Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Reexamination Certificate
1999-09-07
2003-05-13
Mertz, Prema (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
C424S093710, C424S277100, C514S002600, C514S008100, C514S012200, C514S885000
Reexamination Certificate
active
06562334
ABSTRACT:
TECHNICAL FIELD
An object of the present invention is to mass-produce canine interleukin 12 (hereinafter sometimes abbreviated “CaIL12”) with the primary structure of its protein derived from canine genetic information for providing a drug for treatment of animals. Such drugs may be antitumor and antiviral in nature and can act as a vaccine adjuvant. The present invention relates to DNA sequences, recombinant vectors and transformants, CaIL12 itself and a production process thereof.
The present invention also relates to an immune disease remedy and preventive agent for dogs and cats comprising canine interleukin 12 with the primary structure of its protein derived from canine genetic information, and an immune disease treatment method and preventive method for dogs and cats using the remedy or preventive agent.
BACKGROUND ART
Interleukin 12 is a heterodimer consisting of a protein with a molecular weight of about 40 kD (hereinafter sometimes abbreviated “P40” or “P40 subunit”) and a protein with a molecular weight of about 35 kD (hereinafter sometimes abbreviated “P35” or “P35 subunit”), and is a cytokine with such bioactivity as to activate natural killer cells and type 1 helper T cells, being abbreviated as IL12. Concerning IL12, some literature has been published. In addition to human IL12, the cDNAs of mouse IL12 (Shoenhaut et al:
J. Immunol.
148, 3433-3440 (1992) and bovine IL12 (GenBank Database Registration Nos. U11815 and U14416) were cloned by gene manipulation techniques, and their application as remedies against cancers, viral diseases, etc. are being researched and developed.
For IL12, treatment effects in mouse model experiments against tumors, infectious diseases, allergies, and the like especially by its activity in boosting the cell—mediated immune response have been reported. in the literature such as Nastala et al:
J. Immunol.
153, 1697-1706 (1994), Gazzinelli et al:
Proc. Natl. Acad. Sci.
USA 90, 6115-6119 (1993) and Chirgwin et al:
Biochemistry
18, 5294 (1979), and clinical trials of IL12 as a remedy against human cancer and human infectious disease has already started. However, it is not yet reported that canine IL12 has been cloned.
Dogs are known to suffer from various cancers such as mammary gland tumor, various viral diseases such as Parvovirus infectious disease and distemper infectious disease, allergic dermatitis and the like. Therefore, if canine IL12 were to become easily available, it is expected that its application as a canine antitumor drug, antiallergic drug and antiviral drug would be attempted.
Presently, few remedies and preventive agents effective against these canine diseases are available. For example, most dogs suffering from cancer come to hospitals after their tumors have grown, and even if the tumors are ablated by surgery, they soon die after the operation because of metastasis. Also, for skin diseases often seen with dogs, they cannot be cured in most cases even if steroids are administered repetitively for long periods of time as a treatment. As a consequence, fast and continuously acting remedies are being demanded. It is expected that new applications will be attempted for these canine diseases now left without any effective remedy if canine IL12 becomes available.
SUMMARY OF THE INVENTION
The invention includes cloning CaIL12 cDNA for mass-production and for providing a preparation containing CaIL12 as a remedy or preventive agent for dogs and cats suffering from immune diseases. The invention particularly includes cloning cDNAs coding for P40 and P35 of CaIL12 from canine cDNA and, furthermore, producing cells capable of producing CaIL12 using two expression plasmids linked to these cDNAs, respectively, and in producing recombinant Baculovirus containing both the genes.
Thus, the invention has established a method for mass-producing CaIL12 and found that if a preparation containing CaIL12 is administered to dogs and cats suffering from diseases difficult to treat by conventional therapeutic methods or if lymphocytes isolated from the peripheral blood of a sick dog or cat are stimulated in vitro by a preparation containing CaIL12 and returned into the body of the dog or cat again, the disease can be surprisingly remarkably improved.
The present invention provides plasmids capable of producing CaIL12, a transformant of
Escherichia coli
having these plasmids, animal cells transformed by these plasmids, CaIL12 obtained from these transformants, Baculovirus capable of producing CaIL12, CaIL12 produced by the Baculovirus, an immune disease remedy and preventive agent for dogs and cats containing CaIL12 as a main effective ingredient, and an immune disease treatment method and preventive method for dogs and cats. In addition, the present invention also provides cDNAs respectively coding for two subunits of CaIL12 protein.
Important aspects of the present invention include:
(1) Canine interleukin 12 comprises a P40 subunit and a P35 subunit and has at least one ability selected from 1) an ability to induce an antiviral active factor acting on canine leukocytes and a factor to intensify the expression of class II mouse histocompatability antigens (MHC) of canine tumor cells, 2) an ability to promote the proliferation of canine lymphoblasts, and 3) an ability to damage canine tumor cells by activating canine leukocytes.
(2) Canine interleukin 12, stated in paragraph (1) above, wherein the P40 subunit has an amino acid sequence substantially the same as SEQ ID NO:1 or SEQ ID NO:11 or corresponding to part of either of those sequences.
(3) Canine interleukin 12, stated in paragraph (1) above, wherein the P35 subunit has an amino acid sequence substantially the same as SEQ ID NO:2 or SEQ ID NO:12 or corresponding to part of either of those sequences.
(4) An immune disease remedy and preventive agent for dogs and cats comprising canine interleukin 12 and a treatment method and preventive method using the remedy or preventive agent.
(5) A DNA sequence coding for the canine interleukin 12 stated in any one of paragraphs (1), (2) and (3) above, a recombinant vector containing it, a transformant obtained by transforming host cells by the recombinant vector, and a method of producing canine interleukin 12 by culturing the transformant or infecting insect cells or larvae with the transformant for culturing or raising.
BRIEF DESCRIPTION OF SEQUENCE LISTING
SEQ ID No:1 sets forth the the P40 subunit of canine interleukin 12 and the corresponding amino acid sequence.
SEQ ID No:2 sets forth the nucleotide sequence encoding the P35 subunit of canine interleukin 12 and the corresponding amino acid sequence.
SEQ ID NO:3 sets forth the nucleotide sequence for one of the primers used in the cloning of CaIL12 P40 cDNA.
SEQ ID NO:4 sets forth the nucleotide sequence for one of the primers used in the cloning of CaIL12 P40 cDNA.
SEQ ID NO:5 sets forth the nucleotide sequence for one of the primers used in the cloning of CaIL12 P35 cDNA.
SEQ ID NO:6 sets forth the nucleotide sequence for one of the primers used in the cloning of CaIL12 P35 cDNA.
SEQ ID NO:7 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:8 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:9 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:10 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID No:1 sets forth the nucleotide sequence encoding the P40 subunit of canine interleukin 12 and the corresponding amino acid sequence.
SEQ ID No:2 sets forth the nucleotide sequence encoding the P35 subunit of canine interleukin 12 and the corresponding amino acid sequence.
SEQ ID NO:13 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:14 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:15 sets forth the nucleotide sequence
Okano Fumiyoshi
Satoh Masahiro
Yamada Katsushige
Mertz Prema
Piper Rudnick LLP
Toray Industries Inc.
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