Methods and compositions concerning canine interleukin 5

Details Methods and compositions concerning canine interleukin 5 Methods and compositions concerning canine interleukin 5

1999-08-10

2003-03-25

Kemmerer, Elizabeth (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...

C435S069100, C435S320100, C435S325000, C435S252300, C435S254110, C435S069500, C536S023100, C536S023500

Reexamination Certificate

active

06537781

ABSTRACT:

FIELD OF THE INVENTION
This application reports the isolation and characterization of canine interleukin-5 (“IL-5”) and includes nucleic and amino acid sequences therefor. More particularly, it concerns a nucleic acid sequence which comprises DNA which encodes canine interleukin-5.
BACKGROUND
Interleukin 5
Interleukin-5 (IL-5) has been studied in both human and murine systems. It was initially designated T cell-replacing factor or B cell growth factor II (BCGF II). IL-5 is understood to induce or mediate multiple effects. It promotes the proliferation of activated B lymphocytes as well as the generation of both IgM and IgG responses. IL-5 also promotes IgA secretion, apparently by acting on cells that already express surface IgA.
IL-5 cDNA clones from mouse and human species have been isolated. See Takatsu, K., and Tominaga, A., “Interleukin 5 and its Receptor”,
Progress in Growth Factor Research
, 3: 87-102 (1991), incorporated herein by reference. Mouse IL-5 consists of 133 amino acid residues, including a signal sequence of 21 residues and three sites for N-glycosylation. In contrast, human IL-5 consists of 139 amino acid residues, including a signal sequence of 22 residues and two sites for N-glycosylation. Both mouse and human IL-5 exist as a dimer linked by disulfide bond. Further, mouse and human IL-5 are 71% homologous at the amino acid level. However, while human IL-5 is capable of stimulating mouse cells, mouse IL-5 is only weakly cross-reactive with human cells.
Prior to this invention, no canine IL-5 DNA or amino acid sequence has been reported. It is unknown whether canine IL-5 will cross react with human or mouse cells.
In Vitro Activities
In humans IL-5 is known to exert most of its biological activities on hematopoietic lineages outside the lymphoid compartment. This cytokine acts as an eosinophil stimulating factor; e.g., it augments the number of eosinophil colonies that develop in semisolid bone marrow cultures. IL-5 is a potent regulator of eosinophilia and appears to act on relatively mature progenitors, causing them to proliferate and to differentiate into mature effector cells. In fact, eosinophils induced in vitro by IL-5 are fully functional and have been demonstrated to kill antibody-coated schistosomula of
Schistosoma mansoni
and antibody-coated tumor cells.
Since IL-5 plays a major role in the regulation of eosinophils that are prominently involved in allergic inflammation, IL-5 inhibition may have potential therapeutic benefits for various allergies. See Cuss, “Inhibition of Interleukin-5 with a Monoclonal Antibody Attenuates Allergic Inflammation,”
Allergy
, 52: 787-794 (1997), incorporated herein by reference.
IL-5 may also act in concert with other hemtopoietic cytokines such as IL-3 and GM-CSF. The three are known for increasing oxidative metabolism, membrane receptor expression and the release of granule proteins as well as for their role in inducing eosinophilopoiesis. In vitro data shows that IL-5 acts in synergy with other activation signals, as in the case of IL-5 and immune complexes. See Desremeux, P. and Capron, M., “Eosinophils in Allergic Reactions”,
Current Opin. in Immunol
., 8: 790-795 (1996), incorporated herein by reference.
The other known activities of IL-5 all relate to regulation of B-cell immune responses. IL-5 was first described as a T-cell activity that induced antigen-stimulated murine B cells to differentiate into both IgM- and IgG-secreting plasma cells. See Takatsu, K., and Tominaga, A., supra. Subsequent studies have shown that IL-5 is the major factor inducing differentiation to immunoglobulin (Ig) production in B cells which were activated by contact with activated T helper cells. Murine IL-5 also augments proliferation of and induces secretion of Ig in a number of B-cell lines. Further, it has similar activity on “in vivo-activated” normal B cells. Murine IL-5 can also enhance the production of IgA in LPS-stimulated B-cell cultures; however, its principal activity is not as a switch-inducing factor, and it does not specifically enhance IgA in T-cell stimulated cultures. Murine IL-5 induces expression of the P55 chain of the IL-2 receptor on normal B cells and, in combination with IL-4, renders these cells responsive to IL-2 stimulation.
Presently, however, the role of IL-5 in human B-cell growth and differentiation remains controversial. IL-5 is inactive in many of the culture systems commonly used to assay human B-cell growth factors and differentiation factors. However, in other assay systems, IL-5 has been shown to be active on human B cells activated with mitogens or activated T-cell clones. Thus, the contribution of IL-5 to the helper activity of T cells in humans is presently not understood.
In vitro biological activities similar to those described above in humans can be postulated for IL-5 in dogs.
In Vivo Activities
In humans, IL-5 appears to be the most specific cytokine for activation of eosinophils. IL-5, IL-3, and GM-CSF may also act in concert to activate eosinophils and basophils, as these three cytokines cross-react on receptors because each cytokine shares a common chain. These cytokines have been identified in late phase reactions in humans. See Desremaux, P., and Capron, M, “Eosinophils in Allergic Reactions”,
Current Opin. in Immunol
., 8: 790-795 (1996).
IL-5 is involved in the eosinophilia that develops during parasitic infections and allergic reactions, but not in the IgG1 or IgE antibody responses associated with these infections. Administration of anti-IL-5 monoclonal antibody blocks the development of both blood and tissue eosinophil responses, but fails to affect IgG1 or IgE secretion. See Cuss, D., “Inhibition of Interleukin-5 with a Monoclonal Antibody Attenuates Allergic Inflammation”,
Allergy
, 52: 787-794 (1997).
In vitro experiments suggested that IL-5 is an eosinophil regulator, and numerous in vivo experiments indicate that IL-5 is, in fact, a predominant regulator of eosinophilia. This was readily seen in a series of experiments where the administration of anti-IL-5 antibodies to mice infected with
Nippostrongylus brasiliensis, Schistosoma mansoni, Heligmosomoides polygyrus
, or
Strongyloides venezuelensis
totally blocked the development of eosinophilia. Consistent with these data, mice that have over expressed IL-5 due to the introduction of IL-5 transgenes or IL-5 retroviral constructs are characterized by dramatic increases in eosin counts. In contrast to IL-5 expressing mice, mice that have been genetically engineered to over express either IL-3 or GM-CSF live only a few weeks due to massive tissue infiltration and destruction by the greatly increased numbers of myeloid cells.
In vivo biological activities similar to those described above in humans can be postulated for IL-5 in dogs.
SUMMARY OF THE INVENTION
The present invention relates in part to recombinant DNA molecules, and conservative variants thereof, that encode canine IL-5. Disclosed is an isolated polynucleotide comprising the nucleotide sequence of canine IL-5 of
FIG. 1
(SEQ ID NO:1) or its complement.
Also disclosed is an isolated canine IL-5 nucleic acid sequence comprising around 80%, preferably around 85%, preferably around 90%, more preferably around 95%, even more preferably around 97% and most preferably around 99% homology to a nucleic acid sequence of canine IL-5 of
FIG. 1
(SEQ ID NO:1) or its complement.
Further disclosed is an isolate canine IL-5 nucleic acid comprising the nucleotide sequence encoding the polypeptide comprising the amino acid sequence of FIG.
1
.
Also disclosed is an isolated canine IL-5 polynucleotide comprising around 80%, preferably around 85%, preferably around 90%, more preferably around 95%, even more preferably around 97% and most preferably around 99% homology to a nucleic acid comprising the nucleotide sequence encoding the polypeptide comprising the amino acid sequence of FIG.
1
.
Further disclosed is a canine IL-5 polynucleotide comprising at least about 20 consecutive nucleotides, preferably at least about 30 consecutive nucleotides, more preferably at leas

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