Tnf receptor-like molecules and uses thereof   

This application is a continuation of application Ser. No. 11/436,207, filed May 17, 2006, which is a continuation of application Ser. No. 09/948,018, filed Sep. 5, 2001 which is now U.S. Pat. No. 7,153,669, which claims the benefit of U.S. Provisional Application No. 60/230,191 filed Sep. 5, 2000, which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to novel TNF receptor (TNFr)-like polypeptides and nucleic acid molecules encoding the same, termed “MK61” herein.

The invention also relates to vectors, host cells, pharmaceutical compositions, selective binding agents and methods for producing MK61 polypeptides. Also provided for are methods for the diagnosis, treatment, amelioration, and/or prevention of diseases associated with MK61 polypeptides.

BACKGROUND OF THE INVENTION

Technical advances in identification, cloning, expression and manipulation of nucleic acid molecules and deciphering of the human genome have greatly accelerated discovery of novel therapeutics based upon deciphering of the human genome. Rapid nucleic acid sequencing techniques can now generate sequence information at unprecedented rates and, coupled with computational analyses, allow the assembly of overlapping sequences into the partial and entire genomes as well as identification of polypeptide-encoding regions. A comparison of a predicted amino acid sequence against a database compilation of known amino acid sequences allows one to determine the extent of homology to previously identified sequences and/or structural landmarks. The cloning and expression of a polypeptide-encoding region of a nucleic acid molecule provides a polypeptide product for structural and functional analyses. The manipulation of nucleic acid molecules and encoded polypeptides to create variant and derivatives thereof may confer advantageous properties on a product for use as a therapeutic.

In spite of significant technical advances in genome research over the past decade, the potential for development of novel therapeutics based on the human genome is still largely unrealized. Many genes encoding potentially beneficial polypeptide therapeutics or those encoding polypeptides, which may act as “targets” for therapeutic molecules, have still not been identified.

Accordingly, it is an object of the invention to identify novel polypeptides, and nucleic acid molecules encoding the same, which have diagnostic or therapeutic benefit.

After years of study in necrosis of tumors, tumor necrosis factors (TNFs) α and β were finally cloned in 1984. The ensuing years witnessed the emergence of a superfamily of TNF cytokines, including fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), CD40 ligand (CD40L), TNF-related apoptosis-inducing ligand (TRAIL, also designated AGP-1), osteoprotegerin binding protein (OPG-BP or OPG ligand), 4-1BB ligand, LIGHT, APRIL, and TALL-1. Smith et al. (1994), Cell, 76: 959-962; Lacey et al. (1998), Cell, 93: 165-176; Chichepotiche et al. (1997), J. Biol. Chem., 272: 32401-32410; Mauri et al. (1998), Immunity, 8: 21-30; Hahne et al. (1998), J. Exp. Med., 188: 1185-90; Shu et al. (1999), J. Leukocyte Biology, 65: 680-3. This family is unified by its structure, particularly at the C-terminus. In addition, most members known to date are expressed in immune compartments, although some members are also expressed in other tissues or organs, as well. Smith et al. (1994), Cell 76: 959-62. All ligand members, with the exception of LT-α, are type II transmembrane proteins, characterized by a conserved 150 amino acid region within C-terminal extracellular domain. Though restricted to only 20-25% identity, the conserved 150 amino acid domain folds into a characteristic β-pleated sheet sandwich and trimerizes. This conserved region can be proteolyticaly released, thus generating a soluble functional form. Banner et al. (1993), Cell, 73: 431-445.

Many members within this ligand family are expressed in lymphoid enriched tissues and play important roles in the immune system development and modulation. Smith et al. (1994). For example, TNFα is mainly synthesized by macrophages and is an important mediator for inflammatory responses and immune defenses. Tracey & Cerami (1994), Annu. Rev. Med., 45: 491-503. Fas-L, predominantly expressed in activated T cell, modulates TCR-mediated apoptosis of thymocyts. Nagata et al. (1995) Immunology Today, 16:39-43; Castrim et al. (1996), Immunity, 5:617-27. CD40L, also expressed by activated T cells, provides an essential signal for B cell survival, proliferation and immunoglobulin isotype switching. Noelle (1996), Immunity, 4: 415-9.

The cognate receptors for most of the TNF ligand family members have been identified. These receptors share characteristic multiple cysteine-rich repeats within their extracellular domains, and do not possess catalytic motifs within cytoplasmic regions. Smith et al. (1994). Two subgroups of TNFR homologues: Fas, TNFR1, DR3, DR4, DR5, and DR6 contains intracellular death domain which bind TRAD or FADD. This leads to activation of caspase 8 and apoptosis. Locksley et al. (2001) Cell 104: 487-501. However, signaling through death-receptors can also be required for proliferation of hepatocytes and T cells. Strasser et al., (1999) Intl. J. Biochem. Cell Biol. 31: 533-537, Yamada et al. (1997), Proc. Natl. Acad. Of Sci. U.S.A, 94: 1441-6. The other group including TNFR2, CD40, or CD30 bind TNF-Receptor Associated Factors (TRAFs), molecular adapters that couple these surface receptors to downstream signaling cascades. This leads to activation of JNK and NFκB which can promote cell growth and survival. These proteins therefore play critical roles in morphogenesis, the control of apoptosis, differentiation, or proliferation. TNF/TNFR superfamily proteins are now extensively studied as targets for therapies against many human diseases such as atherosclerosis, allograft rejection, arthritis, and cancer. Locksley et al. (2001), Williams et al. (2000), Ann. Rhem. Dis. 59: i75-80.

In addition to the membrane associated receptor molecules described above, a number the receptors belonging to the TNF-receptor supergene family exist as soluble ligand binding proteins. Many of the soluble forms of the transmembrane receptors were subsequently identified as containing only the extracellular ligand binding domain(s) of the receptors. For example, a soluble form of TNF receptor has been found in urine and serum (see U.S. Pat. No. 5,843,789 and Nophar et al., EMBO J., 9(10):3269-3278, 1990), and have been shown to arise by proteolytic cleavage of cell surface TNF-receptors (Wallach et al., Agents Actions Suppl., 35:51-57, 1991). These soluble forms of receptor molecules have been implicated in the modulation of TNF activity by not only interfering with TNF binding to its receptor, but also by stabilizing the TNF structure and preserving its activity, thus prolonging some of its effects (Aderka et al, Cytokine & Growth Factor Reviews, 7(3):231-240, 1996).

Members of the tumor necrosis factor superfamilies of ligands and cell-surface receptors regulate immune function and most TNF/TNFR superfamily proteins, such as FASL/FAS, CD40L/CD40, TNF/TNFR, or LTβ/LTβR to name a few, are expressed in the immune system, where the coordinate immune cell homeostasis, activation induced cell death, T cells priming, functions and survival of dendritic cells, or the formation of germinal centers and lymphoid organs such as Peyer\'s patches and lymph nodes. Fu et al. (1999), Ann. Rev. Immunol. 17: 399-433, Grewal et al. (1998), Ann. Rev. Immunol. 166: 111-135. Recently, novel members of this large families have been identified that have critical functions in immunity and couple lymphoid cells with other organ systems such as bone morphogenesis and mammary gland formation in pregnancy.

Because of the crucial role that members of the TNF family of ligands and their receptors (membrane-associated and soluble) play in the immunological system and in a variety of disease processes, a need exists to identify and characterize novel members of these families, for use to improve diagnosis and therapy.

SUMMARY

The present invention relates to novel MK61 nucleic acid molecules and encoded polypeptides.

In accordance with the invention, a number of human MK61 isoforms are described herein: “hMK61T1”, “hMK61T2”, “hMK61T3”, “hMK61T4”, “hMK61T5”, and “hMK61T6”. Additionally, a mouse isoform (“mMK61”) and an Fc-fusion polypeptide thereof (“mMK61-Fc” and “hMK61-Fc”) are described herein.

The invention provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15;

(b) a nucleotide sequence encoding the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(c) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of (a) or (b), wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; and

(d) a nucleotide sequence complementary to any of (a) through (c).

The invention also provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide that is at least about 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99 percent identical to the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, OR SEQ ID NO:16, wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(b) a nucleotide sequence encoding an allelic variant or splice variant of the nucleotide sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15, wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(c) a nucleotide sequence of SEQ ID NO:1, (a), or (b) encoding a polypeptide fragment of at least about 25 amino acid residues, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(d) a nucleotide sequence encoding a polypeptide that has at least one amino acid substitution and/or deletion of the amino sequence set forth in any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(e) a nucleotide sequence of SEQ ID NO:1, or (a)-(d) comprising a fragment of at least about 16 nucleotides;

(f) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(e), wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, OR SEQ ID NO:16; and

(g) a nucleotide sequence complementary to any of (a)-(e).

The invention further provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 with at least one conservative amino acid substitution, wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(b) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 with at least one amino acid insertion, wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(c) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 with at least one amino acid deletion, wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(d) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 which has a C- and/or N-terminal truncation, wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(e) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncation, and N-terminal truncation, wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(f) a nucleotide sequence of (a)-(e) comprising a fragment of at least about 16 nucleotides;

(g) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(f), wherein the encoded polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; and

(h) a nucleotide sequence complementary to any of (a)-(e).

The invention also provides for an expression vector comprising the isolated nucleic acid molecules set forth herein; recombinant host cells (eukaryotic and/or prokaryotic) that comprise the vector; the process for producing a h2520 polypeptide comprising culturing the host cell under suitable conditions to express the polypeptide and optionally isolating the polypeptide from the culture; and the isolated polypeptide produced by this process. The nucleic acid molecule used in this process may also comprise promoter DNA other than the promoter DNA for the native MK61 polypeptide operatively linked to the nucleotide sequence encoding the MK61 polypeptide.

The invention also provides for a nucleic acid molecule as described in the previous paragraphs wherein the percent identity is determined using a computer program selected from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm.

The present invention provides a process for identifying candidate inhibitors and/or stimulators of MK61 polypeptide activity or production comprising exposing a host cell to the candidate inhibitors and/or stimulators, measuring MK61 polypeptide activity or production in the host cell, and comparing this activity with control cells (i.e., cells not exposed to the candidate inhibitor and/or stimulator). In a related aspect, the invention provides for the inhibitors and/or stimulators identified by any of the preceding methods.

The invention also provides for an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID No: 12, SEQ ID NO: 14 or SEQ ID NO: 16.

The invention also provides for an isolated polypeptide comprising the amino acid sequence selected from the group consisting of:

(a) the mature amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, and optionally further comprising an amino-terminal methionine;

(b) an amino acid sequence for an ortholog of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(c) an amino acid sequence exhibits at least about 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99 percent identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 as determined using a computer program selected from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit and the Smith-Waterman algorithm;

(d) a fragment of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 comprising at least about 25 amino acid residues, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; and

(e) an amino acid sequence for an allelic variant or splice variant of either the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, or at least one of (a)-(c) wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

The invention further provides for an isolated polypeptide comprising the amino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, with at least one conservative amino acid substitution, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(b) the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, with at least one amino acid insertion, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(c) the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, with at least one amino acid deletion, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16;

(d) the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 which has a C- and/or N-terminal truncation, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; and

(e) the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncation, and N-terminal truncation, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

Analogs of MK61 are provided for in the present invention which result from conservative and non-conservative amino acid substitutions of the MK61 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Such analogs include a MK61 polypeptide wherein the amino acid corresponding to position 38, 39 or 51 of SEQ ID NOS: 2, 4, 6, 8, 10 or 12 is cysteine, serine or alanine; a MK61 polypeptide wherein the amino acid corresponding to position 60 or 76 of SEQ ID NOS: 2 or 6 is cysteine, serine or alanine a MK61 polypeptide wherein the amino acid corresponding to position 41, 42, 54, 63 or 79 of SEQ ID NOS: 14 or 16 is cysteine, serine or alanine; a MK61 polypeptide wherein the amino acid corresponding to position 171 or 172 of SEQ ID NO: 2 is leucine, norleucine, valine, methionine, alanine or phenylalanine; a MK61 polypeptide wherein the amino acid corresponding to position 178 or 180 of SEQ ID NOS: 14 or 16 is leucine, norleucine, valine, methionine, alanine or phenylalanine; a MK61 polypeptide wherein the amino acid corresponding to position 141 of SEQ ID NOS: 14 or 16 is glycine, proline or alanine.

The invention also provides methods of inhibiting MK61 receptor and/or ligand activity in a mammal, which comprises administering at least one polypeptide set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

Also provided are fusion polypeptides comprising the amino acid sequences of (a)-(e) above. In addition, the invention encompasses fusion polypeptides comprising the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 36 and SEQ ID NO: 39.

The present invention also provides for an expression vector comprising the isolated nucleic acid molecules as set forth herein, recombinant host cells comprising recombinant nucleic acid molecules as set forth herein, and a method of producing an MK61 polypeptide comprising culturing the host cells and optionally isolating the polypeptide so produced.

A transgenic non-human animal comprising a nucleic acid molecule encoding an MK61 polypeptide is also encompassed by the invention. The MK61 nucleic acid molecules are introduced into the animal in a manner that allows expression and increased levels of the MK61 polypeptide, which may include increased circulating levels. The transgenic non-human animal is preferably a mammal.

Also provided are derivatives of the MK61 polypeptides of the present invention.

The present invention further provides for an antibody or fragment thereof that specifically binds an MK61 polypeptide as set forth herein. This antibody can be polyclonal or monoclonal, and can be produced by immunizing an animal with a peptide comprising an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.

Also provided is the hybridoma that produces a monoclonal antibody that binds to a peptide comprising an amino acid sequence of SEQ ID NO: 2

The present invention also provides for a method of detecting or quantitating the amount of MK61 polypeptide in a sample comprising contacting a sample suspected of containing MK61 polypeptide with the anti-MK61 antibody or antibody fragment set forth herein and detecting the binding of said antibody or antibody fragment.

Additionally provided by the invention are selective binding agents or fragments thereof that are capable of specifically binding the MK61 polypeptides, derivatives, variants, and fragments (preferably having sequences of at least about 25 amino acids) thereof. These selective binding agents may be antibodies such as humanized antibodies, human antibodies, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, complementarity determining region (CDR)-grafted antibodies, anti-idiotypic antibodies, and fragments thereof. Furthermore, the selective binding agents may be antibody variable region fragments, such as Fab or Fab′ fragments, or fragments thereof, and may comprise at least one complementarity determining region with specificity for a MK61 polypeptide set forth herein. The selective binding agent may also be bound to a detectable label, such as a radiolabel, a fluorescent label, an enzyme label, or any other label known in the art. Further, the selective binding agent may antagonize MK61 polypeptide biological activity, and/or be produced by immunizing an animal with a MK61 polypeptide as set forth herein.

The present invention also provides for a hybridoma that produces a selective binding agent capable of binding MK61 polypeptide as set forth herein.

Also provided is a method for treating, preventing, or ameliorating a disease, condition, or disorder comprising administering to a patient an effective amount of a selective binding agent as set forth herein. An effective amount, or a therapeutically effective amount, is an amount sufficient to result in a detectable change in the course or magnitude of the disease, condition or disorder, such as the intensity or duration of presentment of any symptom associated therewith.

Pharmaceutical compositions comprising the above-described nucleic acid molecules, polypeptides or selective binding agents and one or more pharmaceutically acceptable formulation agents are also encompassed by the invention. The pharmaceutical acceptable formulation agent may be a carrier, adjuvant, soubilizer, stabilizer, or anti-oxidant. The nucleic acid molecules of the present invention may be contained in viral vectors. The compositions are used to provide therapeutically effective amounts of the nucleic acid molecules or polypeptides of the present invention. The invention is also directed to methods of using the polypeptides, nucleic acid molecules, and selective binding agents.

Also provided are derivatives of the MK61 polypeptides of the present invention. These polypeptides may be covalently modified with a water-soluble polymer wherein the water-soluble polymer is selected from the group consisting of polyethylene glycol, monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols, and polyvinyl alcohol.

The present invention also provides for fusion polypeptides comprising the polypeptide sequences set forth herein fused to a heterologous amino acid sequence, which may be an IgG constant domain or fragment thereof.

Methods for treating, preventing or ameliorating a medical condition, such as cancer, in a mammal resulting from decreased levels of MK61 polypeptide are also included in the present invention. These methods include administering to a patient a therapeutically effective amount of an antagonist selected from the group consisting of selective binding agents, small molecules, peptides, peptide derivatives and antisense oligonucleotides. These medical conditions may include those characterized by immune system stimulation such as autoimmune diseases and leukemias and lymphomas.

Methods for treating, preventing or ameliorating a medical condition in a mammal resulting from increased levels of MK61 polypeptide are also included in the present invention. These methods comprise administering to a patient a therapeutically effective amount of a MK61 polypeptide; a nucleic acid molecule encoding a MK61 polypeptide; or a nucleic acid molecule comprising elements that regulate or modulate the expression of a MK61 polypeptide. Examples of these methods include gene therapy and cell therapy and are further described herein. These medical conditions may include those characterized by immune system suppression such as AIDs and cancers.

The invention encompasses methods of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject caused by or resulting from abnormal levels of MK61 polypeptide comprising determining the presence or amount of expression of the MK61 polypeptide in a biological, tissue, or cellular sample; and comparing the level of said polypeptide in a biological, tissue, or cellular sample from either normal subjects or the subject at a different time, wherein susceptibility to a pathological condition is based on the presence or amount of expression of the polypeptide.

The MK61 polypeptides and nucleic acid molecules of the present invention may be used to treat, prevent, ameliorate and/or detect diseases and disorders, including those recited herein.

The present invention also provides a method of identifying compounds which bind to a MK61 polypeptide. The method comprises contacting an MK61 polypeptide with a test molecule and determining the extent of binding of the test molecule to the polypeptide. The method may further comprise determining whether such test molecules are agonists or antagonists of an MK61 polypeptide. The present invention further provides a method of testing the impact of molecules on the expression of an MK61 polypeptide or on the activity of an MK61 polypeptide.

Methods of regulating expression and modulating (i.e., increasing or decreasing) levels of an MK61 polypeptide are also encompassed by the invention. One method comprises administering to an animal a nucleic acid molecule encoding an MK61 polypeptide. In another method, a nucleic acid molecule comprising elements that regulate or modulate the expression of an MK61 polypeptide may be administered. Examples of these methods include gene therapy, cell therapy and anti-sense therapy as further described herein.

The present invention further provides a method of modulating levels of a MK61 polypeptide in an animal comprising administering to the animal the nucleic acid molecule set forth herein.

A transgenic non-human animal comprising a nucleic acid molecule encoding a MK61 polypeptide is also encompassed by the invention. The MK61 nucleic acid molecule is introduced into the animal in a manner that allows expression and increased levels of the MK61 polypeptide, which may include increased circulating levels. The transgenic non-human animal is preferably a mammal.

The present invention provides for a diagnostic reagent comprising a detectably labeled polynucleotide encoding the amino acid sequence set out in SEQ ID NO: 2, or a fragment, variant or homolog thereof, including allelic variants and spliced variants thereof. The detectably labeled polynucleotide may be a first-strand cDNA, DNA, or RNA.

The invention also provides a method for detecting the presence of MK61 nucleic acid molecules in a biological sample comprising the steps of:

(a) providing a biological sample suspected of containing MK61 nucleic acid molecules;

(b) contacting the biological sample with a diagnostic reagent under conditions wherein the diagnostic reagent will hybridize with MK61 nucleic acid molecules contained in said biological sample;

(c) detecting hybridization between MK61 nucleic acid molecules in the biological sample and the diagnostic reagent; and

(d) comparing the level of hybridization between the biological sample and diagnostic reagent with the level of hybridization between a known concentration of MK61 nucleic acid molecules and the diagnostic reagent.

The invention also provides a method for detecting the presence of MK61 nucleic acid molecules in a tissue or cellular sample comprising the steps of:

(a) providing a tissue or cellular sample suspected of containing MK61 nucleic acid molecules;

(b) contacting the tissue or cellular sample with a diagnostic reagent under conditions wherein the diagnostic reagent will hybridize with MK61 nucleic acid molecules;

(c) detecting hybridization between MK61 nucleic acid molecules in the tissue or cellular sample and the diagnostic reagent; and

(d) comparing the level of hybridization between the tissue or cellular sample and diagnostic reagent with the level of hybridization between a known concentration of MK61 nucleic acid molecules and the diagnostic reagent.

The invention provides for methods of inhibiting MK61 receptor activity in a mammal comprising administering at least one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 36 and 38.

The invention provides for methods of inhibiting MK61 ligand activity in a mammal comprising administering at least one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 36 and 38.

The invention provides for methods of stimulating an immune response in a mammal by administering a negative regulator of MK61 receptor signaling. A negative regulator is a molecule which inhibits the signaling of the MK61 receptor. Negative regulators include but are not limited to fusion proteins, such as those set out in SEQ ID NOS: 16, 36 and 39, antibodies, small molecules, peptides and peptide derivatives.

The invention also provides for methods of inhibiting an immune response comprising administering a positive regulator or MK61 receptor signaling. A positive regulator is a molecule which activates the signaling of MK61 receptor. Positive regulators include MK61 ligands and agonistic antibodies.

The invention provides for methods of stimulating reverse signaling through a cell surface bound MK61 ligand comprising a positive regulator of MK61 ligand reverse signaling. The positive regulators include but are not limited to MK61 fusion proteins, antibodies, small molecules and peptide derivatives. The term “reverse signaling” refers to activation of cellular signaling induced by a molecule binding to a cell surface bound ligand such as binding by the ligand\'s receptor or an anti-ligand antibody.

The invention also provides for methods of inhibiting reverse signaling through a cell surface bound MK61 ligand comprising a negative regulator of MK61 ligand reverse signaling. The negative regulators include but are not limited to MK61 fusion proteins, antibodies, small molecules and peptide derivatives.

The invention provides for methods of treating a B cell or T cell lymphoproliferative disorder, an autoimmune disease or an inflammatory disease in a mammal comprising administering a therapeutically effective amount of MK61-Fc fusion protein, an anti-MK-61 antibody, an antisense oligonucleotide, a MK61 ligand, or a anti-MK61 ligand antibody to said mammal. The lymphoproliferative diseases that may be treated include but are not limited to myeloma; B lymphoma, leukemia; and non-hodgkins lymphoma. The autoimmune diseases include but are not limited to rheumatoid arthritis, systemic lupus erythematosus, intestinal bowel disease and Crohn\'s Disease. The inflammatory diseases include but are not limited to rheumatoid arthritis, sepsis, intestinal bowel disease and Crohn\'s Disease.

The invention also encompasses a polypeptide fragment having an amino acid sequence comprising the cysteine rich domain residues 26-60 of SEQ ID NO: 36. The cysteine rich domain matches the TNFR superfamily cysteine-rich region signature as defined in Madry et. al (Intl. Immunol. 10:1693-1702, 1998) and references therein and is expected to encompass the MK61 ligand-binding domain.

The MK61 polypeptides can be used for identifying ligands thereof. Various forms of “expression cloning” have been used for cloning ligands for receptors, see e.g., Davis et al., Cell, 87:1161-1169 (1996). These and other MK61 ligand cloning experiments are described in greater detail herein. Isolation of the MK61 ligand(s) allows for the identification or development of novel agonists and/or antagonists of the MK61 signaling pathway. Such agonists and antagonists include MK61 ligand(s), anti-MK61 ligand antibodies and derivatives thereof, small molecules, carbohydrates, lipid, polynucleotides (including antisense oligonucleotides), any of which can be used for potentially treating one or more diseases or disorders, including those recited herein.

It will be understood that in the figures described below, the nucleotides 5′ to those nucleotides encoding the signal peptide are part of the 5′-untranslated (5′-UTR) flanking sequence. Additionally, nucleotides 3′ to the stop codon represent the 3′-untranslated (3′-UTR) sequence.

FIG. 1 depicts a nucleic acid sequence (SEQ ID NO:1) encoding human MK61T1 (hMK61T1). Also depicted is the amino acid sequence (SEQ ID NO:2) of human hMK61T1. hMK61T1 is a cell surface receptor which contains a signal peptide (SP), one TNFr type cysteine rich domain (CRD), spacer, transmembrane domain (TM), and a long intracellular domain with two regions highly conserved between species. The predicted signal peptide is underlined in this figure, and the stop codon in SEQ ID NO:1 is double-underlined.

FIG. 2 depicts a nucleic acid sequence (SEQ ID NO:3) encoding human MK61T2 (hMK61T2), believed to be a soluble receptor. Also depicted is the amino acid sequence (SEQ ID NO:4) of human hMK61T2. The predicted signal peptide is underlined in this figure, and the stop codon in SEQ ID NO:3 is double-underlined.

FIG. 3 depicts a nucleic acid sequence (SEQ ID NO:5) encoding human MK61T3 (hMK61T3). Also depicted is the amino acid sequence (SEQ ID NO:6) of human hMK61T3. hMK61T3 is believed to be a soluble receptor, having a signal peptide and TNFr-type CRD. The predicted signal peptide is underlined in this figure, and the stop codon in SEQ ID NO:5 is double-underlined.

FIG. 4 depicts a nucleic acid sequence (SEQ ID NO:7) and amino acid sequence (SEQ ID NO:8) encoding human MK61T4 (hMK61T4), believed to be a soluble receptor. The predicted signal peptide is underlined in this figure, and the stop codon in SEQ ID NO:7 is double-underlined.

FIG. 5 depicts a nucleic acid sequence (SEQ ID NO:9) and amino acid sequence (SEQ ID NO:10) encoding human MK61T5 (hMK61T5), believed to be a soluble receptor. The predicted signal peptide is underlined in this figure, and the stop codon in SEQ ID NO:9 is double-underlined.

FIG. 6 depicts a nucleic acid sequence (SEQ ID NO:11) and amino acid sequence (SEQ ID NO:12) encoding human MK61T6 (hMK61T6), believed to be a soluble receptor. The predicted signal peptide is underlined in this figure, and the stop codon in SEQ ID NO:11 is double-underlined.

FIG. 7 depicts the nucleic acid sequence (SEQ ID NO:13) and amino acid sequence (SEQ ID NO:14) encoding mouse MK61 (mMK61), also called “Smil2-00051-F3”, or “Smil2-00051”. In this figure, the predicted signal peptide is underlined, and the stop codon in SEQ ID NO:13 is double-underlined.

FIG. 8 depicts the nucleic acid sequence (SEQ ID NO:15) and amino acid sequence (SEQ ID NO:16) encoding the mouse mMK61-Fc fusion polypeptide (mMK61-Fc). In this figure, the predicted signal peptide is underlined, the Fc portion of the sequence is double-underlined, the NotI restrict site for joining MK61 to Fc is in bold, and the Kozak consensus sequence (which is not translated) is in italics.

FIG. 9 sets forth a Western Blot showing that the mMK61 Fc fusion protein (mMK61-Fc) is capable of being secreted from mammalian cells.

FIG. 10 sets forth an amino acid comparison of mMK61 (SEQ ID NO:14) with an OPG receptor, Mrank, (SEQ ID NO:17), a known TNFr family member. Mrank is the mouse OPG (osteoprotegerin) receptor precursor.

FIG. 11 sets forth an amino acid comparison of mMK61 (SEQ ID NO:14) with the Fas ligand receptor (mfas), (SEQ ID NO:18). mfas is an apoptosis-mediating surface antigen receptor precursor.

FIG. 12 sets forth an amino acid comparison of mMK61 (SEQ ID NO:14) with a known mouse lymphotoxin-beta receptor (Tnfrc), SEQ ID NO:19. Tnfrc is a lymphotoxin-beta receptor precursor, and is also called “tumor necrosis factor receptor 2 related protein” or “tumor necrosis factor-c receptor precursor”.

FIGS. 13 and 14 depict multiple tissue Northern blots which were probed with a random primed human MK61 radioactive probe. These blots demonstrate that human MK61 mRNA is expressed in human lymphoid tissues.

FIG. 15 depicts a histogram comparing human MK61 mRNA expression in various human tissues and cell lines as measured by quantitative PCR.

FIG. 16 depicts histograms quantitating the binding of the MK61-Fc fusion protein on the surface of human cells as measured by FACS analysis. The histograms indicate that MK61-Fc fusion protein binds to the cell surface of U937 and Jurkat cells.

FIG. 17 displays histograms demonstrating enhanced binding of the MK61-Fc fusion protein on the cell surface of Jurkat and U937 cells after treatment with interferon gamma.

FIG. 18 depicts histograms quantitating the production of IgG (top panel) and IgA (bottom panel) in mouse splenocyte cultures after treatment with MK61-Fc fusion protein.

FIG. 19 depicts histograms quantitating the effect of MK61-Fc fusion protein on spleen weights in mice (top panel) and spleen lymphocytes (bottom panel). These histograms demonstrate that treatment with the MK61-Fc fusion protein increased the spleen weight and the number of spleen lymphocytes.

FIG. 20 depicts the histological analysis of the spleens of MK61-Fc treated mice. The histological analysis indicated the presence of lymphoid hyperplasia.

FIG. 21 depicts histograms quantitating the numbers of spleen B and T cells in mice treated with MK61-Fc fusion protein.

FIG. 22 depicts histograms quantitating plasma immunoglobulin levels in mice treated with MK61-Fc fusion protein.

FIG. 23 depicts histograms quantitating the generation of anti-KLH specific antibodies in mice treated with MK61-Fc fusion protein.

FIG. 24 sets out the amino acid sequence of the human MK61-delta Fc CHO (SEQ ID NO: 36).

FIG. 25 sets out the amino acid sequence of the human MK61-Fc CHO (SEQ ID NO: 39).

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited in this application are expressly incorporated by reference herein.

The hMK61T1 isoform is a cell-surface receptor having a signal peptide, a TNF receptor (TNFR) cysteine rich domain (CRD), a transmembrane domain (TM), and a long and highly conserved intracellular domain.

The remaining five human isoforms (hMK61T2, hMK61T3, hMK61T4, hMK61T5, and hMK61T6) are believed to be soluble receptor forms of MK61. hMK61T3 and hMK61T5 each contain a complete TNFr CRD, and are likely naturally-occurring inhibitors of the hMK61T1 mediated signal transduction. The hMK61T2, hMK61T4, and hMK61T6 isoforms each contain partial CRD\'s.

mMK61 is a mouse MK61 isoform, and mMK61-Fc is an Fc-fusion polypeptide thereof.

The terms “MK61 gene” or “MK61 nucleic acid molecule” or “polynucleotide” refers to a nucleic acid molecule comprising or consisting of a nucleotide sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15, a nucleotide sequence encoding the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, and nucleic acid molecules as defined herein.

The term “MK61 polypeptide” refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, and related polypeptides. Related polypeptides include: MK61 polypeptide allelic variants, MK61 polypeptide orthologs, MK61 polypeptide splice variants, MK61 polypeptide variants and MK61 polypeptide derivatives. MK61 polypeptides may be mature polypeptides, as defined herein, and may or may not have an amino terminal methionine residue, depending on the method by which they are prepared.

The term “MK61 polypeptide allelic variant” refers to the polypeptide encoded by one of several possible naturally occurring alternate forms of a gene occupying a given locus on a chromosome of an organism or a population of organisms.

The term “MK61 polypeptide derivatives” refers to a polypeptide having the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, MK61 polypeptide allelic variants, MK61 polypeptide orthologs, MK61 polypeptide splice variants, or MK61 polypeptide variants, as defined herein, that have been chemically modified.

The term “MK61 polypeptide fragment” refers to a polypeptide that comprises a truncation at the amino terminus (with or without a leader sequence) and/or a truncation at the carboxy terminus of the polypeptide whose sequence is as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, MK61 polypeptide allelic variants, MK61 polypeptide orthologs, MK61 polypeptide splice variants and/or an MK61 polypeptide variant having one or more amino acid additions or substitutions or internal deletions

(wherein the resulting polypeptide is at least six (6) amino acids or more in length) as compared to the MK61 polypeptide amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. MK61 polypeptide fragments may result from alternative RNA splicing or from in vivo protease activity. For transmembrane or membrane-bound forms of the MK61 polypeptides, preferred fragments include soluble forms such as those lacking a transmembrane or membrane-binding domain.

In preferred embodiments, truncations comprise about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids. The polypeptide fragments so produced will comprise about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids. Such MK61 polypeptide fragments may optionally comprise an amino terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies to MK61 polypeptides.

The term “MK61 fusion polypeptide” refers to a fusion of one or more amino acids (such as a heterologous peptide or polypeptide) at the amino or carboxy terminus of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, MK61 polypeptide allelic variants, MK61 polypeptide orthologs, MK61 polypeptide splice variants, or MK61 polypeptide variants having one or more amino acid deletions, substitutions or internal additions as compared to the MK61 polypeptide amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

The term “MK61 polypeptide ortholog” refers to a polypeptide from another species that corresponds to an MK61 polypeptide amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. For example, mouse and human MK61 polypeptides are considered orthologs of each other.

The term “MK61 polypeptide splice variant” refers to a nucleic acid molecule, usually RNA, which is generated by alternative processing of intron sequences in an RNA primary transcript containing the non-contiguous coding region of the MK61 polypeptide amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

The term “MK61 polypeptide variants” refers to MK61 polypeptides comprising amino acid sequences having one or more amino acid sequence substitutions, deletions (such as internal deletions and/or MK61 polypeptide fragments), and/or additions (such as internal additions and/or MK61 fusion polypeptides) as compared to the MK61 polypeptide amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 (with or without a leader sequence). Variants may be naturally occurring (e.g., MK61 polypeptide allelic variants, MK61 polypeptide orthologs and MK61 polypeptide splice variants) or may be artificially constructed. Such MK61 polypeptide variants may be prepared from the corresponding nucleic acid molecules having a DNA sequence that varies accordingly from the DNA sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 amino acid substitutions, insertions, additions and/or deletions, wherein the substitutions may be conservative, or non-conservative, or any combination thereof.

The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of each antigen. An antigen may have one or more epitopes.

The term “biologically active MK61 polypeptides” refers to MK61 polypeptides having at least one activity characteristic of the polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

The terms “effective amount” and “therapeutically effective amount” each refer to the amount of a MK61 polypeptide or MK61 nucleic acid molecule used to support an observable level of one or more biological activities of the MK61 polypeptides as set forth herein.

The term “expression vector” refers to a vector which is suitable for use in a host cell and contains nucleic acid sequences which direct and/or control the expression of heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.

The term “host cell” is used to refer to a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing a selected gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present.

The term “identity” as known in the art refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”).

The term “similarity” is a related concept but, in contrast to “identity”, refers to a measure of similarity which includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. If, in the same example, there are five more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% (15/20). Therefore, in cases where there are conservative substitutions, the degree of percent similarity between two polypeptides will be higher than the percent identity between those two polypeptides.

The term “isolated nucleic acid molecule” refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates or other materials with which it is naturally found when total DNA is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the “isolated nucleic acid molecule” is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.

The term “isolated polypeptide” refers to a polypeptide of the present invention that (1) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates or other materials with which it is naturally found when isolated from the source cell, (2) is not linked (by covalent or noncovalent interaction) to all or a portion of a polypeptide to which the “isolated polypeptide” is linked in nature, (3) is operably linked (by covalent or noncovalent interaction) to a polypeptide with which it is not linked in nature, or (4) does not occur in nature. Preferably, the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research use.

The term “mature MK61 polypeptide” refers to an MK61 polypeptide lacking a leader sequence. A mature MK61 polypeptide may also include other modifications such as proteolytic processing of the amino terminus (with or without a leader sequence) and/or the carboxy terminus, cleavage of a smaller polypeptide from a larger precursor, N-linked and/or O-linked glycosylation, and the like.

An exemplary mature MK61 polypeptide is depicted by amino acid residue 24 through amino acid residue 355 of SEQ ID NO:2; by amino acid residue 24 through amino acid residue 85 of SEQ ID NO:4; by amino acid residue 24 through amino acid residue 136 of SEQ ID NO:6; by amino acid residue 24 through amino acid residue 187 of SEQ ID NO:8; by amino acid residue 24 through amino acid residue 71 of SEQ ID NO:10; by amino acid residue 24 through amino acid residue 167 of SEQ ID NO:12; by amino acid residue 22 through amino acid residue 345 of SEQ ID NO:14; and by amino acid residue 22 through amino acid residue 404 of SEQ ID NO:16.

The terms “nucleic acid sequence” or “nucleic acid molecule” refer to a DNA or RNA sequence. The terms encompass molecules formed from any of the known base analogs of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenine, aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

The term “naturally occurring” or “native” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refer to materials which are found in nature and are not manipulated by man. Similarly, “non-naturally occurring” or “non-native” as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.

The term “operably linked” is used herein to refer to a method of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function. Thus, a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of that coding sequence. A flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence.

The terms “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refer to one or more formulation materials suitable for accomplishing or enhancing the delivery of the MK61 polypeptide, MK61 nucleic acid molecule or MK61 selective binding agent as a pharmaceutical composition.

The term “selective binding agent” refers to a molecule or molecules having specificity for an MK61 polypeptide. As used herein the terms, “specific” and “specificity” refer to the ability of the selective binding agents to bind to human MK61 polypeptides and not to bind to human non-MK61 polypeptides. It will be appreciated, however, that the selective binding agents may also bind orthologs of the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, that is, interspecies versions thereof, such as mouse and rat polypeptides.

The term “transduction” is used to refer to the transfer of nucleic acids from one bacterium to another, usually by a phage. “Transduction” also refers to the acquisition and transfer of eukaryotic cellular sequences by retroviruses.

The term “transfection” is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell has been “transfected” when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., Virology, 52:456 (1973); Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York, (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier, (1986); and Chu et al., Gene, 13:197 (1981). Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

The term “transformation” as used herein refers to a change in a cells genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transfection or transduction, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, it may be maintained transiently as an episomal element without being replicated, or it may replicate independently as a plasmid. A cell is considered to have been stably transformed when the DNA is replicated with the division of the cell.

The term “vector” is used to refer to any molecule (e.g., nucleic acid, plasmid or virus) used to transfer coding information to a host cell.

Relatedness of Nucleic Acid Molecules and/or Polypeptides

It is understood that related nucleic acid molecules include allelic or splice variants of the nucleic acid molecule of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15, and include sequences which are complementary to any of the above nucleotide sequences. Related nucleic acid molecules also include a nucleotide sequence encoding a polypeptide comprising or consisting essentially of a substitution, modification, addition and/or deletion of one or more amino acid residues compared to the polypeptide in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

Fragments include molecules which encode a polypeptide of at least about 25 amino acid residues, or about 50, or about 75, or about 100, or greater than about 100, amino acid residues of the polypeptide of SEQ ID NO:2.

In addition, related MK61 nucleic acid molecules include those molecules which comprise nucleotide sequences which hybridize under moderately or highly stringent conditions as defined herein with the fully complementary sequence of the nucleic acid molecule of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15, or of a molecule encoding a polypeptide, which polypeptide comprises the amino acid sequence as shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, or of a nucleic acid fragment as defined herein, or of a nucleic acid fragment encoding a polypeptide as defined herein. Hybridization probes may be prepared using the MK61 sequences provided herein to screen cDNA, genomic or synthetic DNA libraries for related sequences. Regions of the DNA and/or amino acid sequence of MK61 polypeptide that exhibit significant identity to known sequences are readily determined using sequence alignment algorithms as described herein, and those regions may be used to design probes for screening.

The term “highly stringent conditions” refers to those conditions that are designed to permit hybridization of DNA strands whose sequences are highly complementary, and to exclude hybridization of significantly mismatched DNAs. Hybridization stringency is principally determined by temperature, ionic strength and the concentration of denaturing agents such as formamide. Examples of “highly stringent conditions” for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate at 65-68° C. or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and Anderson et al., Nucleic Acid Hybridization: a practical approach, Ch. 4, IRL Press Limited, Oxford, England (1985).

More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used; however, the degree of hybridization will be affected. Other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate (NaDodSO4 or SDS), ficoll, Denhardt\'s solution, sonicated salmon sperm DNA (or another non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al., Nucleic Acid Hybridization: a Practical Approach, Ch. 4, IRL Press Limited, Oxford, England (1985).

Factors affecting the stability of a DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one skilled in the art in order to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:

Tm(° C.)=81.5+16.6(log [Na+])+0.41(% G+C)−600/N−0.72(% formamide)

where N is the length of the duplex formed in nucleotides, [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution, and % G+C is the percentage of (guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids, the melting temperature is reduced by approximately 1° C. for each 1% mismatch.

The term “moderately stringent conditions” refers to conditions under which a DNA duplex with a greater degree of base pair mismatching than could occur under “highly stringent conditions” is able to form. Examples of typical “moderately stringent conditions” are 0.015M sodium chloride, 0.0015M sodium citrate at 50-65° C. or 0.015M sodium chloride, 0.0015M sodium citrate, and 20% formamide at 37-50° C. By way of example, a “moderately stringent” condition of 50° C. in 0.015 M sodium ion will allow about a 21% mismatch.

It will be appreciated by those skilled in the art that there is no absolute distinction between “highly” and “moderately” stringent conditions. For example, at 0.015M sodium ion (no formamide), the melting temperature of perfectly matched long DNA is about 71° C. With a wash at 65° C. (at the same ionic strength), this would allow for approximately a 6% mismatch. To capture more distantly related sequences, one skilled in the art can simply lower the temperature or raise the ionic strength.

A good estimate of the melting temperature in 1M NaCl* for oligonucleotide probes up to about 20 nucleotides is given by:

Tm=2° C. per A-T base pair+4° C. per G-C base pair

*The sodium ion concentration in 6× salt sodium citrate (SSC) is 1M. See Suggs et al., Developmental Biology Using Purified Genes, p. 683, Brown and Fox (eds.) (1981).

High stringency washing conditions for oligonucleotides are usually at a temperature of 0-5° C. below the Tm of the oligonucleotide in 6×SSC, 0.1% SDS for longer oligonucleotides.

In another embodiment, related nucleic acid molecules comprise or consist of a nucleotide sequence that is about 70 percent (70%) identical to the nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15, or comprise or consist essentially of a nucleotide sequence encoding a polypeptide that is about 70 percent (70%) identical to the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. In preferred embodiments, the nucleotide sequences are about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the nucleotide sequence as shown in SEQ ID NO:1, or the nucleotide sequences encode a polypeptide that is about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the polypeptide sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

Differences in the nucleic acid sequence may result in conservative and/or non-conservative modifications of the amino acid sequence relative to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

Conservative modifications to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 (and corresponding modifications to the encoding nucleotides) will produce MK61 polypeptides having functional and chemical characteristics similar to those of a naturally occurring MK61 polypeptide. In contrast, substantial modifications in the functional and/or chemical characteristics of MK61 polypeptides may be accomplished by selecting substitutions in the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for “alanine scanning mutagenesis.”

Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

Naturally occurring residues may be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the human MK61 polypeptide that are homologous, or similar, with non-human MK61 polypeptide orthologs, or into the non-homologous regions of the molecule.

In making such changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional equivalent protein or peptide thereby created is intended, in part, for use in immunological embodiments, as in the present case. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5) and tryptophan (−3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”

Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the MK61 polypeptide, or to increase or decrease the affinity of the MK61 polypeptides for their substrates, described herein.

Exemplary amino acid substitutions are set forth in Table I.

TABLE I Amino Acid Substitutions Original Exemplary Preferred

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