DCR5, a BMP-binding protein, and applications thereof

Details DCR5, a BMP-binding protein, and applications thereof DCR5, a BMP-binding protein, and applications thereof

2001-02-14

2003-12-09

Eyler, Yvonne (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S325000, C536S023500, C536S024300, C530S350000, C424S198100

Reexamination Certificate

active

06660499

ABSTRACT:

FIELD OF THE INVENTION
The field of this invention is proteins which regulate cell function, and in particular, antagonize bone morphogenetic proteins.
BACKGROUND
Natural regulators of cellular growth, differentiation and function have provided important pharmaceuticals, clinical and laboratory tools, and targets for therapeutic intervention. A variety of such regulators have been shown to have profound effects on basic cellular differentiation and developmental pathways. For example, the recently cloned cerberus protein induces the formation of head structures in anterior endoderm of vertebrate embryos. Similarly, the Noggin protein induces head structures in vertebrate embryos, and can redirect mesodermal fates from ventral fates, such as blood and mesenchyme, to dorsal fates such as muscle and notochord and can redirect epidermal fates to anterior neural fates. The activities of chordin are similar to those of Noggin, reflecting a common mechanism of action—namely antagonizing bone morphogenetic proteins (BMP) and thereby preventing their function. BMPs have diverse biological activities in different biological contexts, including the induction of cartilage, bone and connective tissue, and roles in kidney, tooth, gut, skin and hair development.
Different members of the TGF&bgr; superfamily can instruct cells to follow different fates, for example TGF&bgr; induces neural crest to form smooth muscle, while BMP2 induces the same cells to become neurons. In Xenopus experiments, dissociated animal cap cells (prospective ectoderm) become epidermis in response to BMP4 but become mesoderm in response to activin.
Since the sequence identity between activin and BMP4 is low, it is not surprising that they induce different fates. It is more surprising that members of the BMP subfamily, which are quite closely related in sequence, can induce distinct fates. A striking example results from implantation of a matrix impregnated with a BMP into muscle; when the effects are monitored histologically, BMP2, 4 and 7 induce endochondral bone formation, whereas a related molecule BMP12/GDF7 induces connective tissue similar to tendon. Similarly, BMP4 can induce cell death in the hindbrain neural crest, while the related protein dorsalin does not.
Since different BMP family members can induce different fates, then BMP antagonists that have specificity in blocking subsets of BMPs could change the balance of BMPs that are presented to a cell, thus altering cell fate. In view of the importance of relative BMP expression in human health and disease, regulators of cellular function and BMP function in particular, such as Noggin and cerberus, provide valuable reagents with a host of clinical and biotechnological applications. The present invention relates to a new family of regulators of cellular function.
Relevant Literature
Bouwmeester, et al. (1996) Nature 382: 595-601 describe the cloning of Xenopus cerberus gene; Lamb, T. M., et al. (1993) Science 262: 713-718; Smith, W. C., et al. (1992) Cell 70: 829-840; Smith, W. C., et al. (1993) Nature 361: 547-549; and Zimmerman, L. B., et al. (1996) Cell 86: 599-606 describe the isolation and function of the Noggin protein. Piccolo, S., et al. (1996) Cell 86: 589-598; Sasai, Y., et al. (1995) Nature 376: 333-336; and Sasai, Y., et al. (1994) Cell 79: 779-790 relate to the chordin protein. Enomoto et al. (1994) Oncogene 9: 2785-2791 and Ozaki, et al. (1996) Jpn. J. Cancer Res. 87: 58-61 describe human and murine homologs of the DAN gene. Hsu, et al. (1998) Mol Cell 1:673-683 describing Gremlin from a variety of species, including human; Minabe-Saegusa, C., et al. (1998) Dev Growth Differ 40:343-353 which describes mouse PRDC.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to DCR5, a protein related to Gremlin, DAN (Differential-screening-selected gene Aberrative in Neuroblastoma) and Cerberus, and related nucleic acids. Included are natural DCR5 homologs from different species, as well as proteins comprising a DCR5 domain and having DCR5-specific activity, particularly the ability to antagonize a bone morphogenetic protein. The proteins may be produced recombinantly from transformed host cells with the subject nucleic acids. The invention provides isolated hybridization probes and primers capable of specifically hybridizing with the disclosed genes, specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g., genetic hybridization screens for DCR5 transcripts), therapy (e.g., gene therapy to modulate DCR5 gene expression) and in the biopharmaceutical industry (e.g., reagents for screening chemical libraries for lead pharmacological agents).
Preferred applications of the subject DCR5 proteins include modifying the physiology of a cell comprising an extracellular surface by contacting the cell or medium surrounding the cell with an exogenous DCR5 protein under conditions whereby the added protein specifically interacts with a component of the medium and/or the extracellular surface to effect a change in the physiology of the cell. Also preferred are methods for screening for biologically active agents, which methods involve incubating a DCR5 protein in the presence of an extracellular DCR5 protein-specific binding target and a candidate agent, under conditions whereby, but for the presence of the agent, the protein specifically binds the binding target at a reference affinity; detecting the binding affinity of the protein to the binding target to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that the agent modulates the binding of the protein to the binding target.
Another preferred embodiment of the invention is a method of treatment of a human or animal body by administering a therapeutic dosage of a human DCR5 polypeptide as wherein the treatment is regulation of cartilage and bone growth.
An additional preferred embodiment of the invention is a ligandbody which comprises human DCR5 fused to an immunoglobulin constant region, wherein the immunoglobulin constant region is the Fc portion of human IgG1.
In a preferred embodiment, a ligandbody may be used in a method of treatment of the human or animal body, or in a method of diagnosis.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides DCR5 proteins which include natural DCR5 proteins and recombinant proteins comprising a DCR5 amino acid sequence, or a functional DCR5 protein domain thereof having an assay-discemable DCR5-specific activity. Accordingly, the proteins may be deletion mutants of the disclosed natural DCR5 proteins and may be provided as fusion products, e.g., with non-DCR5 polypeptides. The subject DCR5 protein domains have DCR5-specific activity or function and are functionally distinct from each other and from DAN, cerberus, Gremlin and Noggin homologs. Such domains include at least 6 and preferably at least 8 consecutive residues of a natural DCR5 protein (See DAN sequence reported by Enomoto, et al. (1994) Oncogene 9: 2785-2791). Preferred DCR5 proteins comprise a DCR5 sequence conserved across species.
The DCR5 proteins described herein are structurally and functionally related to DAN and Cerberus in that they are extracellularly active as antagonists of certain morphogenetic proteins such as BMPs. DCR5-specific activity or function may be determined by convenient in vitro cell-based, or in vivo assays—e.g., in vitro binding assays, cell culture assays, in animals (e.g., immune response, gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the specific molecular interaction of a DCR5 protein with a binding target is evaluated. The binding target may be a natural binding target such as a TGF&bgr; protein, a morphogenetic protein, preferably a bone morphogenetic protein such as BMP2 or BMP4, a chaperon, or other regulator that directly modulates DCR5 activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or a DCR5 s

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