in vitro expression systems of viral origin, such as Baculovirus, in particular propagated on insect cells, or cells of prokaryotic origin (for example
Escherichia coli
) or eukaryotic origin, in particular yeasts, especially
Saccharomiyces cerevisiae
, mammalian eukaryotic cells, especially hamster cells (for example hamster ovary cells or CHO) and horse cells. The invention therefore also covers expression systems transformed by a sequence according to the invention, the equine GM-CSF proteins thus produced and their use as adjuvant for vaccine and nonspecific immunity stimulant.
Preferably, the sequence according to the invention is introduced into in vivo expression vectors under conditions allowing the expression, in horses, of a functional equine GM-CSF protein, and possibly a nucleotide sequence encoding at least one immunogen or at least one immunogenically active fragment or at least one epitope of an immunogen. These expression vectors may be plasmids, viral vectors, such as poxviruses, for example the vaccinia virus, avipoxviruses (canarypox, fowlpox), including the species-specific poxviruses (swinepox, raccoonpox and camelpox), adenoviruses and herpesviruses, such as the equine herpesviruses.
The term plasmid is intended to cover any DNA transcription unit in the form of a polynucleotide sequence comprising the sequence of the equine GM-CSF gene and the elements necessary for its expression in vivo. The circular plasmid form, supercoiled or otherwise, is preferred. The linear form also falls within the scope of this invention.
Each plasmid comprises a promoter capable of ensuring, in the host cells, the expression of the gene inserted under its control. It is in general a strong eukaryotic promoter and in particular a cytomegalovirus early promoter CMV-IE, of human or murine origin, or optionally of other origin such as rat or guinea pig. More generally, the promoter is either of viral origin or of cellular origin. As a viral promote
Andreoni Christine Michèle Pierrette
Bublot Michel
Perez Jennifer Maria
Frommer William S.
Frommer & Lawrence & Haug LLP
Kowalski Thomas J.
Merial Limited
Spector Lorraine
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