Mammalian cytokine receptor subunit proteins, related reagents and methods

Description of the invention

The invention relates to a substantially pure or recombinant cytokine receptor related polypeptide, an isolated or recombinant nucleic acid, an expression vector, a host cell, a method of producing a polypeptide, a binding compound comprising an antibody or antigen binding fragment thereof, a method of producing an antigen: antibody complex, compositions, and uses. .

The solutions according to the invention have an impact on the physiology of mammals, including the functioning of their immune system. In particular, they make it possible to regulate the development and / or functioning of the immune system. Diagnostic and therapeutic uses are also disclosed herein.

Recombinant DNA technology generally relates to techniques for integrating genetic information from a donor source into vectors and its further processing, such as introduction into a host, whereby this information is reproduced and / or expressed in a new environment. Typically, the genetic information exists in the form of complementary DNA (cDNA) obtained from messenger RNA (mRNA) encoding the desired protein product. The carrier is often a plasmid that has the ability to integrate cDNA and then replicate it in host cells and, in some cases, control cDNA expression and thus synthesize the encoded product in the host. See, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed.) Vol. 1-3, CSH Press, NY.

It has been known for some time that the immune response of mammals is based on a series of complex cellular interactions known as the "immune network". Modern research has provided new data on the interaction within this network. While it is clear that a major part of the immune response is related to the interplay of lymphocytes, macrophages, granulocytes, and other cells, immunologists now believe that soluble proteins such as lymphokines, cytokines, and monokines play a key role in controlling these intercellular interactions. Hence, there is a relatively high interest in isolating, characterizing and explaining the mechanisms of action of factors regulating cell activity, which will enable significant progress in the diagnosis and therapy of numerous diseases, e.g. disorders of the immune system.

Lymphokines obviously regulate cellular activity in many ways. See, e.g., Paul (1996 eds) Fundamental Immunology ed. III, Raven Press, New York; and Thomson (1994 eds) The Cytokine Handbook ed. II, Academic Press, San Diego. They have been shown to support the proliferation, growth and / or differentiation of undifferentiated bone marrow stem cells into a variety of precursor cells, including cell lines that form a complex immune system. Proper and balanced interactions between cellular components are essential for a healthy immune response. Different cell lines often respond differently to lymphokines when administered with other agents.

Cell lines of particular importance in the immune response contain two classes of lymphocytes: B cells, which can produce and secrete immunoglobulins (proteins capable of recognizing and binding foreign substances in order to remove them) and various types of T cells that secrete lymphokines and stimulate or inhibit B cells as well as a variety of other cells (including other T cells) forming the immune network. These lymphocytes interact with many other types of cells.

Research to better understand and treat disorders of the immune system has been hampered by the inherent inability to cultivate many cells of the immune system in vitro. Immunologists have found that many cell types can be cultivated using supernatants derived from T cells and other cell types that contain a variety of growth factors, including many lymphokines.

There are many different growth and regulatory factors that influence morphogenetic development. Many cytokine receptors are known. Typically, a functional receptor consists of at least two substantial subunits. See, e.g., Heinrich et al. (1998) Biochem. J. 334: 297-314; Gonda and D'Andrea (1997) Blood 89: 355-369; Presky, et al. (1996) Proc. Nat'l Acad. Sci. USA 93: 14002-14007; Drachman and Kaushansky (1995) Curr. Opin. Hematol. 2: 22-28; Theze (1994) Eur. Cytokine Netw. 5: 353-368; and Lemmon and Schlessinger (1994) Trends Biochem. Sci. 19: 459-463.

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It follows from this discussion that the discovery and study of new soluble proteins and their receptors, including lymphokine-like factors, should lead to new methods of treating a number of disorders, directly or indirectly related to the development, differentiation or functioning of e.g. the immune system. and / or marrow stem cells. In particular, it would be of great benefit to disclose and study new receptors for lymphokine-like molecules that enhance the beneficial effects of other lymphokines. The present invention relates to novel ligand receptors showing similarity to cytokine-like and related compounds, and methods of using them.

The present invention relates to novel receptors related to cytokine receptors, e.g., the molecular, cytokine receptor-like structures in primates, termed DNAX Cytokine Receptor Subunit (DCRS) subunits, and their biological activities. In particular, the present describes one such subunit as DCRS5. The nucleic acid sequences encoding polypeptides and the methods of their production and use are presented. The nucleic acids of the invention are partially characterized by their homology to the cloned complementary DNA (cDNA) sequences contained herein. Herein, the alignment of p40 / IL-B30 ligand with the DCRS5 and IL12Rei receptor subunits was demonstrated. It provides information on the use of agonists and antagonists based on the reagents they are targeted at.

The invention relates to a substantially pure or recombinant polypeptide comprising amino acid residues 1 to 606 of SEQ ID NO. 2, and preferably, amino acid residues -23 to 606 of SEQ ID NO: ID NO .: 2.

The recombinant polypeptide may consist of the mature sequence described in Table 1; can be a non-glycosylated polypeptide; it may be of human origin; may be a natural polymorphic variant of SEQ ID. ID NO .: 2; it may display at least 2 non-overlapping epitopes that are specific for primate DCRS5; in naturally glycosylated form it may have a molecular weight of at least 30 kD; may be a synthetic polypeptide; it can be sterile; it can be in an aqueous or buffered solution; it can be bound to a solid support; it can be conjugated to another chemical moiety; or it may be in a form physically associated with the IL-12Re1 polypeptide. The polypeptide according to the invention may further comprise an epitope permitting its purification or detection.

Various compositions are also provided, including, for example, a substantially pure polypeptide in combination with an IL-12Re1 protein. The invention relates to a heterodimeric composition including

a) a polypeptide comprising amino acid residues 1 to 606 of SEQ ID NO. ID NO .: 2; and

b) IL-12Re1.

Preferably the composition of the invention is capable of binding IL-B30 / p40.

The polypeptide of the invention may be delivered in a carrier which may be: an aqueous solution, in particular a saline solution, and / or a buffer; and / or formulated for oral, rectal, nasal, topical or parenteral administration.

Also described herein are kits comprising a polypeptide of the invention and: a compartment containing the polypeptide; a compartment containing the IL12Re1 polypeptide; a compartment containing p40, IL-B30, or p40 / IL-B30 polypeptide; or instructions on the use or disposal of the kit reagents.

Antibodies and other binding compounds are provided, eg, including an antigen binding site from an antibody that specifically binds to the intracellular portion of DCRS5. In particular, the invention relates to a binding compound comprising an antibody, or antigen-binding fragment thereof, that specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO. 2. Preferably, the binding compound of the invention specifically binds to a polypeptide comprising residues 1 to 606 of SEQ ID NO: ID NO: 2, more preferably with a polypeptide comprising residues 356 to 606 of SEQ ID NO. ID NO: 2. In a preferred embodiment, the binding compound of the invention is a monoclonal antibody or an antigen-binding fragment thereof. It is also preferred that a binding compound of the invention is a Fab, Fab2 or Fv fragment.

The binding compound may be conjugated to another chemical moiety. It may be an antibody made in response to the peptide sequence of a mature polypeptide shown in Table 1, in response to mature DCRS5, or in response to purified human DCRS5. A binding compound of the invention can be obtained by immunoselection.

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It can be a polyclonal antibody or bind to denatured DCRS5. In addition, it can bind an antigen with a Kd binding constant of at least 30 µm. The binding compound may be bonded to a solid support such as beads or a plastic membrane. The binding compound of the invention may be formulated into a sterile composition. It is also possible to label it with a radioactive or fluorescent tag in a way that enables its detection.

Also described herein are kits comprising a binding compound and: a compartment containing a binding compound; a compartment containing: p40 polypeptide; IL-B30 polypeptide; DCRS5 polypeptide; and / or IL-12Re1 polypeptide; a compartment containing an antibody that specifically binds to: a p40 polypeptide; IL-B30 polypeptide; DCRS5 polypeptide; and / or an IL-12Re1 polypeptide; or instructions for using or disposing of the kit reagents.

The invention also relates to a method of producing an antigen-antibody complex comprising contacting a DCRS5 polypeptide of the invention with a binding compound of the invention, e.g., an antibody, under suitable conditions that allow the complex to form. In this method, the complex can be separated from other cytokine receptors; the complex can be separated from other antibodies; the contacting may take place with a sample containing interferon; contacting allows quantification of the antigen; the contacting may take place with a sample containing the antibody; or contacting allows quantification of the antibody.

Other compositions are also provided, for example compositions comprising: a sterile binding compound or binding compound and a carrier, wherein the carrier is: an aqueous solution, including a saline solution, and / or a buffer. Such compositions may be formulated for oral, rectal, nasal, topical or parenteral administration.

The invention also provides purified or recombinant nucleic acid encoding a DCRS5 polypeptide. The invention relates to an isolated or recombinant nucleic acid encoding a polypeptide of the invention. Preferably, an isolated or recombinant nucleic acid of the invention comprises nucleotide residues 188-2005 of SEQ ID NO. NO: 1. or the nucleotide residues 119-2005 of SEQ ID NO: ID NO. : 1.

A nucleic acid according to the invention may contain an origin of replication; come from a natural source; be detectably labeled; include a synthetic nucleotide sequence; be less than 6 kb in length or less than 3 kb in length; come from primates; include a full-length natural coding sequence; be a hybridization probe for the gene encoding DCRS5; or be a PCR primer, PCR product or mutagenesis primer.

The invention also relates to an expression vector comprising a nucleic acid according to the invention and comprising such a host cell vector. Cells containing the recombinant nucleic acid can be: prokaryotic cells; eukaryotic cells; bacterial cells; yeast cells; insect cells; mammalian cells; mouse cells; primate cells; or with human cells.

The invention also relates to a method of producing a polypeptide comprising:

a) culturing the host cells of the invention under conditions suitable for expression of the polypeptide; and

b) isolating or purifying said polypeptide.

Also described herein are kits that include a nucleic acid and: a compartment containing a nucleic acid encoding: a p40 polypeptide; IL-B30 polypeptide; DCRS5 polypeptide; and / or IL-12Re1 polypeptide; a compartment containing: p40 polypeptide; IL-B30 polypeptide; DCRS5 polypeptide; and / or IL-12Re1 polypeptide; a compartment containing an antibody that specifically binds to: a p40 polypeptide; IL-B30 polypeptide; DCRS5 polypeptide; and / or an IL-12Re1 polypeptide; or instructions on how to use or dispose of the kit reagents.

Nucleic acids may include acids that hybridize to a portion of SEQ ID on a 30-minute wash at 30 ° C and salt concentrations of less than 2M. ID NO: 1 encoding intracellular portion of the polypeptide. Preferably, such a nucleic acid hybridizes at a temperature of 45 ° C and / or at a salt concentration of 500 mM; either at 55 ° C and / or at a concentration of 150 mM.

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The invention also provides therapeutic uses.

The invention therefore provides a composition comprising an antagonist antibody, or an antigen binding fragment thereof, which binds to a polypeptide comprising amino acids 1-328 of SEQ ID NO. ID NO: 2 for use as a medicament.

The invention also provides a composition comprising an antagonist antibody, or an antigen binding fragment thereof, which binds to a polypeptide comprising amino acids 1-328 of SEQ ID NO. ID NO: 2 for use as a medicament.

The invention also provides a composition comprising an antagonist antibody or antigen-binding fragment thereof that binds to a complex comprising:

i) a polypeptide comprising the amino acids 1-328 of SEQ ID NO. ID NO .: 2; and ii) the extracellular portion of IL-12Re1 for use as a medicament.

The invention further relates to a composition comprising a complex comprising:

i) a polypeptide comprising the amino acids 1-328 of SEQ ID NO. ID NO .: 2; and ii) the extracellular portion of IL-12Re1 for use as a medicament.

The invention also relates to a composition comprising an antisense nucleic acid for a polynucleotide which encodes a polypeptide comprising the sequence of SEQ ID NOs. ID NO: 2, for use as a medicament.

Preferably, a composition of the invention may be used in combination with an IL-12 antagonist; IL-18; TNF; or IFNy.

The invention relates to the use of a composition according to the invention as defined above, for the manufacture of a medicament for the treatment of a patient with a chronic Th1 mediated disease; with multiple sclerosis; with rheumatoid arthritis; with osteoarthritis; with inflammatory bowel disease; with diabetes; with psoriasis; with sepsis; or with an allogeneic transplant.

The invention further provides a composition comprising an agonist antibody, or antigen binding fragment thereof, which binds to a complex comprising:

i) a polypeptide comprising the amino acids 1-328 of SEQ. ID NO .: 2; and ii) the extracellular portion of IL-12Re1 for use as a medicament.

Preferably a composition of the invention is in combination with: IL-12; IL-18; TNF; or IFNy.

The invention also relates to the use of a composition of the invention as described above in the manufacture of a medicament for the treatment of a patient with a chronic Th2 response; with cancer; with virus infection; with fungal infection; or with an allergic reaction.

The invention also relates to the use of a composition according to the invention as defined above, in the manufacture of a medicament for the treatment of a patient receiving a vaccine.

Detailed Description of the Invention

Synopsis

I. General issues

II. Activities

III. Nucleic acids

A. coding fragments, sequences, probes

B. mutations, chimeras, fusions

C. preparation of nucleic acids

D. vectors, cells containing them

IV. Proteins, peptides

A. fragments, sequences, immunogens, antigens

B. muteins

C. agonists / antagonists, functional equivalents

D. preparation of proteins

V. Production of nucleic acids and proteins

A. synthetic

B. recombinant

C. from natural sources

VI. Antibodies

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A. polyclonal

B. monoclonal

C. fragments; Kd

D. anti-idiotypic antibodies

E. hybridoma cell lines

VII. Kits, diagnostics and quantification

A. ELISA

B. determination of the mRNA coding

C. qualitative / quantitative

D. sets

VIII. Therapeutic compositions, methods

A. combined compositions

B. dosage unit

C. feeding

IX. Screening tests

I. General issues

The present invention provides the amino acid and DNA sequences of mammalian, including primate, cytokine receptor-like subunit molecules, termed a DNAX cytokine receptor subunit, having specific specific properties, both structural and biological. Various cDNAs encoding these molecules have been obtained from primate, e.g. human, cDNA sequence libraries. Equivalents from other primates or other mammals would also be desirable.

Here, it is shown the alignment of p40 / IL-330 ligand with the DCRS5 and IL-12Rei receptor subunits, which provides information on the use of agonists and antagonists based on the reagents to which they are targeted.

Some of the standard methods that may find use herein are described or cited, for example, in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Cold Spring Harbor Press; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed.), Mp 1-3, CSH Press, NY; Ausubel et al., Biology, Greene Publishing Associates, Brooklyn, NY; or Ausubel et al. (1987 with periodic additions) Current Protocols in Molecular Biology, Greene / Wiley, New York.

The nucleotide sequence (SEQ ID NO: 1) and the corresponding amino acid sequence (SEQ ID NO: 2) of the primate DCRS5 coding portion, e.g. of a human, is shown in Table 1. The predicted signal sequence is highlighted but may depend on depending on the type of cell, or it may be several amino acid residues longer or shorter. Potential N-glycosylation sites are asparagine residues at positions 6, 24, 58, 118, 157, 209 and 250. The disulfide bridges possibly can be located between the cysteine ​​residues at positions 29 and 78; the conserved C_CXW pattern is found in positions 110/121/123. Note the rest of the tryptophan at positions 219 and the WxxWS motif at positions 281-285. The segment comprising positions about 1-101 is an Ig domain; from about residues 102-195 there is cytokine binding domain 1; from about residues 196-297 there is cytokine binding domain 2; there is a link between about 298-330; between about 329-354 there is a transmembrane portion; and there is an intracellular domain between about 356-606. The intracellular portion contains a putative SH2 binding site at positions Y374-I377, Y461-Q464, and Y588-Q591; and potentially important tyrosine residues at positions 406, 427, 440, and 453. These sites and ranges deserve special attention.

The ORF contains a putative signal sequence which is predicted to be digested at ... CHG / GIT ... as shown above. Behind the predicted extracellular domain of 328 amino acids is a putative transmembrane segment followed by a cytoplasmic domain of approximately 252 amino acids. The extracellular domain is predicted to perform ligand binding functions. The nucleic acid sequence obtained after the reverse translation is shown in Table 2.

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Table a. 1: Nucleotide and polypeptide sequence of the cytokine DNAK receptor subunit (DCRS5). Primate, e.g., human sequences (see SEQ ID NOs: 1 and 2). The predicted signal sequence is indicated, but its length may vary by several positions and is dependent on the type of cell. The identified variable positions are at nucleotide positions 127 and 563; which occur paired with G and G (translated into combinations of Q and G) or T and A (translated into H and R).

gtggtacggg aattccattg tgttgggcag ccaacaaggg tggcagcctg gctctgaagt 60 ggaattatgt gcttcaaaca ggttgaaaga gggaaacagt cttttcctgc ttccagac 118

<td>atg</td><td>aat</td><td>yes</td><td>gtc</td><td>act</td><td>att</td><td>all</td><td>tgg</td><td>gen</td><td>gca</td><td>gta</td><td>ata</td><td>gcc</td><td>ctt</td><td>trays</td><td>ata</td><td> 166</td>

<td>Underworld</td><td>Asn</td><td>Xaa</td><td>Val -twenty</td><td>Thr</td><td>How much</td><td>Gin</td><td>Trp</td><td>Asp “15</td><td>Ala</td><td>Val</td><td>How much</td><td>Ala</td><td>Leu -10</td><td>Tyr</td><td>How much</td><td></td>

<td>ctc</td><td>ttc</td><td>agc</td><td>tgg</td><td>tgt</td><td>cat</td><td>gga</td><td>gga</td><td>att</td><td>aca</td><td>aat</td><td>ata</td><td>aac</td><td>tgc</td><td>tct</td><td>ggc</td><td> 214</td>

<td>Leu</td><td>Phe</td><td>Cheese -5</td><td>Trp</td><td>Cys</td><td>His</td><td>Gly -1</td><td>Gly 1</td><td>How much</td><td>Thr</td><td>Asn</td><td>How much 5</td><td>Asn</td><td>Cys</td><td>Cheese</td><td>Giy</td><td></td>

<td>cac</td><td>atc</td><td>tgg</td><td>Sta</td><td>gaa</td><td>cca</td><td>gcc</td><td>aca</td><td>att</td><td>ttt</td><td>aag</td><td>atg</td><td>ggt</td><td>atg</td><td>aat</td><td>atc</td><td> 2 62</td>

<td>His 10</td><td>How much</td><td>Trp</td><td>Val</td><td>Glu</td><td>Pro 15</td><td>Ala</td><td>Thr</td><td>How much</td><td>Phe</td><td>Lys twenty</td><td>Underworld</td><td>Gly</td><td>Underworld</td><td>Asn</td><td>How much 25</td><td></td>

<td>tct</td><td>ata</td><td>tat</td><td>tgc</td><td>all</td><td>gca</td><td>gca</td><td>att</td><td>aag</td><td>aac</td><td>tgc</td><td>all</td><td>cca</td><td>agg</td><td>aaa</td><td>ctt</td><td> 310</td>

<td>Cheese</td><td>How much</td><td>Tyr</td><td>cys</td><td>Gin thirty</td><td>Ala</td><td>Ala</td><td>How much</td><td>Lys</td><td>Asn 35</td><td>Cys</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Lys 40</td><td>Leu</td><td></td>

<td>cat</td><td>ttt</td><td>tat</td><td>aaa</td><td>aat</td><td>ggc</td><td>atc</td><td>aaa</td><td>gaa</td><td>aga</td><td>ttt</td><td>all</td><td>atc</td><td>aca</td><td>agg</td><td>att</td><td> 358</td>

<td>His</td><td>Phe</td><td>Tyr</td><td>Lys 45</td><td>Asn</td><td>Gly</td><td>How much</td><td>Lys</td><td>Glu 50</td><td>Arg</td><td>Phe</td><td>Gin </td><td>How much</td><td>Thr 55</td><td>Arg</td><td>How much</td><td></td>

<td>aat</td><td>aaa</td><td>aca</td><td>aca</td><td>gct</td><td>cgg</td><td>ctt</td><td>tgg</td><td>tat</td><td>aaa</td><td>aac</td><td>ttt</td><td>ctg</td><td>gaa</td><td>cca</td><td>cat</td><td> 406</td>

<td>Asn</td><td>Lys</td><td>Thr 60</td><td>Thr</td><td>Ala</td><td>Arg</td><td>Leu</td><td>Trp 65</td><td>Tyr</td><td>Lys</td><td>Asn</td><td>Phe</td><td>Leu 70</td><td>Glu</td><td>Pro</td><td>His</td><td></td>

<td>gct</td><td>tct</td><td>atg</td><td>trays</td><td>tgc</td><td>act</td><td>gct</td><td>gaa</td><td>tgt</td><td>ccc</td><td>aaa</td><td>cat</td><td>ttt</td><td>all</td><td>gag</td><td>aca</td><td> 454</td>

<td>Ala</td><td>Cheese 75</td><td>Underworld</td><td>Tyr</td><td>Cys</td><td>Thr</td><td>Ala 80</td><td>Glu</td><td>Cys</td><td>Pro</td><td>Lys</td><td>His 85</td><td>Phe</td><td>Gin</td><td>Glu</td><td>Thr</td><td></td>

<td>ctg</td><td>ata</td><td>tgt</td><td>gga</td><td>aaa</td><td>jacket</td><td>att</td><td>tct</td><td>tct</td><td>gga</td><td>tat</td><td>ccg</td><td>cca</td><td>gen</td><td>att</td><td>cct</td><td> 502</td>

<td>Leu SO</td><td>How much</td><td>Cys</td><td>Gly</td><td>Lys</td><td>Asp 95</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Gly</td><td>Tyr 100</td><td>Pro</td><td>Pro</td><td>Asp</td><td>How much</td><td>Pro 105</td><td></td>

<td>gen</td><td>gaa</td><td>gta</td><td>acc</td><td>tgt</td><td>gtc</td><td>att</td><td>tat</td><td>gaa</td><td>tat</td><td>tca</td><td>ggc</td><td>aac</td><td>atg</td><td>act</td><td>tgc</td><td> 550</td>

<td>Asp</td><td>Glu</td><td>Val</td><td>Thr</td><td>Cys 110</td><td>Val</td><td>How much</td><td>Tyr</td><td>Glu</td><td>Tyr 115</td><td>Cheese</td><td>Gly</td><td>Asn</td><td>Underworld</td><td>Thr 12 0</td><td>Cys</td><td></td>

<td>acc</td><td>tgg</td><td>aat</td><td>gct</td><td>rgg</td><td>aag</td><td>ctc</td><td>acc</td><td>trays</td><td>ata</td><td>jacket</td><td>aca</td><td>aaa</td><td>trays</td><td>gtg</td><td>gta</td><td> 598</td>

<td>Thr</td><td>Trp</td><td>Asn</td><td>Ala 125</td><td>Xaa</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Tyr 130</td><td>How much</td><td>Asp</td><td>Thr</td><td>Lys</td><td>Tyr 135</td><td>Val</td><td>Val</td><td></td>

<td>cat</td><td>gtg</td><td>aag</td><td>agt</td><td>tta</td><td>gag</td><td>aca</td><td>gaa</td><td>gaa</td><td>gag</td><td>all</td><td>cag</td><td>tat</td><td>ctc</td><td>acc</td><td>tca</td><td> 646</td>

<td>His</td><td>Val</td><td>Lys 14 0</td><td>Cheese</td><td>Leu</td><td>Glu</td><td>Thr</td><td>Glu 145</td><td>Glu</td><td>Glu</td><td>Gin</td><td>Gin</td><td>Tyr 150</td><td>Leu</td><td>Thr</td><td>Cheese</td><td></td>

PL 209 128 B1

<td>age</td><td>tat</td><td>att</td><td>aac</td><td>atc</td><td>tcc</td><td>act</td><td>gen</td><td>tea</td>

<td>Cheese</td><td>Tyr 155</td><td>How much</td><td>Asn</td><td>How much</td><td>Cheese</td><td>Thr 160</td><td>Asp</td><td>Cheese</td>

<td>ttg</td><td>gtt</td><td>tgg</td><td>gtc</td><td>all</td><td>gca</td><td>gca</td><td>aac</td><td>gca</td>

<td>Leu 170</td><td>Val</td><td>Trp</td><td>Val</td><td>Gin</td><td>Ala 175</td><td>Ala</td><td>Asn</td><td>Ala</td>

<td>all</td><td>ctg</td><td>all</td><td>att</td><td>cac</td><td>ctg</td><td>gen</td><td>gen</td><td>ata</td>

<td>Gin</td><td>Leu</td><td>Gin</td><td>How much</td><td>His 190</td><td>Leu</td><td>Asp</td><td>Asp</td><td>How much</td>

<td>att</td><td>tcc</td><td>agg</td><td>get</td><td>gag</td><td>act</td><td>ata</td><td>aat</td><td>get</td>

<td>How much</td><td>Cheese</td><td>Arg</td><td>Ala 205</td><td>Glu</td><td>Thr</td><td>How much</td><td>Asn</td><td>Ala 210</td>

<td>tat</td><td>tgg</td><td>gen</td><td>agt</td><td>all</td><td>aca</td><td>aca</td><td>att</td><td>gaa</td>

<td>Tyr</td><td>Trp</td><td>Asp 220</td><td>Cheese</td><td>Gin</td><td>Thr</td><td>Thr</td><td>How much 225</td><td>Glu</td>

<td>trays</td><td>aag</td><td>get</td><td>aca</td><td>aca</td><td>aac</td><td>all</td><td>act</td><td>tgg</td>

<td>Tyr</td><td>Lys 235</td><td>Ala</td><td>Thr</td><td>Thr</td><td>Asn</td><td>Gin 240</td><td>Thr</td><td>Trp</td>

<td>aat</td><td>ttt</td><td>aca</td><td>tat</td><td>gtg</td><td>all</td><td>cag</td><td>tea</td><td>gaa</td>

<td>Asn 250</td><td>Phe</td><td>Thr</td><td>Tyr</td><td>Val</td><td>Gin 255</td><td>Gin</td><td>Cheese</td><td>Glu</td>

<td>aag</td><td>trays</td><td>gta</td><td>ttt</td><td>all</td><td>gtg</td><td>aga</td><td>tgt</td><td>all</td>

<td>Lys</td><td>Tyr</td><td>Val</td><td>Phe</td><td>Gin 270</td><td>Val</td><td>Arg</td><td>Cys</td><td>Gin</td>

<td>cag</td><td>cct</td><td>tgg</td><td>agt</td><td>tea</td><td>ccg</td><td>ttt</td><td>ttt</td><td>cat</td>

<td>Gin</td><td>Pro</td><td>Trp</td><td>Cheese 2S5</td><td>Cheese</td><td>Pro</td><td>Phe</td><td>Phe</td><td>His 290</td>

<td>cag</td><td>gtc</td><td>aca</td><td>tea</td><td>aaa</td><td>gca</td><td>ttc</td><td>all</td><td>cat</td>

<td>Gin</td><td>Val</td><td>Thr 300</td><td>Cheese</td><td>Lys</td><td>Ala</td><td>Phe</td><td>Gin 305</td><td>His</td>

<td>aca</td><td>gtt</td><td>get</td><td>tcc</td><td>atc</td><td>tet</td><td>aca</td><td>hhg</td><td>cac</td>

<td>Thr</td><td>Val 315</td><td>Ala</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Thr 320</td><td>Gly</td><td>His</td>

<td>jacket</td><td>att</td><td>gga</td><td>ctt</td><td>tta</td><td>ttg</td><td>gga</td><td>atg</td><td>atc</td>

<td>Asp 330</td><td>How much</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Leu 335</td><td>Gly</td><td>Underworld</td><td>How much</td>

<td>att</td><td>ctt</td><td>tet</td><td>ttg</td><td>att</td><td>hhg</td><td>ata</td><td>ttt</td><td>aac</td>

<td>How much</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>How much 350</td><td>Gly</td><td>How much</td><td>Phe</td><td>Asn</td>

<td>aaa</td><td>aga</td><td>agg</td><td>atc</td><td>tta</td><td>ttg</td><td>tta</td><td>ata</td><td>cca</td>

<td>Lys</td><td>Arg</td><td>Arg</td><td>How much 365</td><td>Leu</td><td>Leu</td><td>Leu</td><td>How much</td><td>Pro 370</td>

tta caa ggt ggc aag aag tac 694 Leu Gin Gly Gly Lys Lys Tyr

165 eta ggc atg gaa gag tea aaa 742 Leu Gly Met Glu Glu Ser Lys

180 185 gtg ata cet tet gca gee gtc 790 Val Ile Pro Ser Ala Ala Val 195 200 aca gtg ccc aag acc ata att 838 Thr Val Pro Lys Thr Ile Ile

215 aag gtt tcc tgt gaa atg aga 886 Lys Val Ser Cys Glu Met Arg

230 aat gtt aaa gaa ttt gac acc 934 Asn Val Lys Glu Phe Asp Thr

245 ttc tac ttg gag cca aac att 982 Phe Tyr Leu Glu Pro Asn Ile

260 265 gaa aca ggc aaa agg tac tgg 1030 Glu Thr Gly Lys Arg Tyr Trp 275 280 aaa aca cct gaa aca gtt ccc 1078 Lys Thr Pro Glu Thr Val Pro

295 gac aca tgg aat tet ggg eta 1126 Asp Thr Trp Asn Ser Gly Leu

310 ctt act tet gac aac aga gga 1174 Leu Thr Ser Asp Asn Arg Gly

325 gtc ttt get gtt atg ttg tea 1222 Val Phe Ala Val Met Leu Ser

340 345 aga tea ttc ega act ggg att 1270 Arg Ser Phe Arg Thr Gly Ile 355 360 aag tgg ctt tat gaa gat att 1318 Lys Trp Leu Tyr Glu Asp Ile

375

PL 209 128 B1

<td rowspan="2">cct Pro</td><td rowspan="2">aat Asn</td><td colspan="2">atg aaa</td><td colspan="2">aac agc aat</td><td rowspan="2">gtt gtg Val Val 385</td><td rowspan="2">aaa Lys</td><td rowspan="2">atg Underworld</td><td rowspan="2">eta Leu</td><td rowspan="2">cag gaa Gin Glu 390</td><td rowspan="2">aat Asn</td><td rowspan="2">agt Cheese</td><td rowspan="2"> 1366</td>

<td>Underworld 380</td><td>Lys</td><td>Asn</td><td>Asn cheese</td>

<td>gaa</td><td>ctt</td><td>atg</td><td>aat</td><td>aat</td><td>aat tcc</td><td>agt gag</td><td>cag</td><td>gtc</td><td>eta</td><td>tat gtt</td><td>gen</td><td>ccc</td><td> 1414</td>

<td>Glu</td><td>Leu 395</td><td>Underworld</td><td>Asn</td><td>Asn</td><td>Asn Ser 400</td><td>Glu cheese</td><td>Gin</td><td>Val</td><td>Leu 405</td><td>Tire Val</td><td>Asp</td><td>Pro</td><td></td>

<td>atg</td><td>att</td><td>aca</td><td>gag</td><td>ata</td><td>aaa gaa</td><td>atc ttc</td><td>atc</td><td>cca</td><td>gaa</td><td>cac aag</td><td>cct</td><td>aca</td><td> 1462</td>

<td>Underworld 410</td><td>How much</td><td>Thr</td><td>Glu</td><td>How much</td><td>Lys Glu 415</td><td>How Much Phe</td><td>How much</td><td>Pro 420</td><td>Glu</td><td>His Lys</td><td>Pro</td><td>Thr 425</td><td></td>

<td>jacket</td><td>trays</td><td>aag</td><td>aag</td><td>gag</td><td>aat aca</td><td>gga ccc</td><td>ctg</td><td>gag</td><td>aca</td><td>aga gac</td><td>trays</td><td>ccg</td><td> 1510</td>

<td>Asp</td><td>Tyr</td><td>Lys</td><td>Lys</td><td>Glu 430</td><td>Asn Thr</td><td>Gly Pro</td><td>Leu 435</td><td>Glu</td><td>Thr</td><td>Arg Asp</td><td>Tyr 440</td><td>Pro</td><td></td>

<td>all</td><td>aac</td><td>tcg</td><td>eta</td><td>ttc</td><td>gac aat</td><td>act aca</td><td>gtt</td><td>gta</td><td>tat</td><td>att cct</td><td>gen</td><td>ctc</td><td> 1558</td>

<td>Gin</td><td>Asn</td><td>Cheese</td><td>Leu 445</td><td>Phe</td><td>Asp Asn</td><td>Thr Thr 450</td><td>Val</td><td>Val</td><td>Tyr</td><td>How much Pro 455</td><td>Asp</td><td>Leu</td><td></td>

<td>aac</td><td>act</td><td>gga</td><td>tat</td><td>aaa</td><td>ccc caa</td><td>att tca</td><td>aat</td><td>ttt</td><td>ctg</td><td>cct gag</td><td>gga</td><td>agc</td><td> 1606</td>

<td>Asn</td><td>Thr</td><td>Gly 460</td><td>Tyr</td><td>Lys</td><td>Pro Gin</td><td>How Much Cheese 465</td><td>Asn</td><td>Phe</td><td>Leu</td><td>Pro Glu 470</td><td>Gly</td><td>Cheese</td><td></td>

<td>cat</td><td>ctc</td><td>agc</td><td>aat</td><td>aat</td><td>aat gaa</td><td>att act</td><td>tcc</td><td>tta</td><td>aca</td><td>ctt aaa</td><td>cca</td><td>cca</td><td> 1654</td>

<td>His</td><td>Leu 475</td><td>Cheese</td><td>Asn</td><td>Asn</td><td>Asn Olu 480</td><td>How Much Thr</td><td>Cheese</td><td>Leu</td><td>Thr 485</td><td>Leu Lys</td><td>Pro</td><td>Pro</td><td></td>

<td>gtt</td><td>gen</td><td>tcc</td><td>tta</td><td>jacket</td><td>tca gga</td><td>aat aat</td><td>ccc</td><td>agg</td><td>tta</td><td>all aag</td><td>cat</td><td>cct</td><td> 1702</td>

<td>Val 490</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>Asp</td><td>Gly Cheese 495</td><td>Asn Asn</td><td>Pro</td><td>Arg 500</td><td>Leu</td><td>Gin Lys</td><td>His</td><td>Pro 505</td><td></td>

<td>aat</td><td>ttt</td><td>gct</td><td>ttt</td><td>tet</td><td>gtt tca</td><td>agt gtg</td><td>aat</td><td>tca</td><td>eta</td><td>agc aac</td><td>aca</td><td>ata</td><td> 1750</td>

<td>Asn</td><td>Phe</td><td>Ala</td><td>Phe</td><td>Cheese 510</td><td>Val Ser</td><td>Val cheese</td><td>Asn 515</td><td>Cheese</td><td>Leu</td><td>Asn cheese</td><td>Thr 520</td><td>How much</td><td></td>

<td>ttt</td><td>ctt</td><td>gga</td><td>gaa</td><td>tta</td><td>agc ctc</td><td>ata tta</td><td>aat</td><td>all</td><td>gga</td><td>gaa tgc</td><td>agt</td><td>tet</td><td> 1798</td>

<td>Phe</td><td>Leu</td><td>Gly</td><td>Glu 525</td><td>Leu</td><td>Leu cheese</td><td>How much Leu 530</td><td>Asn</td><td>Gin</td><td>Gly</td><td>Glu Cys 535</td><td>Cheese</td><td>Cheese</td><td></td>

<td>cct</td><td>jacket</td><td>ata</td><td>all</td><td>aac</td><td>tca gta</td><td>gag gag</td><td>gaa</td><td>acc</td><td>acc</td><td>atg ctt</td><td>ttg</td><td>gaa</td><td> 1846</td>

<td>Pro</td><td>Asp</td><td>How much 540</td><td>Gin</td><td>Asn</td><td>Val cheese</td><td>Glu Glu 545</td><td>Glu</td><td>Thr</td><td>Thr</td><td>Met Leu 550</td><td>Leu</td><td>Glu</td><td></td>

<td>aat</td><td>gen</td><td>tca</td><td>ccc</td><td>agt</td><td>gaa act</td><td>att cca</td><td>gaa</td><td>cag</td><td>acc</td><td>ctg ctt</td><td>cct</td><td>gen</td><td> 1894</td>

<td>Asn</td><td>Asp 555</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Glu Thr 560</td><td>How much Pro</td><td>Glu</td><td>Gin</td><td>Thr 565</td><td>Leu Leu</td><td>Pro</td><td>Asp</td><td></td>

<td>gaa</td><td>ttt</td><td>gtc</td><td>tcc</td><td>tgt</td><td>ttg hgg</td><td>atc gtg</td><td>aat</td><td>gag</td><td>gag</td><td>ttg cca</td><td>tet</td><td>att</td><td> 1942</td>

<td>Glu 570</td><td>Phe</td><td>Val</td><td>Cheese</td><td>Cys</td><td>Leu Gly 575</td><td>How many Val</td><td>Asn</td><td>Glu 580</td><td>Glu</td><td>Leu Pro</td><td>Cheese</td><td>How much 585</td><td></td>

<td>aat</td><td>act</td><td>tat</td><td>ttt</td><td>cca</td><td>all aat</td><td>att ttg</td><td>gaa</td><td>agc</td><td>cac</td><td>ttc aat</td><td>agg</td><td>att</td><td> 1990</td>

<td>Asn</td><td>Thr</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Gin Asn</td><td>How much Leu</td><td>Glu</td><td>Cheese</td><td>His</td><td>Phe Asn</td><td>Arg</td><td>How much</td><td></td>

590 595 600

PL 209 128 B1 tca etc ttg gaa aag tagagctgtg tggtcaaaat caatatgaga aagctgcctt 2045 Cheese Leu Leu Olu Lys

605

<td>gcaatctgaa</td><td>cttgggtttt</td><td>ccctgcaata</td><td>gaaattgaat</td><td>tctgcctctt</td><td>tttgaaaaaa</td><td> 2105</td>

<td>atgtattcac</td><td>atacaaatct</td><td>tcacatggac</td><td>acatgttttc</td><td>atttcccttg</td><td>gataaatacc</td><td> 2165</td>

<td>taggtagggg</td><td>attgctgggc</td><td>catatgataa</td><td>gcatatgttt</td><td>cagttctacc</td><td>aatcttgttt</td><td> 2225</td>

<td>ccagagtagt</td><td>gacatttctg</td><td>tgctcctacc</td><td>atcaccatgt</td><td>aagaattccc</td><td>gggagctcca</td><td> 2285</td>

<td>tgccttttta</td><td>attttagcca</td><td>ttcttctgcc</td><td>tmatttctta</td><td>aaattagaga</td><td>attaaggtcc</td><td> 2345</td>

<td>cgaaggtgga</td><td>acatgcttca</td><td>tggtcacaca</td><td>tacaggcaca</td><td>aaaacagcat</td><td>tatgtggacg</td><td> 2405</td>

<td>cctcatgtat</td><td>tttttataga</td><td>gtcaactatt</td><td>tcctctttat</td><td>tttccctcat</td><td>tgaaagatgc</td><td> 2465</td>

<td>aaaacagctc</td><td>tctattgtgt</td><td>acagaaaggg</td><td>taaataatgc</td><td>aaaatacctg</td><td>gtagtaaaat</td><td> 2525</td>

<td>aaatgctgaa</td><td>aattttcctt</td><td>taaaatagaa</td><td>tcattaggcc</td><td>aggcgtggtg</td><td>gctcatgctt</td><td> 2585</td>

<td>gtaatcccag</td><td>cactttggta</td><td>ggctgaggtr</td><td>ggtggatcac</td><td>ctgaggtcag</td><td>gagttcgagt</td><td> 2645</td>

<td>ccagcctggc</td><td>caatatgctg</td><td>aaaccctgtc</td><td>tctactaaaa</td><td>ttacaaaaat</td><td>tagccggcca</td><td> 2705</td>

<td>tggtggcagg</td><td>tgcttgtaat</td><td>cccagctact</td><td>tgggaggctg</td><td>aggcaggaga</td><td>atcacttgaa</td><td> 2765</td>

<td>ccaggaaggc</td><td>agaggttgca</td><td>ctgagctgag</td><td>attgtgccac</td><td>tgcactccag</td><td>cctgggcaac</td><td> 2825</td>

<td>aagagcaaaa</td><td>ctctgtctgg</td><td>aaaaaaaaaa</td><td>aaaa</td><td></td><td></td><td> 2859</td>

MN (O / H) VTIOWDAVIALYXI.FSWCHGGXTNINCSGHIWVEPATIFKMGMNISI YCOAAXKNCQPRKLHF YKNGIKERFQITRINKTTARLWYKNPLEPHASMYCTAECPKHFQETbICGKDISSGYPPDIPDHVTCViy EYSGNMTCTWNA (G / R) KLTYIDTKYWHVKSLETEEEQQYLTSSYINISTDSLQGGKKYIiVWVQAANAL GMEBSKQLQIHLDDI VIPSAAVISRAETINATVPKTIIYWDSGTTIEKVSCEMRYKATTNQTWNVKEFDT NFTYVQQSEFYLEPNXKYVFQVRCQETGKRYWQPWSSPFFHKTPBTVPQVTSKAFQHDTWNSGLTVASIS TGHLTSDNRGDIGLL1, GMIVFAVMLSILSLIGIFNRSFRTGIKRRILLLIPKWLYEPXPNMKNSNWKML QENSELMNNNSSEQVL ¥ VDPMITEIKEIFIPBHKPTDYKKENTGPLETRDYPQNSLFDNTTWYIPDLNT GrKPOISNFLPBGSHLSNNNEITSLTLKPPVDSLDSGNNPRLQKHPNFAFSVSSVNSLSNTIFLGBLSLI LNQGECSSPDIQNSVEEETTMLLENDSPSETIPEQTLLPDEFVSCLGIVNBBLPSINTYFP0! LXXJBSHFN RISLLEK

PL 209 128 B1

Table 2: DCRS5 reverse translation from primate e.g. human (SEQ ID NO: 3):

ATGAAYCAYGTNACNATHCARTGGGAYGCnGTNATHGCNYTNTAYATEYTNTTYWSNTGGTGYCAYGGNGGNAT

HACNAAYATHAAYTGYWSNGGHCAYATHTGGGTNGARCCNGCKACaiATETTYAARATGGGHATGAAYATHWSNA

THTAYTGYCARGCNGCNATHAARAAYTGYCARCCEMGNAARYTKiCAYTTYTAYAARAAYGGNATHAARGARMGN

TTYCARATHACNMGNATHAAYAARACNACHGCNMGHYTNTGGTAYAARAAYTTYYTNGARCCNCAYGCNWSNAT

GTAYTGYACTGCTGARTGYCCNAARCAYTTYCARGARACNYTHATHTGYGGNAARGAYATHWSNWSNGGNTAYC

CNCCNGAYATHCCNGAYGARGTNACNTGYGTNATHTAYGARTAYWSNGGHTUiYATGACNTGYACHTGGAAYGCN

MGNAARYTNACNTAYATHGAYACNAARTAYGTNGTNCAYGTEAARWSNYTNGARACNGARGARGARCARTA

YYTNACNWSNWSKTAYATHAAYATHWSNACNGAYWSHYTNCARGGHGGHAARAARTAYYTIIGTNTGGGTNCARG

CNGCNAAYGCNYTNGGNATGGARGARWSNAARCARYTNCARATHCAYYTNGAYGAYATHGHiATHCCHWSNGCN

GCNGTNATHWSNMGNGCKrGARACNATHAAYGCKACNGTNGCNAARACilATHATETAYTGGGAYWSNCARACNAC

KATHGARAARGTKWSNTGYGARATGMGNTA.YAARGCNAOIACliIAAYCARACNTGGAAYGTłtAARGARTTYGAYA

CajAAYTTYACNTAYGTNCARCARWSNGARTTYTAYYTNGARCCNAAYATHAARTAYGTNTTYCARGTNMGKTGY

CARGARACNGGNAARMGKTAYTGGCARCCHTGGWSNWSNCCNTTYTTYCAYAARACliCCNGARACNGTNCCNCA

RGTNACNWSNAARGCNTTYCARCAYGAYACNTGGAAYWSNGGNYTNACNGTNGCNWSNATHWSWACNGGNCAYY

TNACNWSNGAYAAYMGNGGNGAYATHGGNYTNYTNYTNGGNATGATHGTNTTYGCNGTNATGYTNWSNATHYTH

WSNYTNATHGGNATHTTYAAYMGNWSNTTYMGNACNGGNATEAARMGNMGNATHYTNYTNYTRATHCCNAARTG

GYTOTAYGARGAYATHCCNAAYATGAARAAYWSNAAYGTOGTNAARATGYTNCARGARAAYWSHGARYTNATGA

AYAAYAAYWSłJWSNGARCARGTNYTNTAYGTNGAYCCNATGATHACNGARATHAARGARATHTTYATHCCIIGAR

CAYAARCCNACNGAYTAYAARAARGARAAYACłTGGIJCCNYTNGARACNMGNGAYTAYCCNCARAAYWSNYTNTT

YGAYAAYACNAGNGTNGTNTAYATHCCErGAYYTNAAYACNGGNTAYAARCCNCARATHWSNAAYTTYYTNCCNG arggnwsncayytktwsnaayaayaaygarathacnwsnythacnytnaarccnccngtwgaywsnythgaywsnccngtwgaywsnythgaywsn

GaNAAYAAYCCNMGNYTNCARAARCAYCCNAAYTTYGCIŚiTTYWSNGTNWSKWSNGTNAAYWSMYTNWSNAAYAC

EATHTTYYTNGGNGARYTNWSNYTNATHYTNAAYCARGGNGARTGYWSHWSNCCWGAYATHCARAAYWSNGTEG

ARGARGARACNACKATGYTNYTNGARAAYGAYWSSCCNWSNGARACNATHCCNGARCARACNYTNYTNCCHGAY

GARTTYGTHWSNTGYYTNGGNATHGTNAAYGARGARYTWCCNWSNATHAAYACNTAYTTYGCNCARAAYATHYT

NGARWSNCAYTTYAAYMGNATHWSNYTNYTNGARAAR

Table 3: Summary of the subunits of various cytokine receptors. Protein gpl30 human IL-6 receptor - SEQ. ID NO: 4 (GenBank M57230); beta2 subunit of the human IL-12 receptor - SEQ. ID NO: 5 (GenBank U64198).

luUlIi-12R 2 1 hugpl30 1 huDCRS5 1 huIL-12R 2 49 hugpl30 46 huDCRSS 50 huIL-12R 2 96 hugpl30 96 huDCRS5 94 huIL-12R 2 143 hugpi30 144 huDCRS5 144 huIL-12R 2 193 hugpl30 191

MAHTFROCSLAFMFIITWLLI KAKIDACKRGDVTVKPSHVII, LGSTVN MLTLQTWWQALFIFLTTESTGELLDPCG- - -YISPESPWQLHSNFT

MNHVTIQMDAVIAXxYILFSWCHGGITNINCS-GHIWVEPATIFKMGMlJIS *. * ....

ITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLG- - AVCVLKEKCMDYFHVNANYIVWKTHHFTIPKEQYTIINRTASSVTFTDIA IYCQAAIKN - CQP --- RKLIKHFYKLQNGINKHKTARQNGINKHKTARQN . . *. .

- - TTLFVCKLACXHSD - EIQX CGAEIFVGVAPEQPQNLSCIQKGEQGTVA susiiQLTcasriLTFGQi, -EQNVYGXTiiSGi.pPEKPKNLSCivii-EGKKMR EPHASMYCTAECŁPTYKDEGISSTCPTYKDEGISSTCPTYKDEGISSTCPTYKDEGISSTCPTYKDEGISSTCPKHFQETG *, ****. * .. *.

CTWERGRDTHLYTEYTLQLSGPKNI₁TWQKQCKDIYCDYIiDFGIliLTPESP CEWDGGRETHLETNFTLKS - - EWATHKFADCKAKRDTPTSCTTO YS - TVY

CTWŃARKLTYIDTKYWHVKSLETEEEQQYLTSSYINISTDSLQGG --- *** · * ....

ESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRC

FVNIEVWVBAENALGKVTSDHTNFDPVYKVKPNPPHNLSVINSEELSSXL

45

142

143 143

192

190

189

242

240

GB 209 128 B1 -KKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKT huDCRS5 190 238 * * * huIL-12R 2 243 TLYWRD EGLVLLNRLRYRPSNSRLWNM \ / N ντΚΑΚΠΡΠπτ.Τ.ητ, Κ KLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLK 285 hugpl30 241 290 239 huDCRSS IIYWDS - QTTIBKVSCEMRYKATTNQTWNVKEFD TNFTYVQQSEFYLE-285. *. . . *. *. HuIL-12R 2 28S PFTBYBFOXSSKLHLYKGSWSDWSESLRAOTPEEEPTnMT.nvwvi * nrenTD 335 hugpl30 291 PFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSKAPSKAPSKAPSFWYKIDPSH 340--------QPSSV * BTWQPPSH3 , "★ * ** * * * huIL-12R 2 336 YS-RQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWT 384 hugpl30 341 TQGYRTVQLVWKTLPPFEANGKILDYEVT LTRWKSHLQNYTVNATKL 387 huDCRSS --- 321 ----- ------ QVTSKAFQHDTWNSGLTVASISTG HLTSDN - RGDIGLL 357 huIL-12R 2 385 TVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGM 434 hugpl30 388 TVNLTNDRYLATLTVRNLVGKSDAAVLTIP-ACDFQATHPVMDLKAFPKD 436 huDCRSS 358 LGMIVFAVMLSILSLIGIFHRSFRTGIKRR -------------------- 387 huIL-12R 2 435 DNSAWIDEWTWQPpl-12R 2 435 DNSAWIDEWTWQPpl-12R 2 435 DNSAWIDEWTWQPpl-12R 2 435 DNPRILVTRA 437 NMLWVEWTTPRE SVKKYILEWCVLS --- --- 4 80 DKAPCITDWQQEDGTVHRT huDCRSS 388 ---------------- ILIiLIPKWLYEDIPNMKNSNWKMLQEN ---- SE 417 huIL-12R 2 484 LISENIKSYICYEIRVYALSGDQ-GGCSSILGNSKHXAPLSGPHINAITB 532 hugpl30 481 YLRGNIiAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKV 530 huDCRSS 418 LMNNNSSE -------- QVLYVDP ----- MITEIKEIFIPEHKPTDYKKE- 453. *. * * * * huIL-12R 2 533 EKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNS 582 hugpl30 531 GKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGN ---- ETATRSNVDSNTGNNVDSNTGNNVDSNS --- . * *. . . * huIL-12R 2 583 HPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKAN-WMAFVAPSI 631 hugpl30 577 YTLSSLTSDTLYMVRMAAYTDBG-GKDGPEFTFTTPKFAQGEIEAIWPV 625 -------------------- FLRSS 491 -------------- 495 huIL-12R 2 632 CIAIIMVGIFSTHYFQQKVFVIiLAALRP ----------- QWCSREIPDPA 670 hugpl30 626 CL ^ FLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPH----TPPRHN 6756 --- SHLSNNN-EITSLTLKP -------------- PVDSLDSG 519 huIL-12R 2 671 NSTCAKKYPIAEEKTQLPLDRLLID-WPTPHDPEPLVIS - EVLHQVTPV 717 hugpl30 676 FNSKDQMYSDGNFTDVSWEIEANDKKPFPEDLKSLDLFKKEKINTEGHS 725 huDCRSS 520 NNPRLQKHPN-FAFSVSSVNSLSNT ------------- --- 1 FLGELSLI 552 huIL-12R 2 718 767 FRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLY hugpl3 0 726 775 SGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTWHSGYRHQVPSVQ huDCRSS LNQGECS 553 --- --- S-PDIQNSVEEETTMLLENDSP -------------- 580. ★ ★ *

PL 209 128 B1

<td colspan="2">huIL-12R 2768</td>

<td>hu9pl30 huDCRS5</td><td> 776 581</td>

<td>huIL-12R</td><td> 2 815</td>

<td>hugpl30</td><td> 826</td>

<td>huDCRS5</td><td> 624</td>

<td>huIL-12R</td><td> 2 846</td>

<td>hugpl30</td><td> 876</td>

<td>huDCRS5</td><td> 630</td>

KVLESRGSDPKPENPACPWTVLPAGDI »PTHDGYLPSN --- IDDLPSHEAP

VFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESS --SETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQN ILESHFNR- LADSLEELEPQHISLS ----- VSSPSSSLHPLQVFSMISCF ----- VSSPSSSLHPLQTFSVISHF --QFPSSSLHPLQVFSKSFEV--QVSAKSFE

--ISLLEK * *

---------- DKLTLDQLKMRCDSLML 862

AFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 918

629

814

825

623

845

875

629

The extracellular domain "IL30R is most closely related to the IL-6 signaling protein gp130 and IL12Re2. It is slightly less related to the GCSF receptor, the leptin receptor, the leukemia inhibitor factor receptor and the CNTF receptor. Thus, “IL-30R belongs to class I within the cytokine receptor superfamily and is closely related to the 6R / IL-12R family.

Table 3 compares the available primate subunit sequences with primate, e.g. human, DCRS5 (IL-30R). DCRS5 shows similarity to the gp130 subunit of the IL-6 receptor (e.g., the IL-6R subunit) and to the IL-12e2 subunit. DCRS5 exhibits structural features of the beta subunit, but the proper course of protein interaction and signaling remains unknown.

As used herein, the name DCRS5 will be used to describe a protein comprising the amino acid sequence shown in Table 1. In many instances, a significant portion will be functionally or structurally equivalent to it, including, for example, additional extra-cellular portions. Also described herein is a protein variant of the corresponding DCRS5 allele, the sequence of which is provided, e.g., muteins and other constructs. Typically such variants will exhibit less than 10% sequence differences in the target region and thus often have 1- to 11-fold substitutions, e.g., 2-, 3-, 5-, 7-fold and others. Allelic and other variants, e.g., natural polymorphisms of the described protein, are described herein. Typically, this protein binds to its respective natural ligand, possibly as a dimer, to the alpha subunit of the receptor with high affinity, e.g. at least about 100 nM, usually better than about 30 nM, preferably better than 10 nM, more preferably better than 3 nM. This name will also be used in this application to describe naturally occurring forms, e.g., alleles, polymorphs, and metabolic variants of a mammalian protein. The desired forms of the receptor complexes will bind the appropriate ligand with a strength and specificity appropriate to the ligand-receptor interaction.

Embodiments of the invention also relate to combinations of proteins or peptides that exhibit significant amino acid sequence similarity to the amino acid sequence of Table 1. Also included herein are sequence variants with relatively few substitutions, e.g., preferably with less than about 3-5.

A substantial "fragment or" polypeptide segment is a stretch of amino acid residues of at least about 8 amino acids, generally at least 10 amino acids, more generally at least 12 amino acids, often at least 14 amino acids, more usually at least 16 amino acids, more typically at least 18 amino acids, more typically at least 20 amino acids. usually at least 22 amino acids, more than usually at least 24 amino acids, preferably at least 26 amino acids, more preferably at least 28 amino acids, and, in particularly desirable embodiments, at least about 30 or more amino acids. The sequence of segments of different proteins may be compared to each other over stretches of appropriate length. In many cases, fragments may exhibit the functional properties of whole subunits, e.g., the extracellular domain of a transmembrane receptor may retain

The ligand binding capacity i can be used to prepare a soluble complex similar to the receptor complex.

Amino acid sequence homology or sequence similarity is determined by the best alignment of the positions of the individual residues. In some comparisons, you can enter spaces as needed. See, e.g., Needleham et al., (1970) J. Mol. Biol. 48: 443-453; Sankoff et al. (1983) ch. 1 in: Time Warps, String Edits, and Macromolecules: The Theory and Practice of Sequence Comparison, Addison-Wesley, Reading, MA; and programs developed by IntelliGenetics, Mountain View, CA and the University of Wisconsin Genetics Computer Group (GCG), Madison, WI. This changes when considering conservative substitutions as matches. Conservative substitutions typically include substitutions in the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Homologous amino acid sequences should contain natural allelic and interspecies variants in the cytokine sequence. Typical homologous proteins or peptides will show from 50-100% homology (if spacing can be introduced into the sequence) to 60-100% homology (if conservative substitutions are considered) to a portion of the amino acid sequence in Table 1. The degree of homology will be at least about 70%, generally at least 76%, more generally at least 81%, often at least 85%, more usually at least 88%, typically at least 90%, more typically at least 92%, usually at least 94%, more usually at least at least 95%, preferably at least 96%, and more preferably at least 97%, and in particularly desirable embodiments, at least 98% or more. The degree of homology will depend on the length of the fragments being compared. Homologous proteins or peptides, such as allelic variants, will exhibit mostly the same biological activities as shown in Table 1, particularly in the intracellular portion.

The term "biological activity is used herein to describe, but is not limited to, the effects of cytokine-like ligands on signaling, inflammatory responses, innate immune responses, and / or morphogenetic development. For example, these receptors should mediate phosphatase or phosphorylase activity, which activities can be readily measured using standard methods. See, e.g., Hardie et al. (1995 ed.) The Protein Kinase FactBook vol. I and II, Academic Press, San Diego, CA; Hanks et al. (1991) Meth. Enzymol. 200: 38-62; Hunter et al. (1992) Cell 70: 375-388; Lewin (1990) Cell 61: 743-752; Pines et al. (1991) Cold Spring Harbor Symp. Quant. Biol. 56: 449-463; and Parker et al. (1993) Nature 363: 736-738. Receptors or parts thereof can be used as phosphate-joining enzymes to label specific or non-specific substrates. The subunits may also exhibit immunogenic properties and give rise to antibodies recognizing them, or they may be antigens capable of binding antibodies.

The terms ligand, agonist, antagonist, and analog, e.g., DCRS5, refer to the interaction of a ligand with a receptor, e.g., wherein the receptor is a natural receptor or an antibody. The cellular response is likely to be mediated by a tyrosine kinase receptor,

Also, the ligand is a molecule that either functions as a natural ligand to which the above-mentioned receptor or analog thereof binds, or a molecule that is a functional analog of the natural ligand. The functional analog may be a ligand containing structural modifications, or it may be a completely unrelated molecule whose shape allows it to interact with appropriate ligand binding determinants. Ligands can act as agonists or antagonists, see, e.g., Goodman et al. (eds 1990) Goodman &amp; Gilman's: The Pharmacological Bases of Therapeutics, Pergamon Press, New York.

Rational drug design may also be based on structural studies of the receptor or antibody and other electors or ligands. See, e.g., Herz et al. (1997) J. Recept. Signal Transduct. Res. 17: 671-776; and Chaiken et al. (1996) Trends Biotechnol. 14: 369-375. Effectors can be other proteins that perform different functions in response to ligand binding or other proteins that normally interact with the receptor. One method of determining which sites in a molecule interact with other specific proteins is through the study of the physical structure, e.g. X-ray crystallography or two-dimensional NMR techniques. They can provide clues as to which amino acid residues make up the molecular contact regions. For a detailed description of protein structure determination see, e.g., Blundell and Johnson (1976) Protein Crystallography,

Academic Press, New York, incorporated herein by reference.

II. Activities

Cytokine receptor-like proteins will exhibit a wide variety of biological activities, e.g., intracellular signaling, e.g., by STAT4, affecting cell proliferation or the metabolism of phosphate added or removed from specific substrates, typically proteins. These activities will generally alter inflammatory function, alter the innate immune response, or have a morphological effect. The subunit is likely to bind the ligand with low affinity.

DCRS5 has motifs specific to JAK signaling receptors. See, e.g., Ihle et al. (1997) Stem Cells 15 (Appendix 1): 105-111; Silvennoinen et al. (1997) APMIS 105: 497-509; Levy (1997) Cytokine Growth Factor Review 8: 81-90; Winston and Hunter (1996) Current Biol. 6: 668-671; Barrett (1996) Baillieres Clin. Gastroenterol. 10: 115; and Briscoe et al. (1996) Philos. Trance. R. Soc. Lond. B. Biol. Sci. 351: 167-171. The SH2 binding motifs described above are of particular interest.

The biological activities of the cytokine receptor subunits are associated with the addition or removal of phosphate moieties to substrates, usually in a specific, but sometimes non-specific manner. It will be possible to identify substrates as well as determine conditions of enzymatic activity by standard methods, e.g., as described in Hardie et al. (Ed. 1995) The Protein Kinase FactBook vol. II, Academic Press, San Diego, CA; Hanks et al. (1991) Meth. Enzymol. 200: 38-62; Hunter, et al. (1992) Cell 70: 375-388; Lewin (1990) Cell 61: 743-752; Pines, et al. (1991) Cold Spring Harbor Symp. Quant. Biol. 56: 449-463; and Parker, et al. (1993) Nature 363: 736-738.

Receptor subunits can associate and form functional complexes, eg, that can be used to bind ligand or generate antibodies. This will have an important diagnostic application, for example for detection or quantification. The functional association of the receptor with the p40 / IL-B30 ligand provides important information about the clinical indications for which this receptor will be used. Thus, antagonists and agonists will have certain functional effects.

III. Nucleic acids

The embodiments of the invention relate to the use of isolated nucleic acids or fragments thereof, e.g. encoding these or closely related proteins or fragments thereof, e.g. encoding a suitable polypeptide, preferably one that exhibits biological activity. It is possible to use isolated or recombinant DNA which codes for combinations of proteins or polypeptides with characteristic sequences, e.g. DCRS5 alone or in combination with others such as the IL-12Rei subunit (see Showe et al. (1996) Ann. NY Acad. Sci. 795: 413-425; Gately et al. (1998) Ann. Rev. Immunol. 16: 495-521; GenBank U03187, NM005535). Typically, a nucleic acid may hybridize under suitable conditions to a nucleic acid having the sequence shown in Table 1, but preferably not to the corresponding fragment of other receptors described in Table 3. The aforementioned biologically active protein or polypeptide may be a full-length protein or a fragment, and will typically contain a segment of an amino acid sequence that is highly homologous, e.g., having significant stretches of identity, to those shown in Table 1. Further, the present invention encompasses the use of isolated or recombinant nucleic acids or the same. fragments which code for proteins containing portions corresponding to DCRS5 proteins, e.g. intracellular fragments thereof. Isolated nucleic acids may contain appropriate regulatory sequences at their 5 'and 3' ends, e.g., promoters, transcription enhancers, polyadenylation signals, and others derived from a natural gene. Combinations as described are also provided, e.g., the fusion of DCRS5 with IL-12Re1 or extracellular ligand binding fragments thereof, acting as ligand antagonists. The diagnostic importance of, for example, polymorphic and other variants is also undisputed.

"An isolated nucleic acid is a nucleic acid, e.g. RNA, DNA or mixed polymer that is substantially pure, e.g. separated from other components that naturally accompany nucleic acids, such as ribosomes, polymerases, and species-specific flanking sequences." The term includes a nucleic acid that has been obtained from its natural environment, and also includes recombinant or cloned DNA, which is therefore indistinguishable from naturally occurring compositions, as well as chemically synthesized analogs or ana16

Biologically Synthesized Logs in Heterologous Systems. Essentially pure molecule means the isolated forms of the molecule, both completely and essentially pure.

An isolated nucleic acid is generally a homogeneous composition of molecules, but may in some embodiments exhibit heterogeneity, most preferably little. This diversity typically occurs at the ends of the polymer or in portions that are irrelevant to the desired biological function or activity.

"A recombinant nucleic acid is usually defined by the method of its production or its structure. With respect to a method of production, e.g., a product produced by a process, the process is the use of a recombinant nucleic acid technique, e.g. involving human intervention in a nucleotide sequence. Typically, this intervention involves in vitro manipulation, although under certain conditions it may employ more classical animal breeding techniques. Alternatively, it may be a nucleic acid obtained by producing a sequence comprising the joining of two fragments which do not naturally occur side by side with the aim of eliminating natural products, e.g. naturally occurring mutants discovered in the natural state. Thus, e.g. Included herein are products obtained by transforming cells with a non-natural vector, as well as nucleic acids including a sequence obtained using the synthetic oligonucleotide process. Such a process is often carried out to replace e.g. a codon with a codon of the same meaning encoding the same or a conservative amino acid, thereby introducing or removing a restriction enzyme recognition sequence or for structure-function analysis purposes. Alternatively, the process is performed to link together parts of the nucleic acids with the desired functions to obtain a single genetic entity comprising a desired combination of functions not present in commonly available natural forms, e.g., encoding a fusion protein. Restriction enzyme recognition sites are often the target of such artificial manipulation, but other specific target sites, e.g. promoters, DNA replication sites, regulatory sequences, control sequences, and other useful features can be incorporated by design. A similar approach is intended for a recombinant, e.g., fusion, polypeptide. Included herein is a dimer repeat or a DCRS5 fusion to the subunit

IL-12Rei. Specifically included herein are synthetic nucleic acids which, through the degeneracy of the genetic code, encode polypeptides corresponding to DCRS5 fragments and sequence fusions from various related molecules, e.g., others belonging to the family of cytokine receptors.

"A fragment with respect to a nucleic acid is a contiguous segment of at least about 17 nucleotides in length, generally at least 21 nucleotides in length, more generally at least 25 nucleotides in length, more commonly at least 30 nucleotides, more commonly at least 35 nucleotides, often at least 39 nucleotides in length, more typically at least 45 nucleotides in length, typically at least 50 nucleotides, more typically at least 55 nucleotides, usually at least 60 nucleotides, more often than usually at least 66 nucleotides, preferably at least 72 nucleotides, more preferably at least 79 nucleotides, and in particularly preferred embodiments at least 85 and more nucleotides, including 90, 100, 120, 140, 160, 180, 200, etc. Typically, fragments of different genetic sequences can be compared with each other over appropriate length segments, particularly defined segments such as the domains described below.

A nucleic acid that encodes DCRS5 will be particularly useful in identifying gene types, mRNA and cDNA that encode DCRS5 alone or closely related proteins, as well as DNA that encodes polymorphic, allelic and other genetic variants, e.g., from different individuals or related species. Preferred probes for such searches are receptor regions which are conserved between different polymorphic variants, or which contain nucleotides that lack specificity, and which are most preferably full or nearly full length. In other situations, specific polymorphic variant sequences will be more useful. Combinations of polymorphic DCRS5 variants with IL-12Re1 variants may also be recognized.

The present invention further includes recombinant nucleic acid molecules and fragments having a nucleic acid sequence identical to or highly homologous to the isolated DNA defined herein. In particular, the sequences will be operably linked to DNA segments that control DNA transcription, translation, and replication. These additional segments typically assist in expressing the desired nucleic acid segment.

Homologous or highly similar nucleic acid sequences, e.g., DCRS5, show significant similarity when compared with each other. Nucleic acid homology standards are either a measure of homology generally used in the art for sequence comparison.

PL 209 128 B1 or based on hybridization conditions. Comparative hybridization conditions are described in more detail below.

Substantial similarity in the context of comparing nucleic acid sequences means that either the segments or their complementary strands when compared are identical, when optimally aligned, with the corresponding nucleotide insertions or deletions in at least about 60% of the nucleotides, generally at least 66%, commonly at least 71%, often at least 76%, more usually at least 80%, usually at least 84%, more than usually at least 88%, typically at least 91%, more typically at least about 93%, preferably at least about 95%, more preferably at least about 96% to 98% or more, and in particular embodiments as many as about 99% or more nucleotides, including, e.g., segments encoding the structural domains or other segments described. Alternatively, substantial similarity will exist when segments will hybridize under selective hybridization conditions to the complementary strand or strand, typically using a sequence derived from Table 1. Typically, selective hybridization will occur when the homology over at least about 14 nucleotides is at least about 55%, more typically at least about 65%, preferably at least about 75%, and more preferably at least about 90%. See Kanehisa (1984) Nucl. Acids Res. 12: 203-213. The length of homologous comparisons as described may extend to longer stretches, and in some embodiments such a segment will be at least about 17 nucleotides, generally at least about 20 nucleotides, generally at least about 24 nucleotides, typically at least about 28 nucleotides, typically at least about 32 nucleotides, more typically at least about 32 nucleotides. about 40 nucleotides, preferably at least about 50 nucleotides, more preferably at least about 75 to 100 or more nucleotides, including those also e.g. 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 etc. and other lengths.

Stringent conditions with respect to homology in the context of hybridization will mean stringent conditions resulting from the combination of salt concentration, temperature, organic solvents, and other parameters typically controlled in hybridization reactions. The stringent temperature conditions will typically include temperatures in excess of about 30 °, typically in excess of about 37 °, typically in excess of about 45 °, more typically in excess of about 55 °, preferably in excess of about 65 °, and more preferably in excess of about 70 °. Stringent salt concentration conditions will generally be less than about 500 mM, more typically less than about 400 mM, more typically less than about 300 mM, typically less than about 200 mM, preferably less than about 100 mM, and more preferably less than about 88 mM, and even lower than about 50 or 20 mM. However, the combination of parameters is much more important than the value of any single parameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31: 349-370.

The isolated DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. As a result of these modifications, new DNA sequences that code for this protein or its derivatives are obtained. Modified sequences can be used to produce mutant proteins (muteins) or to enhance the expression of variant types. Enhanced expression can be associated with gene amplification, increased transcription, increased translation, and other mechanisms. Such mutant DCRS5 mimics DCRS5 as defined above, but has an amino acid sequence that differs from that of other naturally-encountered cytokine receptor-like proteins, either by introducing deletions, substitutions or insertions. In particular, a "DCRS5 mutant with a site specific mutation includes a protein having significant sequence similarity with the protein in Table 1 and typically showing most of the biological activities or effects of the forms included in this application. Various natural polymorphic sequence variants will also be identified.

Although the sites for a site specific mutation are predetermined, the mutants need not be site specific. Mammalian DCRS5 mutagenesis can be achieved by introducing amino acid insertions or deletions in a gene in conjunction with expression. Substitutions, deletions, insertions or multiple combinations can be made to arrive at the final construct. Insertions contain either amino- or carboxy-terminal fusions. Random mutagenesis can be performed at the target codon and the expressed mammalian DCRS5 mutants can then be analyzed for the desired activity, providing knowledge of the structure-activity relationship. Methods for obtaining substitutional mutations at specific sites in DNA having a known sequence are well known in the art, e.g., by mutagenesis with M13 primers. See also Sambrook et al. (1989) and Ausubel et al. (1987 with periodic additions). Particularly useful constructs will be the extracellular portions of DCRS5 associated with IL-12Rei segments.

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Mutations in DNA should not normally cause the coding sequences to fall out of reading frames, and preferably should not form complementary regions that could hybridize to produce mRNA secondary structures such as loops or pins.

By the phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22: 1859-1862, appropriate synthetic DNA fragments can be made. The double-stranded fragment will often be obtained, either by synthesizing the complementary strand and joining the strands together under suitable conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Polymerase chain reaction (PCR) techniques can often be used in mutagenesis. Alternatively, mutagenic primers are a commonly used method of generating specific mutations at selected sites. See, e.g., Innis et al. (1990 Ed.) PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA; and Dieffenbach and Dveksler (1995; eds.) PCR Primer: A Laboratory Manual Cold Spring Harbor Press, CSH, NY.

Certain embodiments of the invention pertain to a combination of components comprising the described receptor sequences. In other embodiments, functional parts of the sequence may be joined to encode fusion proteins. In other embodiments, variants of the described sequences may be substituted.

IV. Proteins, peptides

As described above, the present invention includes primate DCRS5, e.g., the sequences of which are provided in Table 1 and described above. Allelic and other variants are also contemplated including, e.g., fusion proteins, linking portions of such sequences to each other, including, e.g., IL-12Re1, epitope tags, and functional domains.

The present invention also relates to recombinant proteins, e.g., proteins that are heterologous fusions using primate or rodent protein segments. A heterologous fusion protein is a fusion of proteins or segments that are not normally joined in this way in nature. Thus, the DCRS5 fusion product with another cytokine receptor is a contiguous protein molecule having sequences linked by conventional peptide bonds, typically produced as a single translation product, and exhibiting properties, e.g. sequence or antigenicity, derived from each source peptide. A similar concept applies to heterologous nucleic acid sequences. Assemblies into complexes of various scheduled proteins are also provided.

Moreover, new constructs can be generated by fusing similar functional or structural domains from other related proteins, e.g. cytokine receptors or Toll receptors, including species variants. For example, ligand binding or other segments may be "interchanged between different novel fusion polypeptides or fragments." See, e.g., Cunningham et al. (1989) Science 243: 1330-1336; and O'Dowd et al. (1988) J. Biol. Chem. 263: 15985-15992. In this way, new chimeric polypeptides exhibiting new combinations of properties will be obtained from the functional combination of the properties of the binding receptors. For example, ligand binding domains from other related receptor molecules may be added to or replaced with other domains of that or related proteins. The resulting protein will often have hybrid functions and properties. For example, a fusion protein may contain a signaling domain that may serve to direct the fusion protein to a particular cell organelle.

Potential fusion participants and sequences can be selected from various sequence databases, e.g., GenBank, IntelliGenetics, Mountain View, CA; and BCG, University of Wisconsin Biotechnology Computing Group, Madison, WI. In particular, combinations of the polypeptide sequences described in Tables 1 and 3 are preferred. Variant forms of these proteins may be used in the described combinations.

Described herein are muteins that bind cytokine-like ligands and / or that are altered in signal transduction. Structural alignment of human DCRS5 with other members of the cytokine receptor family shows conserved features / residues. See Table 3. The alignment of human DCRS5 sequences with other members of the cytokine receptor family shows various structural and functional commonalities. See also Bazan et al. (1996) Nature 379: 591; Lodi et al. (1994) Science 263: 1762-1766; Sayle and Milner-White (1995) TIBS 20: 374-376; and Gronenberg et al. (1991) Protein Engineering 4: 263-269.

Substitutions with murine sequences or human sequences are particularly preferred. In contrast, conservative substitutions outside the ligand binding regions are likely to retain

Most signaling activities; and conservative substitutions outside of the intracellular domains are likely to retain most ligand binding properties.

"The primate DCRS5 derivatives contain amino acid sequence mutants, glycosylation variants, metabolic derivatives, and covalent or associated conjugates with other chemical moieties. Covalent derivatives can be prepared by attaching the substituents to groups which are on the amino acid side chains of DCRS5 or at the N-terminus thereof, e.g. by methods which are well known in the art. These derivatives can include, without limitation, aliphatic esters or carboxy terminal amides or residues containing carboxyl side chains, O-acyl derivatives of hydroxyl-containing residues, and N-acyl derivatives of the amino-terminal amino acid or amino-containing residues, e.g., lysine or arginine. Acyl groups are selected from the group of alkyl moieties, including straight C3 to C18 alkyls, thereby forming alkanoylaroyl moieties.

In particular, changes due to glycosylation are included herein, e.g., caused by modification of the glycosylation patterns of the polypeptide during its synthesis and processing, or during subsequent processing steps. Particularly preferred methods are to treat the peptide with glycosylation enzymes derived from cells that normally process such processing, e.g. mammalian glycosylation enzymes. Deglycosylating enzymes should also be considered. Also included are versions of the same primary amino acid sequence that have other minor modifications, for example phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphotreonine.

The main group of derivatives are covalent conjugates of the receptors or their fragments with other proteins or polypeptides. These derivatives, such as N-terminal fusions, can be produced in recombinant culture, or with the use of factors known in the art for their utility in cross-linking proteins through reactive side chains. Preferred derivatization sites under the influence of cross-linking agents are free amino groups, carboxyl residues, and cysteine ​​residues.

Fusion polypeptides between the receptors and other homologous or heterologous proteins are also provided. The homologous polypeptides can be fusions between different receptors, resulting in, for example, a hybrid protein having binding specificity for a wide variety of cytokine ligands, or a receptor with extended or decreased substrate specificity. Similarly, heterologous fusions can be constructed that will exhibit combinations of the properties or activities of the parent proteins. Typical examples are fusions of a reporter polypeptide, e.g., a luciferase, with a receptor segment or domain, e.g., a ligand-binding segment, which allows the presence and location of the desired ligand to be easily determined. See, e.g., Dull et al., U.S. Patent No. 4,859,609. Other “gene fusion partners include glutathione S-transferase (GST), bacterial β-galactosidase, trpE, α protein, β-lactamase, alpha amylase, alcohol dehydrogenase, and yeast sex factor alpha. See, e.g., Godowski et al. (1988) Science 241: 812-816. The tagged proteins will often be substituted with the protein combinations described. The association of DCRS5 with IL-12Re1 is particularly significant as described.

The phosphoramidite method described by Beaucage and Carruthers (1981) Tetra, Letts. 22: 1859-1862, provides suitable synthetic DNA fragments. The double-stranded fragment will often be obtained, either by synthesizing the complementary strand and joining the strands together under suitable conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Such polypeptides may also contain amino acid residues that have been chemically modified by phosphorylation, sulfonation, biotinylation, or by the addition or removal of other residues, particularly those that have a phosphate-like molecular form. In some embodiments, these modifications will be useful labeling reagents or serve as purification targets, e.g., affinity ligands.

Fusion proteins will typically be produced by recombinant nucleic acid methods, or by polypeptide synthesis. Techniques for the manipulation and expression of nucleic acids are generally described in, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, and Ausubel et al. 1987 with periodic additions) Current Protocols in Molecular Biology, Greene / Wiley, New York. Techniques for synthesizing polypeptides are described, for example, in Merrifield (1963) J. Amer. Chem. Soc. 85: 21492156; Merrifield (1986) Science 232: 341-347; and Atherton et al. (1989) Solid Phase Peptide Synthesis: A Practical Approach, IRL

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Press, Oxford. See also Dawson et al. (1994) Science 266: 776-779 for methods for making larger polypeptides.

The use of DCRS5 derivatives other than amino acid sequence or glycosylation variants is also contemplated herein. Such derivatives may include covalent or aggregate associations with chemical moieties. These derivatives generally fall into three classes: (1) salts, (2) covalent modifications to the side chains and terminal residues, and (3) adsorption complexes, e.g. with cell membranes. Such covalent or aggregate derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as receptor affinity purification or other binding molecule, e.g., antibodies. For example, a cytokine ligand may be covalently bonded to a solid support such as Sepharose, after activation with cyanogen bromide using methods that are well known in the art, or adsorbed onto polyolefin surfaces with or without glutaraldehyde attachment for cytokine receptor antibody assay or purification or the like. molecules. The ligand can also be labeled with a detectable group, e.g. by radioactive iodine by chloramine T, covalently bound to rare earth chelates or attached to another fluorescent moiety for use in diagnostic testing.

A combination, e.g. containing DCRS5 according to the invention, can be used as an immunogen to produce antisera or specific antibodies, e.g. capable of distinguishing between other members of the cytokine receptors for the described combinations. The complexes can be used to screen monoclonal antibodies or antigen-binding fragments prepared by immunization with various forms of partially purified protein preparations. In particular, the term "antibodies also includes antigen-binding fragments of natural antibodies, e.g. Fab, Fab2, Fv, etc." Purified DCRS5 can also be used as a reagent to detect antibodies generated in response to the presence of elevated expression levels or immune disorders that lead to the production of endogenous receptor antibodies. In addition, DCRS5 fragments may also serve as immunogens for the production of the antibodies of the invention as described immediately below. For example, the present invention contemplates antibodies having binding affinity or generated in response to the amino acid sequences shown in Table 1, fragments thereof, or various homologous peptides. In particular, the present invention contemplates antibodies having binding affinity or produced in response to specific fragments that are predicted to be, or actually are, displayed on the outer surface of the native DCRS5 protein. Protein combination complexes will also be useful and antibody preparations made therefor.

In certain other embodiments, soluble constructs, e.g., extracellular segments of DCRS5 ligand binding to IL-e1 may be ligand binding compositions and may be useful as either ligand antagonists or as antigens to block ligand signaling. As such, they may be useful or diagnostically useful, e.g., for histological labeling of a ligand; or therapeutically, e.g. as ligand antagonists.

Blocking the physiological response to receptor ligands may be due to inhibition of ligand binding to the receptor possibly by competitive inhibition. Thus, in vitro studies will often employ the antibodies or antigen-binding segments of these antibodies, soluble receptor constructs, or fragments bound to a solid support. These studies will also enable the diagnostic determination of the effects of both mutations and modifications in the ligand binding region as well as other mutations and modifications, e.g. that affect signal transduction or enzymatic function.

Also contemplated herein is the use of competitive screening for competing drugs, e.g., in which neutralizing antibodies to the receptor complex or fragments thereof compete with a test component for binding to a ligand or other antibody. In this way, neutralizing antibodies or fragments can be used to detect the presence of a polypeptide that has the same one or more binding sites as the receptor, and can also be used to seize receptor binding sites that might otherwise bind ligand. Soluble receptor constructs that link the extracellular or ligand-binding domain of DCRS5 to IL-12Re1 may be useful antagonists for the competitive binding of p40 ligand / IL-B30.

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V. Production of nucleic acids and proteins

DNA encoding a protein or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or by screening genomic libraries obtained from a wide variety of cell lines or tissue samples. Natural sequences can be isolated using standard methods and sequences described herein, e.g. in Table 1. Equivalents in other species can be identified by hybridization techniques or various PCR techniques in conjunction with or by searching sequence databases, e.g.

This DNA can be expressed in a wide variety of host cells for the synthesis of full-length receptor or fragments, which in turn can, for example, be used: for the production of polyclonal or monoclonal antibodies; in binding studies; for construction and expression of modified ligand binding domains or kinase / phosphatase domains; and for structure / function analysis. Variants or fragments can be expressed in host cells that are transformed or transfected with appropriate expression vectors. These molecules may be substantially free of proteins or cellular contaminants other than those derived from the recombinant host, and are therefore particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and / or diluent. A protein or portions thereof can be expressed as fusions with other proteins. Combinations of the described proteins or the nucleic acids encoding them are of particular interest.

Expression vectors are typically self-replicating DNA or RNA constructs containing the desired receptor gene, fragments thereof, or combinations of genes typically operably linked to appropriate genetic control elements that are recognized in an appropriate host cell. These control elements are capable of driving expression in an appropriate host. Multiple genes can be coordinated and can be polycistronic messenger RNA. The specific type of control elements needed to drive expression will depend on the type of host cell used. In general, the genetic control elements may include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically contain a transcriptional promoter, sometimes an operator that controls transcription efficiency, transcription enhancers that increase mRNA expression, a sequence encoding an appropriate ribosome binding site, and transcription and translation termination sequences. Expression vectors also typically contain an origin of replication that allows the vector to replicate independently of the host cells.

The vectors of the invention include those containing DNA encoding a combination of proteins as described or a biologically active equivalent polypeptide. The DNA may be under the control of a viral promoter and may encode a selectable marker. The present invention further contemplates the use of such expression vectors that are capable of expressing eukaryotic cDNAs encoding such proteins in a prokaryotic or eukaryotic host, wherein the vector is compatible with the host and the eukaryotic cDNA is incorporated into the vector such that the growth of the host containing the vector binds to expression. the discussed cDNAs. Typically, expression vectors are designed to be replicated continuously in their host cells, or to be amplified to appreciably multiply the total copy number of the desired gene (s) per cell. It is not always necessary for the expression vector to replicate in the host cells, e.g. it is possible to transiently express a protein or fragments thereof in different hosts using vectors that do not contain an origin of replication recognized by the host cells. It is also possible to use vectors which cause the protein coding portions to be incorporated into the host DNA by recombination.

The vectors used in the present invention include plasmids, viruses, bacteriophages, integratable DNA fragments, and other carriers which enable the DNA fragments to be incorporated into the host genome. Expression vectors are specialized vectors that contain genetic control elements that affect the expression of operably linked genes. Plasmids are the most commonly used forms of vectors, but all other forms of vectors which have an equivalent function and which are, or will become, known in the art are suitable for use herein. See, e.g., Pouwels et al. (1985 and supplements) Cloning Vectors: A Laboratory Manual, Elsevier, NY, and Rodriguez et al. (1988 eds) Vectors: A Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston.

Transformed cells are, most preferably, mammalian cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques. Transformed

Host cells typically express the desired proteins, but for cloning, amplification, and DNA manipulation, expression of the encoded proteins is not needed. The present invention further contemplates culturing transformed cells in a medium, thereby allowing the proteins to accumulate. These proteins can be recovered either from culture or, in some cases, from the medium.

For the purposes of the present invention, nucleic acid sequences are operably linked when their actions are related to each other. For example, DNA for a presequence or secretory sequence is operably linked to a polypeptide if the product is expressed as a preprotein or if it is involved in the targeting of the polypeptide to the plasma membrane or in the secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls transcription of the polypeptide; a ribosome binding site is operably linked to a coding sequence when it is placed in a translational manner. Typically, operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked, but still bind to operator sequences, which in turn controls expression.

Suitable host cells include Prokaryotes, lower Eukaryotes, and higher Eukaryotes. Prokaryotes include both gram negative and gram positive organisms, e.g., E. coli and B. subtilis. Lower eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and species of the genus Dictyostelium. Higher eukaryotes include solid tissue cultures of cell lines from animal cells, both of non-mammalian origin, e.g., insect and avian cells, and of mammalian origin, e.g., human, primate, and rodent.

The vector systems for prokaryotic hosts encompass a wide variety of vectors from many different species. As indicated herein, E. coli and its vectors will generally be used to include equivalent vectors used in other Prokaryotes. An exemplary DNA amplification vector is pBR322 or many of its derivatives. Vectors that can be used to express the receptor or fragments thereof include, but are not limited to, vectors such as those containing the lac promoter (pUC series); trp promoter (pBR322 trp); Ipp promoter (pIN series); lambda pP or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540). See Brosius et al. (1988) Expression Vectors Employing Lambda, and Ipp derived Promoters, in: Vectors: A Survey of Molecular Cloning Vectors and Their Uses, (ed. Rodriguez and Denhardt), Buttersworth, Boston, Chapter 10, pp. 205-236.

Lower eukaryotes, e.g. yeast and Dictyosteolium, can be transformed with vectors containing DCRS5 sequences. For the purposes of the present invention, the most common lower eukaryote host is the baker's yeast, Sacchaomyces cerevisiae. They will be used to generally represent the lower eukaryotes, although many other strains and species are also available. Yeast vectors typically contain an origin of replication (unless they are of the integrative type), a selection gene, a promoter, DNA encoding the receptor or fragments thereof, and translation termination, polyadenylation, and transcription termination sequences. Suitable yeast expression vectors include such constitutive promoters as the 3-phosphoglycerate kinase promoter and promoters of a variety of other glycolytic enzyme genes, or such inducible promoters as the alcohol 2-dehydrogenase promoter or the metallothionin promoter. Suitable vectors include derivatives of the following types: low copy number self-replicating (such as the YRp series), high copy number self-replicating (such as the YEp series); integrative types (such as the YIp series) or minichromosomes (such as the YCp series).

Tissue cultures of higher eukaryotic cells are typically the most preferred host cells for the expression of functionally active interleukins or receptor proteins. In fact, many tissue cultures of higher eukaryotic cell lines are useful, e.g., insect baculovirus expression systems, both of invertebrate and vertebrate origin. However, mammalian cells are most preferred. Transformation or transfection and culturing of such cells has become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines, avian cell lines, and simian cell lines (COS). Expression vectors for such cell lines typically include an origin of replication, a promoter, a translation initiation site, an RNA splice site (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or an amplification gene. Suitable expression vectors may be plasmids, viruses, or retroviruses containing promoters derived, for example, from sources such as adenovirus, SV40, parvovirus, pox or cytomegalovirus. Suitable examples of appropriate expression vectors include pCDNAl; pCD, see

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Okayama et al. (1985) Mol. Cell Biol. 5: 1136 1142; pMClneo PolyA, see Thomas et al. (1987) Cell 51: 503 512; and a baculoviral vector such as pAC 373 or pAC 610.

For secreted proteins and some membrane proteins, the open reading frame usually encodes a polypeptide that consists of a mature or secreted product covalently linked at its N-terminus to a signal peptide. The signal peptide is cleaved before the mature or active polypeptide is externally secreted. The cut site can be predicted with a high degree of accuracy from empirical principles, e.g. von-Heijne (1986) Nucleic Acid Research 14: 4683-4690 and Nielsen et al. (1997) Protein Eng. 10: 1-12, and the precise amino acid structure of a signal peptide often does not appear to be essential for its function, e.g., Randall et al. (1989) Science 243: 1156-1159; Kaiser et al. (1987) Science 235: 312-317. The mature proteins of the present invention can be easily identified using standard methods.

It will often be desirable to express these polypeptides in a system that provides a specific or defined glycosylation pattern. In this case, the usual pattern will be that provided naturally by the expression system. However, the pattern could be modified by treating the polypeptide, e.g., with a non-glycosylated form, with appropriate glycosylating proteins incorporated into a heterologous expression system. For example, the receptor gene can be transformed together with one or more genes encoding mammalian or other glycosylating enzymes. Using this approach, certain mammalian glycosylation patterns will be obtainable in prokaryotic or other cells. Expression in prokaryotic cells will typically lead to non-glycosylated forms of the protein.

The source of DCRS5 may be a eukaryotic or prokaryotic host expressing recombinant DCRS5 as described above. The source may also be a cell line, but other mammalian cell lines are also contemplated, with the most preferred lines being from the human species.

Since primate DCRS5 sequences are known, fragments or derivatives thereof can be made by standard peptide synthesis processes. These include processes such as those described in Stewart and Young (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis, Springer Verlag, New York; and Bodanszky (1984) The Principles of Peptide Synthesis, Springer Verlag, New York. For example, processes involving azides, acid chlorides, acid anhydrides, mixed anhydrides, active esters (e.g., p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester), carbodiimidazole, redox processes, or an addition process using dicyclohexylcarbodiimide (DCCD). Both solid phase and liquid phase synthesis can be used in the processes described above. Similar techniques can be used for partial DCRS5 sequences.

The DCRS5 proteins, fragments or derivatives thereof are suitably prepared according to the above processes as conventionally employed in peptide synthesis, generally either by a so-called step-by-step process which involves condensing an amino acid to a terminal amino acid sequentially one at a time, or by coupling the peptide fragments to a terminal amino acid. Amino groups which are not used in the attachment reaction typically must be protected to prevent misplacement.

When solid phase synthesis is used, the C-terminal amino acid is bound to the insoluble support or support via its carboxyl group. The insoluble carrier is not particularly limited as long as it has a binding capacity to a reactive carboxyl group. Examples of such insoluble carriers include halogenomethyl resins such as chloromethyl resins or bromomethyl resins, hydroxymethyl resins, phenolic resins, tert-alkyloxycarbonyl hydrazidated resins, and the like.

The amino-blocked amino acid is sequentially attached by linking its activated carboxyl group to a reactive amino group of the previously formed peptide or chain to synthesize the peptide in subsequent steps. After the complete sequence is synthesized, the peptide is cleaved from the insoluble support to produce the peptide. The solid phase method is generally described by Merrifield et al. (1963) in J. Am. Chem. Soc. 85: 2149-2156.

The produced protein and its fragments can be isolated and purified from the reaction mixture by peptide separation methods, e.g., extraction, precipitation, electrophoresis, various forms of chromatography, immunoaffinity, and the like. The receptors of the present invention can be obtained with varying degrees of purity depending on the desired applications. Cleansing can

Performed by the protein purification techniques disclosed below, or by the antibodies described herein by the methods of immunoabsorbent affinity chromatography. This immunoabsorbent affinity chromatography is performed by first attaching the antibodies to a solid support and then by combining the bound antibodies with reconstituted lysates of the appropriate cells, lysates of other cells expressing the receptor, or lysates or supernatants of protein-producing cells as a result of DNA techniques, see below.

Generally, the purified protein will be at least about 40% pure, commonly at least about 50% pure, typically at least about 60% pure, typically at least about 70% pure, more typically at least about 80% pure, preferably at least about 90% pure. % pure, and more preferably at least about 95% pure, and in particular embodiments at least about 97% -99% or greater. Purity will usually be judged by weight, but may also be by molarity. Different tests will be applied accordingly. Individual proteins can be purified and then combined.

VI. Antibodies

Antibodies can be obtained against a variety of mammalian, e.g. primates, DCRS5 proteins and fragments thereof, both in naturally occurring native forms and in recombinant forms thereof, with the difference that anti-active receptor antibodies are more likely to recognize epitopes that are present. exclusively in native conformations. Antibodies that recognize epitopes displayed by the combination of DCRS5 with IL-12Re1, eg functionally, are also contemplated. Detection of denatured antigens may also be useful e.g. in Western analysis. Anti-idiotype antibodies are also contemplated which would be useful as agonists or antagonists of the natural receptor or antibody.

Antibodies, including binding fragments and single-chain versions against specific protein fragments, can be obtained by immunizing animals with conjugates of the fragments with immunogenic proteins. Monoclonal antibodies are obtained from cells secreting the desired antibody. These antibodies can be screened for binding to a normal or defective protein, or screened for agonist or antagonist activity. These monoclonal antibodies will typically bind to a Kd of at least about 1 mM, more typically at least about 300 µM, typically at least about 100 µM, more typically at least about 30 µM, preferably at least about 10 µM, and more preferably at least about 3 µM or better.

Antibodies, including antigen-binding fragments, of the invention may have significant diagnostic or therapeutic value. They can be potent antagonists that bind to the receptor and inhibit ligand binding or inhibit the receptor's ability to elicit a biological response, e.g., interact with its substrate. They can also be useful as non-neutralizing antibodies and can be attached to toxins or radioactive isotopes to bind producer cells or cells located close to the interleukin source. Furthermore, these antibodies can be conjugated to drugs or other therapeutic agents, both directly and indirectly via a linker.

The antibodies of the invention may also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can bind to the receptor without inhibiting ligand or substrate binding. As neutralizing antibodies, they may be useful in competitive binding analyzes. They will also be useful in detecting or quantifying the ligand. They can be used as reagents in Western blot analysis or in immunoprecipitation or in immuno purification of the corresponding protein. Likewise, nucleic acids and proteins can be bound to solid supports for affinity purification or detection methods. The substrates can be, for example, solid resin beads or plastic sheets.

Protein fragments may be attached to other materials, particularly polypeptides, as fused or covalently joined polypeptides for use as immunogens. Mammalian cytokine receptors and fragments thereof may fuse or be covalently attached to various immunogens. See Microbiology, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Serological Reactions, Dover Publications, New York; and Williams et al. (1967) Methods in Immunology and Immunochemistry, vol. I, Academic Press, New York, in which methods for the preparation of polyclonal sera are described. A typical method is to hyperimmune the animal with an antigen. The animal's blood is then collected shortly after the repeat immunizations and the gamma globulins are isolated.

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In some cases, it is desirable to prepare monoclonal antibodies from a variety of mammalian hosts such as mice, rodents, primates, humans, etc. A description of techniques for preparing such polyclonal antibodies can be found in, e.g., Stites et al. (Eds.) Basic and Clinical Immunology ( ed. IV.), Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press, New York; and especially in Kohler and Milstein (1975) in Nature 256: 495 497, where one method of making polyclonal antibodies is discussed. In short, this method involves injecting an animal with an immunogen. The animal is then killed and cells from its spleen are harvested and linked to multiple myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The hybridoma cell population is then screened for the isolation of individual clones, each secreting a single type of anti-immunogen antibody. Thus, the individual types of antibodies obtained are the products of immortalized and cloned single B cells from an immunized animal produced in response to a specific recognition site in an immunogenic substance.

Other suitable techniques include in vitro exposure of lymphocytes to antigenic polypeptides or alternatively selection with antibody phage libraries or similar vectors. See Huse et al. (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246: 1275-1281 and Ward et al. (1989) Nature 341: 544-546. The polypeptides and antibodies of the invention can be used with or without modification, including chimeric or humanized antibodies. Often, polypeptides and antibodies will be labeled by attaching both covalently and non-covalently to a substance that provides a detectable signal. A wide variety of labels and attachment techniques are known and widely described in both the scientific and patent literature. Suitable labels include radioactive isotopes, enzymes, substrates, cofactors, inhibitors, fluorescent residues, chemiluminescent residues, magnetic particles, and the like. The use of such tags is described in US Patent Nos. 3,817,837; 3,850,752; 3939350; 3996345; 4,277,437; 4,275,149 and 4,366,241. Recombinant or chimeric immunoglobulins can also be produced, see Cabilly, US Patent No. 4,816,567, or produced in transgenic mice, see Mendez et al. (1997) Nature Genetics 15: 146-156.

The antibodies of the invention may also be used in affinity chromatography to isolate DCRS5 proteins or peptides. Columns may be prepared in which the antibodies are attached to a solid support, e.g. particles such as agarose, Sephadex or the like, and the cell lysate may be passed through the column, then the column may be washed with increasing concentrations of a mild denaturing agent, thereby purified protein will be released. Alternatively, the protein can be used to purify the antibody. Suitable cross-absorption or depletion techniques can be used.

The antibodies can also be used to screen expression libraries for specific expression products. Typically, the antibodies used in such a procedure will be labeled with a moiety allowing the presence of the antigen to be readily detected by antibody binding.

Antibodies raised against the cytokine receptor will also be used to generate anti-idiotype antibodies. They will be useful for detecting or diagnosing a variety of immune conditions associated with the expression of the protein or cells that express the protein. They will also be useful as ligand agonists or antagonists, which may be receptor competing inhibitors or substitutes for naturally occurring ligands. Certain antibodies against the receptor subunits or combinations may serve as activating antibodies which may thus influence signal transduction, e.g. serving as ligand agonists.

A cytokine receptor protein that specifically binds to, or is specifically immunoreactive with, an antibody raised against a particular immunogen, such as an immunogen containing the amino acid sequence of SEQ ID NO: ID NO: 2, is usually determined by an immunoassay. An immunoassay typically uses a polyclonal serum which has been raised e.g. against a protein of SEQ ID NO. ID NO .: 2. The immune serum is selected so that it has low cross-activity against other members of the cytokine receptor family, e.g. the IL-12Re2 receptor subunit or the gp130 subunit of the Il-6 receptor, preferably from the same species and any such cross-activity is removed by immunoabsorption prior to use. in an immunological test.

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To generate an immune serum for use in an immunoassay, the protein, e.g., from SEQ. ID NO: 2, is isolated as described herein. For example, the recombinant protein can be produced in a mammalian cell line. A suitable host, e.g., an inbred mouse strain such as Balb / c, is immunized with the selected protein, typically using a standard adjuvant such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and Lane, supra). Alternatively, a synthetic peptide derived from the sequences disclosed herein, conjugated to a carrier protein, may be used as the immunogen. The polyclonal serum is harvested and titered with an immunogenic protein by an immunoassay, e.g., a solid phase immunoassay, where the immunogen is immobilized on a solid support. Polyclonal sera with a titer of 10⁴ or greater are harvested and tested for cross-activity against other members of the cytokine receptor family, e.g., gp130 or IL-12Re1, using a competitive binding immunoassay such as one described in Harlow and Lane, supra, on page 570- 573. Preferably, at least two members of the cytokine receptor family are used for definition. Such members of the family of cytokine receptors can be made as recombinant proteins and isolated using standard molecular biology and protein chemistry techniques described herein.

Competition binding immunoassays can be used to determine cross-activity. For example, a protein with SEQ. ID NO: 2 can be immobilized on a solid support. Proteins added to the assay compete with the binding of the antisera with the immobilized antigen. The ability of the above proteins to compete with the immobilized protein for binding to antisera is compared to proteins, e.g., gp130 or IL-12Re2. The percentage of cross-activity for the above proteins is calculated using standard calculations. Those antisera which have less than 10% cross-activity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorption with the proteins listed above.

The pooled immunosuppressed immune sera are then used in a competitive binding immunoassay as described above to compare the second protein to an immunogenic protein (e.g., a DCRS5-type protein of SEQ ID NO: 2). To make such a comparison, each of the two proteins is tested over a wide concentration range and the amount of each protein is determined which is required to inhibit 50% of the binding of the serum antibodies to the immobilized protein. If the required amount of the second protein is less than twice the amount of the selected protein or proteins, the second protein is said to specifically bind the antibody raised against the immunogen.

It is understood that the cytokine receptor proteins are members of a family of homologous proteins comprising many of the identified genes. For a particular gene product such as DCRS5, the term refers not only to the amino acid sequences disclosed herein, but also to other proteins that are allelic, non-allelic, or species variants. It is also understood that these terms include unnatural mutations introduced by deliberate mutation using conventional recombinant technology such as single site mutation or the excision of a short length of DNA encoding the relevant proteins, or by substituting new amino acids or adding new amino acids. Such minor changes typically will substantially maintain the immunological identity of the parent molecule and / or its biological activity. Thus, such changes include proteins that specifically cross-interact with the desired naturally-occurring DCRS5 protein. The biological properties of the altered proteins can be determined by producing the protein in an appropriate cell line and measuring the appropriate effect, e.g. on transfected lymphocytes. Some modifications to the protein, considered to be minor, will include conservative amino acid substitutions with similar characteristics as described above for the cytokine receptor family in full. The protein compositions of the invention can be determined by optimally aligning the protein with the cytokine receptor protein and by using the conventional immunoassays described herein to determine immunological identity.

In addition, antibodies directed against the receptor subunits can be administered to sterically block ligand binding to a functional receptor. Such antibodies can be raised for single subunits or for the combination of DCRS5 with IL-12Re1. As a result, antagonist antibodies will be produced.

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VII. Kits, diagnostics and quantification

Both naturally occurring and recombinant forms of the cytokine receptor type molecules of the invention are particularly useful in kits and assay methods. For example, such methods can also be used to screen for binding activity, e.g., ligands for these proteins. In recent years, a number of automated test methods have been developed that allow screening of tens of thousands of compounds per year. See, e.g., a BIOMEK automated workstation, Beckman Instruments, Palo Alto, California, and Fodor et al. (1991) Science 251: 767-773. The latter describes methods of testing binding by a number of specific polymers synthesized on a solid support. The development of suitable assays to search for ligand or homologous antagonist / agonist proteins can greatly facilitate access to the large number of purified, soluble cytokine receptors in the active state as provided according to the invention.

Purified DCRS5 can be directly coated onto plates used for the above-mentioned ligand screening technique. Non-neutralized antibodies to these proteins can be used as capture antibodies to immobilize the respective receptor on a solid phase, which is useful, e.g., in diagnostic applications.

The use of DCRS5, its fragments, peptides and its fusion products in a variety of diagnostic kits and methods for detecting the presence of a protein or its ligand is described herein. Alternatively or in addition, antibodies to the molecules can be incorporated into the kits and methods. Typically the kit will have a container containing the DCRS5 peptide or gene segment or reagent recognizing one or the other. Typically, the recognition reagents will be a receptor or an antibody for a peptide or, in the case of a gene segment, it will usually be a hybridization probe. Other kit components may include other proteins or reagents related to the p40, IL-B30, or IL-12Re1 peptides of the ligand / receptor pair.

A preferred kit for determining the concentration of DCRS5 in a sample will typically include a labeled component, e.g., a ligand or antibody, with a known binding affinity for DCRS5, a source of DCRS5 (naturally occurring or recombinant) as a positive control, and means for separating the labeled bound component from the free , for example a solid phase to immobilize DCRS5 in the test sample. Containers containing reagents and instructions are also normally provided.

Antibodies, including antigen-binding fragments, specific for mammalian DCRS5 or a peptide fragment, or receptor fragments are useful in diagnostic applications to detect the presence of elevated levels of ligand and / or fragments thereof. Diagnostic assays can be homogeneous (without the separation step of free reagent and the antibody-antigen complex) or heterogeneous (with the separation step). There are various commercial tests, such as RIA (radioimmunoassay), ELISA ( enzyme linked immunosorbent assay, EIA (enzyme immunoassay), EMIT (enzyme multiplied immunoassay technique), SLFIA (substrate labeled fluorescent immunoassay) and the like. For example, unlabeled antibodies can be used by using a labeled secondary antibody that recognizes the antibody for a cytokine receptor or a specific fragment thereof. Such tests are also extensively discussed in the literature. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH., And Coligan (1991 Ed. And Periodic Supplements) Current Protocols In Immunology Greene / Wiley, New York.

The anti-idiotype antibodies may have a similar utility as cytokine receptor agonists or antagonists. Under certain circumstances, they may be useful as therapeutic reagents.

Often the diagnostic test reagents are supplied in kits to optimize the sensitivity of the test. Depending on the nature of the assay, a protocol and label, labeled or unlabeled antibody, or labeled ligand can be provided. The assay typically has other additional reagents such as buffers, stabilizers, materials necessary to generate the signal, such as enzyme substrates, and the like.

Preferably, the kit also includes instructions on how to properly use and dispose of the contents after use. Typically, the kit has containers for each useful reagent, and will include instructions for proper use and disposal of the reagents. It is desirable that the reagents be supplied as a dry lyophilized powder and that they can be reconstituted in an aqueous medium at concentrations appropriate for the performance of the assay.

The above-mentioned diagnostic assay components can be used without modification or with modifications by various methods. For example, labeling can

This can be achieved by covalently or non-covalently binding the particle which directly or indirectly provides the signal to be detected. In many such assays, a test component, a cytokine receptor or an antibody directed against it can be directly or indirectly labeled. Direct labels that can be used include label groups: isotope labels such as¹²⁵And enzymes (US Patent No. 3,645,090), such as peroxidase and alkaline phosphatase, and fluorescent labels (US Patent No. 3,940,475) for which a change in fluorescence intensity, wavelength shift, or fluorescence polarization can be monitored. Indirect labeling that may be used involves biotinylating one component followed by binding to an avidin conjugated to one of the above groups of labels.

There are also many methods for separating bound ligand from free, or alternatively bound, test component from unbound. The cytokine receptor can be immobilized on various arrays and then washed. Suitable arrays include plastics such as ELISA plates, filters, and beads. Methods for immobilizing the receptor on the matrix include, but are not limited to, direct adhesion to plastic, use of scavenger antibodies, chemical conjugation, and biotin-avidin. The last step of the test involves the precipitation of the antibody / antigen complex using a variety of methods including those using, for example, an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate. Other suitable separation techniques include, but are not limited to, the fluorescein labeled antibody magnetic bead method described in Rattle et al. (1984) Clin. Chem. 30 (9): 1457 1461 and the dual antibody magnetic bead separation described in US Patent No. 4,659,678.

Methods for linking a protein or fragments with different tags are widely described in the literature and do not need to be discussed in detail. Many of these techniques involve the use of an activated carboxyl group by the use of a carboimide or active esters to form peptide bonds, the formation of thioesters by the reaction of a mercaptan group with an activated halogen such as chloroacetyl or an activated olefin such as maleimide or the like. Fusion proteins may also find use here.

Another diagnostic aspect requires the use of oligonucleotide or polynucleotide sequences derived from cytokine receptor sequences. These sequences can be used as probes to detect the levels of the appropriate cytokine receptor in patients expected to have an immune disorder. Preparations of both RNA and DNA nucleotide sequences, the labeling of these sequences, and the preferred sequence sizes have previously been discussed in the literature. Normally, an oligonucleotide probe should be at least about 14 nucleotides, typically at least about 18 nucleotides, and polynucleotide probes can be up to several thousand base pairs. Various labels can be used, most commonly radionuclides, in particular³²P. Although, other techniques such as the use of biotin-modified nucleotides to introduce into a polynucleotide can also be used. The biotin then serves as a binding site for avidin or antibodies that can be labeled with a variety of labels such as radionuclides, fluorescent compounds, enzymes, or the like. Alternatively, antibodies that recognize specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes, can be used. In turn, the antibodies can be labeled, and the test can be performed while the duplex is bound to the surface, such that once a duplex is formed on the surface, the presence of antibodies bound to the duplex can be detected. Traditional techniques such as hybridization, plus and minus screening, recombinant probing, HRT (hybrid relaased translation), and HART (hybrid arrested translation) can employ novel antisense RNA probes. This also includes amplification techniques such as the polymerase chain reaction (PCR).

Diagnostic kits that test qualitative and quantitative percentages of other markers are also considered. Diagnosis and prediction may depend on a combination of multiple indicators used as markers. Thus, the kits can test combinations of markers. See, e.g., Viallet et al. (1989) Progress in Growth Factor Res. 1: 89-97. Detection of polymorphic variants that may reflect differences in functional receptor signaling may be useful in determining a therapeutic strategy. Variants showing a greater or lesser ligand response may allow patient pools to be subdivided into responders / non-responders.

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VIII. Therapeutic use

The present invention provides reagents of significant therapeutic value. See, e.g., Levitzki (1996) Curr. Opin. Cell Biol. 8: 239-244. Cytokine receptors (naturally occurring or recombinant) fragments thereof, mutein receptors and antibodies together with components identified as having binding affinity for the receptors or antibodies should be useful in the treatment of conditions that abnormally express the receptors or their ligands. Such an abnormality will usually manifest itself through an immune disorder. See WO 01/18051, incorporated herein by reference. Additionally, the present invention should provide therapeutic value for a variety of diseases and disorders associated with aberrant expression or aberrant ligand response. For example, it is suggested that p40 ligand / IL B30 ligand is required for the development of cellular immunity, e.g., anti-tumor activity, enhancement of humoral and cellular immunity, and antiviral responses. In particular, the ligand appears to activate NK cells and T cells. Combination therapy with IL-18, IL-12, TNF, IFN?, Radio / chemotherapy, adjuvants or anti-tumor, anti-viral or anti-fungal agents may be used.

Conversely, antagonists that can be used in combination with antagonists for TNF, IFN?, IL-18 or IL-12 or with IL-10 or steroids may be indicated in chronic Th1-mediated diseases, in autoimmune diseases, or in transplant situations and / or rejection, multiple sclerosis, psoriasis, chronic inflammation, rheumatoid arthritis, osteoarthritis or inflammatory bowel diseases. Antagonists may take the form of antibodies to the receptor subunits, soluble receptor constructs, or antisense nucleic acids to one or more of the receptor subunits. Matching the p40 / IL-B30 ligand to the DCRS5 and IL-12Re1 receptor subunits provides an indication for the use of agonists and antagonists.

Therapeutically, based on the described p40 / IL-B30 activities, cytokinin antagonists can be influenced, e.g., with soluble DCRS5, with or without soluble IL-12Re1, or with an antibody to any receptor subunit. Antagonists may be useful as inhibitors of unwanted immune and inflammatory responses, to target memory T cells, or in combination with IL-12 / IL-12R antagonists or other anti-inflammatory and immunosuppressive compounds. Clinical indications may be chronic inflammation or post-transplant conditions. Various polymorphisms may enhance or weaken receptor function and, if dominant, may be useful as therapeutic agents. The identification of such variants may allow the division of pools of responders and non-responders. The reagents may be useful as detection or labeling reagents, or reagents to cut memory T cells and / or NK cells.

Gene therapy can use populations of desired p40 / IL-B30 ligand responsive cells, e.g., as adjuvants for tumor immunotherapy, to facilitate activation of tumor-infiltrating lymphocytes, T cells, or NK cells. Antisense sequence strategies can be used, e.g., to prevent a receptor response.

Various abnormalities are known in different cell types that have been shown to produce both IL-12 p40 and / or IL-B30 mRNA by Northern blot analysis. See Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck &amp; Co., Rahway, N, J .; Thorn et al. Harrison's Principles of Internal Medicine, McGraw-Hill, NY and Weatherall et al. (Eds) Oxford Textbook of Medicine, Oxford University Press, Oxford. Many other conditions and diseases will respond to treatment with the agonists and antagonists provided herein. See, e.g., Stites and Terr (ed .; 1991) Basic and Clinical Immunology Appleton and Lange, Norwalk, Connecticut and Samter et al. (Eds.) Immunological Diseases Little, Brown and Co. Other similar indications for treatment include bone remodeling, sexual dysfunction, prevention of neurodegenerative diseases, dementia, stress, and others. These problems should be amenable to preventive action and treatment with the compositions provided herein.

Recombinant cytokine receptors, muteins, agonist and antagonist antibodies or antibodies therefor can be purified and administered to a patient. For therapeutic use, these reagents can be used in combination with additional active ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, together with physiologically harmless stabilizers and excipients. These combinations may be sterile, e.g., filtered, and placed in dosage forms as a lyophilisate in a container or stored as stabilized aqueous preparations.

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Also described herein is the use of antibodies or binding fragments thereof that do not bind the complement.

Ligand screening using the cytokine receptor or fragments thereof can be performed to identify molecules having binding affinity for the receptors. Then, biological assays can be used to determine whether the putative ligand is capable of undergoing competitive binding that blocks intrinsic stimulating activity. Receptor fragments can be used as blockers or antagonists to block ligand activity. Likewise, a component having intrinsic stimulatory activity may activate the receptor and thus be an agonist to stimulate ligand activity, e.g., including signal transduction. The invention further contemplates therapeutic use as antibody antagonists to cytokine receptors.

The amounts of reagents necessary for effective therapy will depend on a variety of factors, including the mode of administration, target site, reagent physiological life, pharmacological life, physiological state of the patient, and administration of other drugs. Thus, the therapeutic dosage should be titrated to optimize safety and efficacy. Typically, dosages used in vitro can provide useful guidance on the appropriate amounts of reagents for in situ administration. Animal studies at effective doses for the treatment of specific disorders will provide additional indications for dosing in humans. Various considerations of this type are described, for example, in Gilman et al. (1990 ed.) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press and Remington's Pharmaceutical Sciences, eds. 17. (1990), Mack Publishing Co., Easton, Penn. Methods of administration are discussed herein and below, and include, for example, oral, intravenous, intraperitoneal, or intramuscular administration, skin diffusion, and others. Pharmaceutically acceptable carriers will include water, saline, buffers, and other ingredients as described, for example, in the Merck Index, Merckt &amp; Co., Rahway, New Jersey. Due to the likelihood of high binding affinity or high number of turns between the putative ligand and its receptors, low dosage is expected to be effective. Also, the signal transduction pathway suggests that extremely low amounts of ligand can be effective. Thus, dosage ranges are expected to be generally below concentrations of 1 mM, typically below concentrations of about 10 µM, typically below concentrations of about 100 nM, preferably below concentrations of about 10 pM (picomolar) and most preferably below concentrations of about 1 fM (femtomolar), with appropriate carrier. Often, slow release formulations or slow release devices will be used for continuous administration.

Cytokine receptors, fragments thereof, and antibodies or fragments thereof, antagonists and agonists may be administered directly to the host treated, or depending on the size of the components, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to administration. Therapeutic preparations can be administered in a variety of conventional dosage forms. While it is possible for the active ingredient to be administered alone, it is preferable that it is present as a pharmaceutical preparation. Formulations include at least one active ingredient as defined above together with one or more acceptable carriers. Each carrier must be both pharmaceutically and physiologically acceptable in the sense that it is compatible with the other ingredients and not deleterious to the patient. Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. See, e.g., Gilman et al. (1990 ed.) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, eds. 8, Pergamon Press; and Remington's Pharmaceutical Sciences, ed. 17. (1990), Mack Publishing Co., Easton, Penn .; Avis et al. (1993 Ed.) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY; Lieberman et al. (1990 Ed.) Pharmaceutical Dosage Forms: Tablets Dekker, NY and Lieberman et al. (1990 Ed.) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY. Therapy can be combined or used in combination with other therapeutic agents, in particular with agonists and antagonists of other members of the cytokine receptor family.

IX. Screening tests

Drug screening using DCRS5 or fragments thereof can be performed to identify components having binding affinity for the receptor subunit, including isolating associated components. Then you can use biological tests for

Determining whether the component has intrinsic stimulatory activity and is therefore a blocker or antagonist that blocks ligand activity.

Moreover, matching the p40 / IL-B30 ligand with the functional IL-12Re1 DCRS3 receptor allows the search for antagonists or agonists with positive control of signal transduction.

A search for a small molecule or an antibody can be performed.

One method of drug screening uses eukaryotic or prokaryotic host cells that are stably transformed with a recombinant DNA molecule that expresses DCRS5 in combination with another cytokine receptor subunit, e.g., IL-12Re1. The signaling is expected to use STAT4. You can isolate cells that produce a receptor that is isolated from other functional receptors. Such cells, either live or in fixed form, can be used in standard antibody / antigen or ligand / receptor binding assays. See also, Parce et al. (1989) Science 246: 243-247 and Owicki et al. (1990) Proc. Natl. Acad. Sci. USA 87: 4007-4011, which describes sensitive methods for detecting cellular responses. Competition assays are especially useful in which cells are contacted and incubated with a labeled receptor or with an antibody of known binding affinity for a ligand, such as a labeled antibody. ¹²⁵I and the binding affinity of the test sample to the binding composition is measured. Bound and free labeled binding compositions are then separated to evaluate the degree of ligand binding. The amount of test component bound is inversely proportional to the amount of labeled receptor bound to a known source. Many techniques can be used to separate bound from unbound ligand to assess the level of ligand binding. Such a separation step typically may involve a procedure such as filter adhesion followed by washing, adhesion to plastic followed by washing, or pelleting of cell membranes. Live cells can also be used to study the effects of drugs on cytokine-mediated functions, e.g., STAT4 signaling and others. Some detection methods allow the separation step to be eliminated, e.g., a near sensitive detection system.

The broad scope of the present invention will be best understood with reference to the following examples, which are not intended to limit the invention to specific embodiments.

Examples

I. General methods

Some of the standard methods are described or referenced, e.g., in Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed.), Volumes 1-3, CSH Press, NY or Ausubel et al. (1987 and supplements) Current Protocols in Molecular Biology, Greene / Wiley, New York. Protein purification methods include ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., Ausubel et al. (1987 and periodic supplements); Coligan et al. (Ed. 1996) and periodic supplements, Current Protocols in Protein Science Greene / Wiley, New York; Deutscher (1990) "Guide to Protein Purification in Methods in Enzymology, Vol. 182, and other volumes in this series, and manufacturers' literature on the use of protein purification products, e.g., Pharmacia, Piscataway, NJ or BioRad, Richmond, CA. Combination with recombinant techniques allows for the creation of fusions with the appropriate segments, e.g. a FLAG sequence, or the equivalent thereof, which may be joined by a protease deleted sequence. See, for example, Hochuli (1990) "Purification of Recombinant Protelns with Metal Chelate Absorbent in Setlow (ed.) Genetic Engineering, Principle and Methods 12: 87-98, Plenum Press, NY, and Crowe et al. (1992) QIAexpress: The High Level Expression &amp; Protein Purification System QUIAGEN, Inc., Chatsworth, CA.

Computerized sequence analysis is performed, e.g., using available software, including those from GCG (U. Wisconsin) and GenBank resources. Public databases are also used, e.g. from GenBank and others.

Many methods for IL-10 receptors, as described, e.g., in US Patent No. 5,789,192 (IL-10 receptor), can be applied to DCRS5.

II. Functional cloning

Anti-hIL-12Re1 antibodies were observed to block human T cell responses to p40 / IL-B30 and p40 / IL-B30 bound to IL-12Re1. This suggested that IL-12Re1 is a subunit of the receptor complex for p40 / IL-B30.

A population of murine T cells responsive to p40 / IL-B30 but not to p40 / IL-B30 was identified

IL-12, and another population that responds to IL-12 but not p40 / IL-B30. Additionally, the observation32

It was found that Ba / F3 cells producing recombinant mIL-12Re1 and mIL-12Re2 responded to IL12 but not to p40 / IL-B30. Together, these results indicate that the receptor complex for p40 / ILB30 contains IL-12Re1 and at least one other subunit that is not IL-12Re2. Thus, an expression cloning strategy was developed to isolate the second receptor component.

A cDNA library was prepared from mRNA isolated from Kit225 cells, an IL-2 dependent human T cell line responding to both IL-12 and p40 / IL-B30. The cDNA library was made using a retroviral expression vector, pMX. The cDNA library was infected with Ba / F3 cells producing recombinant hIL-12Re1, allowed to reconstitute for 3-4 days in IL-3, then washed and plated at ~ 15,000 cells / well in 96 well plates with medium containing 50 ng / ml hyper- hp40 / hIL-B30. See WO 01/18051. Cultures were replenished every 5 days with additional hyper-hp40 / hIL-B30. After approximately two weeks, 5-10% of the wells showed cell growth. Cells were recovered from the well, grown singly in larger cultures in hyper-hp40 / hIL-B30, and tested for growth on hyper-hp40 / hIL-B30.

Cells whose growth was dependent on p40 / IL-B30 were analyzed by PCR, examining the retroviral cDNA inserts. Of the more than 40 isolates analyzed, all but one contained cDNA molecules encoding the new DCRS5 receptor. A selected human cDNA was cloned into an expression vector and transfected with Ba / F3 cells expressing hIL-12Re1. Cells became responsive to p40 / IL-B30; thus, we concluded that the new cDNA encodes the desired DCRS5, a functional IL-B30 receptor subunit.

III. Features of full-length DCRS5; chromosomal location

The cytoplasmic domain of DCRS5 is completely related to the other cytoplasmic domains of the cytokine receptor commonly observed in this family of molecules. The cytoplasmic domain contains seven tyr residues, at least three of which are part of recognized SH2 binding motifs: YEDI, YKPQ and YFPQ. The YEDI motif is similar to the identified binding sites for shp2 tyrosine phosphatase. The two further motifs are very similar to the known Stat1 / Stat3 or Stat3 binding sequences, respectively. The YKPQ motif, along with the surrounding sequences, is also highly similar to the motifs in Stat4 and IL-12Re2 known to bind Statl-3. This is consistent with preliminary results suggesting that p40 / IL-B30, like IL-12, activates Stat4.

PCR primers for the DCRS5 sequence are used as probes to analyze a human cDNA library. Sequences may be selected, e.g. from Table 1, preferably those adjacent to the termination sequence. Full-length DCRS5 cDNA molecules for primate, rodent, or other species have been cloned, e.g., by screening by DNA hybridization on phage λgt10. PCR reactions are performed using Taqaplus DNA polymerase from T. aquaticus (Stratagene) under appropriate conditions.

Chromosome smears were prepared. On chromosome preparations obtained from human lymphocytes stimulated with phytohaemagglutinin; cultivated for 72 hours, to which 5-bromodeoxyuridine (60 µg / ml of medium) was added for the last seven hours of culture, in order to ensure good quality chromosome striation after hybridization.

The PCR fragment amplified with the primers was cloned into a suitable vector. The vector was tagged with the nick-translation technique z³H. The labeled probe was hybridized to metaphase smears at a final concentration of 200 ng / ml hybridization solution as described by Mattei et al. (1985) Hum. Genet. 69: 327-331.

After coating with a nuclear track emulsion (KODAK NTB2), the slides were exposed. In order to avoid any displacement of the silver granules during the binding procedure, the chromosome smears are first stained with Giemsa's buffered solution and the metaphase is photographed. R-striation is then performed using the Fluorochromephotolysis-Giemsa (FPG) method and the metaphases are photographed again prior to analysis.

Likewise, suitable methods can be applied to other species.

IV. Localization of the mRNA for DCRS5

Membranes from various human tissues (Cat # 1,2) and tumor cell lines (Cat # 7757-1), containing approximately 2 µg poly (A) + RNA per lane, are purchased from Clontech (Palo Alto, CA).

Probes are labeled with [α-P] dATP, e.g. using the Amersham Rediprime random primer labeling kit (RPN1633). Pre-hybridization and hybridization are performed e.g. at 65 ° C in 0.5 M Na2HPO4, 7% SDS, 0.5 M EDTA (pH 8.0). Rinsing is performed under stringent conditions, for example at 65 ° C; the first two washes in 2 x SSC, 0.1% SDS for 40 min, followed by a wash

PL 209 128 B1 in 0.1 x SSC, 0.1% SDS for 20 min. The membranes are then exposed at -70 ° C to X-ray film (Kodak) and the presence of signal enhancement shutters. More detailed analyzes of the cDNA libraries using the Southern blot technique are performed with the appropriate human DCRS5 clones selected to study their expression in blood stem cells and other cell types.

Alternatively, two suitable primers are selected from Table 1. RT-PCR is used for an appropriate mRNA sample selected for the presence of messenger RNA to obtain a cDNA, e.g., a gene expressing sample.

Full-length clones can be isolated by hybridizing cDNA libraries from appropriate tissues previously selected based on the PCR signal. Northern blot analyzes can be performed.

The messenger RNA of the DCRS5-encoding genes will be examined using appropriate techniques, e.g., PCR, immunoassays, hybridization, or others. Tissue and organ cDNA preparations may be provided, e.g., from Clontech, Mountain View, CA. Understanding the sources of natural expression is useful as described. Understanding the interactions of functional receptor subunits makes it possible to determine which cells express the combination of receptor subunits that will result in a physiological ability to respond to each of the cytokine ligands.

To study distribution in mice, e.g. Southern analysis can be performed: DNA- (5 μg) from the original amplified cDNA library was digested with appropriate restriction enzymes to release the inserts, separated on a 1% agarose gel and transferred to a nylon membrane (Schleicher and Schuell, Keene, NY).

Samples for murine mRNA isolation may include: resting mouse L cell line (C200) fibroblasts; Braf: ER transfected cells (Braf fusion with estrogen receptor), control (C201); T cells, TH1-differentiated (Mel14 bright, CD4 + cells from spleen, differentiated for 7 days with IFN-γ and anti-IL-4; T200); T cells, TH2 differentiated (Mel14 bright, CD4 + cells from spleen, 7 days differentiated with IL-4 and anti-IFN-γ; T201); T cells, highly TH1 differentiated (see Openshaw, et al. (1995) J. Exp. Med. 182: 1357-1367; activated anti-CD3 for 2, 6, 16 h, pooled; T202); T cells, strongly TH2 differentiated (see Openshaw, et al. (1995) J. Exp. Med. 182: 1357-1367; activated with anti-CD3 for 2, 6, 16 hrs. and combined; T203); CD44-CD25 + pre-T cells, derived from thymus (T204); TH1 T cell line D1.1 resting for 3 weeks after last antigen stimulation (T205); line D1.1 of TH1 T cells, stimulated with 10 µg / ml ConA for 15 h. (T206); CDC35 TH2 T cell line resting for 3 weeks after last antigen stimulation (T207); CDC35 line of TH2 T cells, stimulated with 10 µg / ml ConA for 15 h. (T208); naive spleen Me114 + T cells, resting (T209); Me114 + T cells, differentiated into Th1 with IFN-Y / IL-12 / anti-IL-4 for 6, 12, 24 h, pooled (T210); Me114 + T cells, differentiated into Th2 with IL-4 / anti-IFN-Y for 6, 13, 24 h, pooled (T211); unstimulated mature A20 (B200) leukemia line B cells; unstimulated B cells of the CH12 lineage (B201); unstimulated large B cells from spleen (B202); B cells from total spleen, activated with LPS (B203); spleen dendritic cells enriched with metrizamide, resting (D200); Bone marrow dendritic cells, resting (D201); RAW 264.7 monocyte line activated with LPS for 4 h. (M200); bone marrow macrophages obtained with GM and M-CSF (M201); macrophage line J774, resting (M202); macrophage line J774 + LPS + anti-IL-10 for 0.5, 1, 3, 6, 12 h and connected (M203); macrophage line J774 + LPS + IL-10 for 0, 5, 1, 3, 5, 12 h and connected (M204); mouse lung tissue sprayed, Th2 primers, sprayed with OVA for 7, 14, 23 h, pooled (see Garlisi, et al. (1995) Clinical Immunology and Immunopathology 75: 75-83 X206); lung tissue infected with Nippostrongulus (see Coffman et al. (1989) Science 245: 308-310; X200); adult lung complete, normal (O200); total lung, rag-1 (see Schwarz, et al. (1993) Immunodeficiency 4: 249-252; O205); spleen with IL-10 elimination (see Kuhn, et al. (1991) Cell 75: 263-274; X201); complete adult spleen, normal (O201); total spleen, rag-1 (O207); Peyer's patches with elimination of IL-10 (O202); complete Peyer's patches, normal (O210); mesenteric lymph nodes with elimination of IL-10 (X203); total mesenteric lymph nodes, normal (0211); large intestine with IL-10 (X203) elimination; total colon, normal (0212); pancreas of NOD mice (see Makino et al. (1980) Jikken Dobutsu 29: 1-13; X205); total thymus, rag-1 (O208); complete kidney, rag-1 (O209); complete heart, rag-1 (O202); total brain, rag-1 (O203); total nuclei, rag-1 (O204); total liver34

PL 209 128 B1 ba, rag-1 (O206); normal rat articular tissue (O300); and arthritic rat articular tissue (X300).

Samples for isolating human mRNA may include: peripheral blood mononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells), resting (T100); peripheral blood mononuclear cells, activated with anti-CD3 for 2, 6, 12 hours. combined (T101); T cells, TH0 clone Mot 72, resting (T102); T cells, TH0 clone Mot 72, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h. combined (T103); T cells, TH0 clone Mot 72, anergic treated with a specific peptide for 2, 7, 12 h. combined (T104); T cells, TH1 clone HY06, resting (T107); T cells, TH1 clone HY06, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h. combined (T108); T cells, TH1 clone HY06, anergic treated with a specific peptide for 2, 6, 12 h. combined (T109); T cells, TH2 clone HY935, resting (T110); T cells, TH2 clone HY935, activated with anti-CD28 and anti-CD3 for 2, 7, 12 h. combined (T111); CD4 + CD45RO T cells - T cells differentiated for 27 days in anti-CD28, IL-4, and anti IFN-γ, TH2 differentiated, activated with anti-CD3 and anti-CD28 for 4 h. (T116); Jurkat and Hut78 tumor lineage T cells, resting (T117); T cell clones, linked by AD130.2, Tc783.12, Tc783. 13, Tc783.58, Tc782.69, resting (T118); random T cells γδ T cell clones, resting (T119); splenocytes, resting (B100); splenocytes, activated with anti-CD40 and IL-4 (B101); EBV B cells fused with WT49, RSB, JY, CVIR, 721,221, RM3, HSY, resting (B102); JY B cells, activated with PMA and ionomycin for 1.6 h. combined (B 103); NK 20 clone pooled, resting (K100); NK 20 clone pooled, activated with PMA and ionomycin for 6 h. (K101); NKL clone, obtained from the peripheral blood of an LGL leukemia patient, treated with IL-2 (K106); NK cytotoxic clone 640-A30-1, resting (K107); TF1 blood stem cell precursor line, activated with PMA and ionomycin for 1.6 h. combined (C100); premonocytic line U937, resting (M100); premonocytic line U937, activated with PMA and ionomycin for 1.6 h. combined (M101); purified monocytes, activated with LPS, IFN?, anti-IL-10 for 1, 2, 6, 12, 24 h. combined (M102); purified monocytes, activated with LPS, IFN?, IL-10 for 1, 2, 6, 12, 24 h. combined (M103); purified monocytes, activated with LPS, IFN?, anti-IL-10 for 4, 16 h. combined (M106); purified monocytes, activated with LPS, IFN?, IL-10 for 4, 16 h. combined (M107); purified monocytes, activated with LPS for 1 h. (M108); purified monocytes, activated with LPS for 6 h. (M109); DC 70% CDla +, with CD34 + GM-CSF, TNF? For 12 days, resting (D101); DC 70% CDla +, with CD34 + GM-CSF, TNF? For 12 days, activated with PMA and ionomycin for 1 h. (D102); DC 70% CDla +, with CD34 + GM-CSF, TNF? For 12 days, activated with PMA and ionomycin for 6 h (D103); DC 95% CDla +, with CD34 + GM-CSF, TNF? For 12 days FACS sorted, activated with PMA and ionomycin for 1.6 h. combined (D104); DC 95% CD14 +, excluding CD34 + GM-CSF, TNF? For 12 days FACS sorted, activated with PMA and ionomycin for 1.6 h. combined (D105); DC CDla + CD86 +, with CD34 + GM-CSF, TNF? For 12 days FAC sorted, activated with PMA and ionomycin for 1.6 h. combined (K106); DC from GM-CSF monocytes, IL-4 for 5 days, resting (D107); DC from GM-CSF monocytes, IL-4 for 5 days, resting (D108); DC from GM CSF monocytes, IL-4 for 5 days, activated with LPS for 4, 16 h. combined (D109); DC from GM-CSF monocytes, IL-4 for 5 days, activated with TNF?, Monocyte supernatant for 4.16 h. combined (D110); LI 1 leiomyoma from benign tumor (X101); normal M5 myometrium (O115); malignant leiomyosarcoma GSl (X103); pulmonary fibroblasts of sarcoma line MRC5, activated with PMA and ionomycin for 1.6 h. combined (C101); nerve epithelial tumor CHA cells, activated with PMA and ionomycin for 1.6 h. combined (C102); male fetus 28 weeks (O100); male fetal lung 28 weeks (O101); male fetal liver 28 weeks (O102); heart of a male fetus 28 weeks (O103); male fetal brain 28 weeks (O104); male fetus gallbladder 28 weeks (O106); small intestine of male fetus 28 weeks (O107); male fetal adipose tissue 28 weeks (O108); fetal ovary at 25 weeks (O109); female fetus uterus 25 weeks (O110); testicles of male fetus 28 weeks (O111); male fetus spleen 28 weeks (O112); adult placenta at 28 weeks pregnancy (0113); and tonsils, inflamed from the age of 12 (X100).

Similar samples may be taken from other species for evaluation.

V. Cloning of species equivalents of DCRS5

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Various approaches are used to obtain species-specific equivalents of DCRS5, preferably from other primates or rodents. One method is cross-hybridization using DNA probes from closely related species. It may be useful to use evolutionarily similar species as intermediate steps. Another method is to use specific PCR primers based on the identification of blocks of similarity or difference between genes, e.g. regions of a highly conserved or unpreserved polypeptide or nucleotide sequence.

By searching databases, similar sequences can be identified, which will allow the generation of suitable probes.

VI. Production of the mammalian DCRS5 protein

A suitable, e.g., GST, fusion construct is produced for expression, e.g., in E. coli. For example, the murine IGIF pGex plasmid is constructed and transformed into E. coli. Freshly transformed cells are cultured, e.g., in LB medium containing 50 µg / ml ampicillin and induced with IPTG (Sigma, St. Louis, MO). After induction overnight, the bacteria are harvested and the DCRS5 protein-containing pellets are separated. These sediments are homogenized, e.g. in TE buffer (50 mM Tris-base pH 8.0, 10 mM EDTA and 2 mM pefabloc) in a volume of 2 liters. This material is passed through a microfluidizer (Microfluidics, Newton, MA) three times. The liquefied supernatant is centrifuged in a Sorvall GS-3 rotor for 1 hour. at 13,000 rpm. The supernatant thus obtained is passed over a glutathione-SEPHAROSE column equilibrated with 50 mM basic Tris pH 8.0. Fractions containing the DCRS5-GST fusion protein are pooled and digested, e.g., with thrombin (Enzyme Research Laboratories, Inc., South Bend, IN). The digested material is then passed through a QSEPHAROSE column equilibrated in 50 mM basic Tris. Fractions containing DCRS5 are pooled and diluted with cold distilled H 2 O to lower the conductivity and passed only through or through a fresh Q-Sepharose column followed by an antibody immunoaffinity column. The fractions containing DCRS5 are pooled, aliquoted and stored in a freezer at -70 ° C.

A comparison of the CD spectrum with a cytokine receptor protein may indicate that the protein is correctly folded. See Hazuda et al. (1969) J. Biol. Chem. 264: 1689-1693.

VII. Preparation of DCRS5 specific antibodies

Inbred Balb / c mice are immunized intraperitoneally with recombinant forms of protein, e.g. with purified DCRS5 or continuously transfected with NIH-3T3 cells. Animals are immunized with protein at appropriate intervals, with or without additional adjuvant, to further stimulate antibody production. Plasma is obtained or hybridoma cells are produced from the spleen obtained.

Alternatively, Balb / c mice are immunized with cells transformed with the gene or fragments thereof, both endogenous and exogenous cells, or with isolated membranes enriched for antigen expression. Plasma is collected in a timely manner, typically after multiple subsequent administrations. Various gene therapy techniques may be useful, e.g., for generating proteins in situ to elicit an immune response. The plasma or antibody preparations can be cross-absorbed or immunoselected to prepare substantially pure antibodies with defined specificity and high affinity.

Monoclonal antibodies can be prepared. For example, splenocytes are combined with an appropriate fusion partner and hybridoma cells are selected in the culture medium by standard methods. Hybridoma supernatants are screened for the presence of DCRS5 binding antibodies, e.g., by ELISA or other assays. Antibodies that specifically recognize DCRS5 can also be selected or prepared.

According to another method, synthetic peptides or purified proteins are presented to the immune system to produce monoclonal or polyclonal antibodies. See, e.g., Coligan (ed. 1991) Current Protocols in Immunology Wiley / Greene; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press. Where appropriate, the binding reagent is labeled as described above, e.g. by fluorescence or other methods, or combined with a panning medium. Nucleic acids can also be introduced into cells in an animal to produce an antigen that serves to elicit an immune response. See, e.g., Wang et al. (1993) Proc. Natl. Acad. Sci. 90: 4156-4160; Barry et al. (1994) BioTechniques 16: 616-619 and Xiang et al. (1995) Immunity 2: 129-135.

PL 209 128 B1

VIII. Preparation of fusion proteins with DCRS5

Various fusion constructs are made with DCRS5, including the junctions of DCRS5 to the IL-12Re1 sequence. Part of the appropriate gene is attached to an epitope tag, e.g., a FLAG tag or to a two-hybrid system construct. See, e.g., Fields and Song (1989) Nature 340: 245-246.

The epitope tag can be used in an expression cloning procedure with detection with anti-FLAG antibodies to detect a binding partner, e.g. a ligand of an appropriate cytokine receptor. The two-hybrid system can also be used to isolate proteins that specifically bind DCRS5.

IX. Structure - function relationship

The significance information of individual residues is determined using standard procedures and analyzes. Standard mutagenesis analysis is performed, e.g., by generating a plurality of different variants at specific positions, e.g., at the positions identified above, and evaluating the biological activity of the variants. This can be done in terms of finding positions that alter activity, or by selecting specific positions to determine residues that can be substituted to either preserve, inhibit, or alter biological activity.

Alternatively, analysis of natural variants can reveal at which positions natural mutations are tolerated. This can be achieved by population analysis of variability between individuals or between strains or species. Samples from selected individuals are analyzed, e.g., by PCR analysis and sequencing. This allows the assessment of population polymorphisms.

X. Simultaneous expression of DCRS5 and IL-12Re1

The vector or vectors encoding the appropriate genes can be transfected into the cell. Preferably, the vector will contain selectable markers that enable identification of cells that have been successfully transformed. The joint expression of the two genes will enable the products of these genes to interact properly and generate active receptor complexes.

Alternatively, the use of methods for associating functional dimers is available. See, e.g., O'Shea et al. (1989) Science 245: 646-648; Kostelny et al. (1992) J. Immunol. 148: 1547-1553; and Patel et al. (1996) J. Biol. Chem. 271: 30386-30391. Expression of extracellular domains and physical interaction, e.g. caused by the affinity of the Fos / Jun leucine zipper, will yield ligand binding constructs that should act as binding components in diagnostic or therapeutic applications.

Many modifications and variations of the present invention can be made without departing from its spirit and purpose, as will be apparent to those skilled in the art. Specific embodiments described herein are merely illustrative and the invention is defined by the scope of the appended claims along with the full range of equivalents to which those claims also apply. The invention cannot be limited only by the specific embodiments presented herein by way of example.

PL 209 128 B1

Sequence list &lt;110&gt; Schering Corporation &lt;120&gt; Mammalian Receptor Proteins; related reagents and methods &lt;130&gt; DX01074 &lt;150&gt; US 60 / 203,426 &lt;151&gt; 2000-05-10 &lt;160&gt; 5 &lt;170&gt; PatentIn version 2.0 &lt;210&gt; 1 &lt;211&gt; 2859 &lt;212&gt; DNA &lt;213&gt; Unknown &lt; 220&gt;

&lt;223&gt; Description of unknown organism: primate; presumably homo sapiens &lt;220&gt;

&lt;221&gt; CDS &lt;222&gt; (119). . (2005) &lt;220&gt;

&lt;221&gt; mat_peptyd &lt;222&gt; (188) .. (2005) &lt;220&gt;

&lt;221&gt; any_trait &lt;222&gt; (1) .. (2859) &lt;223&gt; Xaa translations depend on the genetic code &lt;400&gt; 1 gtggtacggg aattccattg tgttgggcag ccaacaaggg tggcagcctg gctctgaagt 60 ggaattatgt gttcgaagcaga ggttacgactgaa ggttaaccagagaa ggttaaccagagaa ggttaaccaagtaca

<td>atg Underworld</td><td>aat Asn</td><td>yes Xaa</td><td>gtc Val -twenty</td><td>act Thr</td><td>att How much</td><td>all Gin</td><td>tgg Trp</td><td>gen Asp -15</td><td>gca Ala</td><td>gta Val</td><td>ata How much</td><td>gee Ala</td><td>ctt Leu -10</td><td>trays Tyr</td><td>ata How much</td><td> 166</td>

<td>ctc</td><td>ttc</td><td>age</td><td>tgg</td><td>tgt</td><td>cat</td><td>gga</td><td>gga</td><td>att</td><td>aca</td><td>aat</td><td>ata</td><td>aac</td><td>tgc</td><td>tet</td><td>ggc</td><td> 214</td>

<td>Leu</td><td>Phe</td><td>Cheese</td><td>Trp</td><td>Cys</td><td>His</td><td>Gly</td><td>Gly</td><td>How much</td><td>Thr</td><td>Asn</td><td>How much</td><td>Asn</td><td>Cys</td><td>Cheese</td><td>Gly</td><td></td>

<td></td><td></td><td> -5</td><td></td><td></td><td></td><td> -1</td><td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td></td>

<td>cac</td><td>atc</td><td>tgg</td><td>gta</td><td>gaa</td><td>cca</td><td>gee</td><td>aca</td><td>att</td><td>ttt</td><td>aag</td><td>atg</td><td>ggt</td><td>atg</td><td>aat</td><td>atc</td><td> 262</td>

<td>His</td><td>How much</td><td>Trp</td><td>Val</td><td>Glu</td><td>Pro</td><td>Ala</td><td>Thr</td><td>How much</td><td>Phe</td><td>Lys</td><td>Underworld</td><td>Gly</td><td>Underworld</td><td>Asn</td><td>How much</td><td></td>

<td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td>

<td>tet</td><td>ata</td><td>tat</td><td>tgc</td><td>all</td><td>gca</td><td>gca</td><td>att</td><td>aag</td><td>aac</td><td>tgc</td><td>all</td><td>cca</td><td>agg</td><td>aaa</td><td>ctt</td><td> 310</td>

<td>Cheese</td><td>How much</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Ala</td><td>Ala</td><td>How much</td><td>Lys</td><td>Asn</td><td>Cys</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Lys</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td>

<td>cat</td><td>ttt</td><td>tat</td><td>aaa</td><td>aat</td><td>ggc</td><td>atc</td><td>aaa</td><td>gaa</td><td>aga</td><td>ttt</td><td>all</td><td>atc</td><td>aca</td><td>agg</td><td>att</td><td> 358</td>

<td>His</td><td>Phe</td><td>Tyr</td><td>Lys</td><td>Asn</td><td>Gly</td><td>How much</td><td>Lys</td><td>Glu</td><td>Arg</td><td>Phe</td><td>Gin</td><td>How much</td><td>Thr</td><td>Arg</td><td>How much</td><td></td>

<td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td>

PL 209 128 B1

<td>aat Asn</td><td>aaa Lys</td><td>aca Thr 60</td><td>aca Thr</td><td>gct Ala</td><td>cgg Arg</td><td>ctt Leu</td><td>tgg Trp 65</td><td>tat Tyr</td><td>aaa Lys</td><td>aac Asn</td><td>ttt Phe</td><td>ctg Leu 70</td><td>gaa Glu</td><td>cca Pro</td><td>cat His</td><td> 406</td>

<td>gct</td><td>tct</td><td>atg</td><td>trays</td><td>tgc</td><td>act</td><td>gct</td><td>gaa</td><td>tgt</td><td>ccc</td><td>aaa</td><td>cat</td><td>ttt</td><td>all</td><td>gag</td><td>aca</td><td> 454</td>

<td>Ala</td><td>Cheese 75</td><td>Underworld</td><td>Tyr</td><td>Cys</td><td>Thr</td><td>Ala 80</td><td>Glu</td><td>Cys</td><td>Pro</td><td>Lys</td><td>His 85</td><td>Phe</td><td>Gin</td><td>Glu</td><td>Thr</td><td></td>

<td>ctg</td><td>ata</td><td>tgt</td><td>gga</td><td>aaa</td><td>jacket</td><td>att</td><td>tct</td><td>tct</td><td>gga</td><td>tat</td><td>ccg</td><td>cca</td><td>gen</td><td>att</td><td>cct</td><td> 502</td>

<td>Leu 90</td><td>How much</td><td>Cys</td><td>Gly</td><td>Lys</td><td>Asp 95</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Gly</td><td>Tyr 100</td><td>Pro</td><td>Pro</td><td>Asp</td><td>How much</td><td>Pro 105</td><td></td>

<td>gen</td><td>gaa</td><td>gta</td><td>acc</td><td>tgt</td><td>gtc</td><td>att</td><td>tat</td><td>gaa</td><td>tat</td><td>tca</td><td>ggc</td><td>aac</td><td>atg</td><td>act</td><td>tgc</td><td> 550</td>

<td>Asp</td><td>Glu</td><td>Val</td><td>Thr</td><td>Cys 110</td><td>Val</td><td>How much</td><td>Tyr</td><td>Glu</td><td>Tyr 115</td><td>Cheese</td><td>Gly</td><td>Asn</td><td>Underworld</td><td>Thr 120</td><td>Cys</td><td></td>

<td>acc</td><td>tgg</td><td>aat</td><td>gct</td><td>rgg</td><td>aag</td><td>ctc</td><td>acc</td><td>trays</td><td>ata</td><td>jacket</td><td>aca</td><td>aaa</td><td>trays</td><td>gtg</td><td>gta</td><td> 598</td>

<td>Thr</td><td>Trp</td><td>Asn</td><td>Ala 125</td><td>Xaa</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Tyr 130</td><td>How much</td><td>Asp</td><td>Thr</td><td>Lys</td><td>Tyr 135</td><td>Val</td><td>Val</td><td></td>

<td>cat</td><td>gtg</td><td>aag</td><td>agt</td><td>tta</td><td>gag</td><td>aca</td><td>gaa</td><td>gaa</td><td>gag</td><td>all</td><td>cag</td><td>tat</td><td>ctc</td><td>acc</td><td>tca</td><td> 646</td>

<td>His</td><td>Val</td><td>Lys 140</td><td>Cheese</td><td>Leu</td><td>Glu</td><td>Thr</td><td>Glu 145</td><td>Glu</td><td>Glu</td><td>Gin</td><td>Gin</td><td>Tyr 150</td><td>Leu</td><td>Thr</td><td>Cheese</td><td></td>

<td>agc</td><td>tat</td><td>att</td><td>aac</td><td>atc</td><td>tcc</td><td>act</td><td>gen</td><td>tca</td><td>tta</td><td>all</td><td>ggt</td><td>ggc</td><td>aag</td><td>aag</td><td>trays</td><td> 694</td>

<td>Cheese</td><td>Tyr 155</td><td>How much</td><td>Asn</td><td>How much</td><td>Cheese</td><td>Thr 160</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Gly 165</td><td>Gly</td><td>Lys</td><td>Lys</td><td>Tyr</td><td></td>

<td>ttg</td><td>gtt</td><td>tgg</td><td>gtc</td><td>all</td><td>gca</td><td>gca</td><td>aac</td><td>gca</td><td>eta</td><td>ggc</td><td>atg</td><td>gaa</td><td>gag</td><td>tca</td><td>aaa</td><td> 742</td>

<td>Leu 170</td><td>Val</td><td>Trp</td><td>Val</td><td>Gin</td><td>Ala 175</td><td>Ala</td><td>Asn</td><td>Ala</td><td>Leu</td><td>Gly 180</td><td>Underworld</td><td>Glu</td><td>Glu</td><td>Cheese</td><td>Lys 185</td><td></td>

<td>all</td><td>ctg</td><td>all</td><td>att</td><td>cac</td><td>ctg</td><td>gen</td><td>gen</td><td>ata</td><td>gtg</td><td>ata</td><td>cct</td><td>tct</td><td>gca</td><td>gcc</td><td>gtc</td><td> 790</td>

<td>Gin</td><td>Leu</td><td>Gin</td><td>How much</td><td>His 190</td><td>Leu</td><td>Asp</td><td>Asp</td><td>How much</td><td>Val 195</td><td>How much</td><td>Pro</td><td>Cheese</td><td>Ala</td><td>Ala 200</td><td>Val</td><td></td>

<td>att</td><td>tcc</td><td>agg</td><td>gct</td><td>gag</td><td>act</td><td>ata</td><td>aat</td><td>gct</td><td>aca</td><td>gtg</td><td>ccc</td><td>aag</td><td>acc</td><td>ata</td><td>att</td><td> 838</td>

<td>How much</td><td>Cheese</td><td>Arg</td><td>Ala 205</td><td>Glu</td><td>Thr</td><td>How much</td><td>Asn</td><td>Ala 210</td><td>Thr</td><td>Val</td><td>Pro</td><td>Lys</td><td>Thr 215</td><td>How much</td><td>How much</td><td></td>

<td>tat</td><td>tgg</td><td>gen</td><td>agt</td><td>all</td><td>aca</td><td>aca</td><td>att</td><td>gaa</td><td>aag</td><td>gtt</td><td>tcc</td><td>tgt</td><td>gaa</td><td>atg</td><td>aga</td><td> 886</td>

<td>Tyr</td><td>Trp</td><td>Asp 220</td><td>Cheese</td><td>Gin</td><td>Thr</td><td>Thr</td><td>How much 225</td><td>Glu</td><td>Lys</td><td>Val</td><td>Cheese</td><td>Cys 230</td><td>Glu</td><td>Underworld</td><td>Arg</td><td></td>

<td>trays</td><td>aag</td><td>gct</td><td>aca</td><td>aca</td><td>aac</td><td>all</td><td>act</td><td>tgg</td><td>aat</td><td>gtt</td><td>aaa</td><td>gaa</td><td>ttt</td><td>jacket</td><td>acc</td><td> 934</td>

<td>Tyr</td><td>Lys 235</td><td>Ala</td><td>Thr</td><td>Thr</td><td>Asn</td><td>Gin 240</td><td>Thr</td><td>Trp</td><td>Asn</td><td>Val</td><td>Lys 245</td><td>Glu</td><td>Phe</td><td>Asp</td><td>Thr</td><td></td>

<td>aat</td><td>ttt</td><td>aca</td><td>tat</td><td>gtg</td><td>all</td><td>cag</td><td>tca</td><td>gaa</td><td>ttc</td><td>trays</td><td>ttg</td><td>gag</td><td>cca</td><td>aac</td><td>att</td><td> 982</td>

<td>Asn 250</td><td>Phe</td><td>Thr</td><td>Tyr</td><td>Val</td><td>Gin 255</td><td>Gin</td><td>Cheese</td><td>Glu</td><td>Phe</td><td>Tyr 260</td><td>Leu</td><td>Glu</td><td>Pro</td><td>Asn</td><td>How much 265</td><td></td>

<td>aag</td><td>trays</td><td>gta</td><td>ttt</td><td>all</td><td>gtg</td><td>aga</td><td>tgt</td><td>all</td><td>gaa</td><td>aca</td><td>ggc</td><td>aaa</td><td>agg</td><td>trays</td><td>tgg</td><td> 1030</td>

<td>Lys</td><td>Tyr</td><td>Val</td><td>Phe</td><td>Gin 270</td><td>Val</td><td>Arg</td><td>Cys</td><td>Gin</td><td>Glu 275</td><td>Thr</td><td>Gly</td><td>Lys</td><td>Arg</td><td>Tyr 280</td><td>Trp</td><td></td>

<td>cag</td><td>cct</td><td>tgg</td><td>agt</td><td>tca</td><td>ccg</td><td>ttt</td><td>ttt</td><td>cat</td><td>aaa</td><td>aca</td><td>cct</td><td>gaa</td><td>aca</td><td>gtt</td><td>ccc</td><td> 1078</td>

<td>Gin</td><td>Pro</td><td>Trp</td><td>Cheese 285</td><td>Cheese</td><td>Pro</td><td>Phe</td><td>Phe</td><td>His 290</td><td>Lys</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Thr 295</td><td>Val</td><td>Pro</td><td></td>

<td>cag</td><td>gtc</td><td>aca</td><td>tca</td><td>aaa</td><td>gca</td><td>ttc</td><td>all</td><td>cat</td><td>jacket</td><td>aca</td><td>tgg</td><td>aat</td><td>tct</td><td>hhg</td><td>eta</td><td> 1126</td>

PL 209 128 B1

<td>Gin</td><td>Val</td><td>Thr 300</td><td>Cheese</td><td>Lys</td><td>Ala</td><td>Phe</td><td>Gin 305</td><td>His</td><td>Asp</td><td>Thr</td><td>Trp</td><td>Asn 310</td><td>Cheese</td><td>Gly</td><td colspan="2">Leu</td>

<td>aca</td><td>gtt</td><td>gct</td><td>tcc</td><td>atc</td><td>tet</td><td>aca</td><td>hhg</td><td>cac</td><td>ctt</td><td>act</td><td>tet</td><td>jacket</td><td>aac</td><td>aga</td><td>gga</td><td> 1174</td>

<td>Thr</td><td>Val 315</td><td>Ala</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Thr 320</td><td>Gly</td><td>His</td><td>Leu</td><td>Thr</td><td>Cheese 325</td><td>Asp</td><td>Asn</td><td>Arg</td><td>Gly</td><td></td>

<td>jacket</td><td>att</td><td>gga</td><td>ctt</td><td>tta</td><td>ttg</td><td>gga</td><td>atg</td><td>atc</td><td>gtc</td><td>ttt</td><td>gct</td><td>gtt</td><td>atg</td><td>ttg</td><td>tca</td><td> 1222</td>

<td>Asp 330</td><td>How much</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Leu 335</td><td>Gly</td><td>Underworld</td><td>How much</td><td>Val</td><td>Phe 340</td><td>Ala</td><td>Val</td><td>Underworld</td><td>Leu</td><td>Cheese 345</td><td></td>

<td>att</td><td>ctt</td><td>tet</td><td>ttg</td><td>att</td><td>hhg</td><td>ata</td><td>ttt</td><td>aac</td><td>aga</td><td>tca</td><td>ttc</td><td>ega</td><td>act</td><td>hhg</td><td>att</td><td> 1270</td>

<td>How much</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>How much 350</td><td>Gly</td><td>How much</td><td>Phe</td><td>Asn</td><td>Arg 355</td><td>Cheese</td><td>Phe</td><td>Arg</td><td>Thr</td><td>Gly 360</td><td>How much</td><td></td>

<td>aaa</td><td>aga</td><td>agg</td><td>atc</td><td>tta</td><td>ttg</td><td>tta</td><td>ata</td><td>cca</td><td>aag</td><td>tgg</td><td>ctt</td><td>tat</td><td>gaa</td><td>gen</td><td>att</td><td> 1318</td>

<td>Lys</td><td>Arg</td><td>Arg</td><td>How much 365</td><td>Leu</td><td>Leu</td><td>Leu</td><td>How much</td><td>Pro 370</td><td>Lys</td><td>Trp</td><td>Leu</td><td>Tyr</td><td>Glu 375</td><td>Asp</td><td>How much</td><td></td>

<td>cct</td><td>aat</td><td>atg</td><td>aaa</td><td>aac</td><td>agc</td><td>aat</td><td>gtt</td><td>gtg</td><td>aaa</td><td>atg</td><td>eta</td><td>cag</td><td>gaa</td><td>aat</td><td>agt</td><td> 1366</td>

<td>Pro</td><td>Asn</td><td>Underworld 380</td><td>Lys</td><td>Asn</td><td>Cheese</td><td>Asn</td><td>Val 385</td><td>Val</td><td>Lys</td><td>Underworld</td><td>Leu</td><td>Gin 390</td><td>Glu</td><td>Asn</td><td>Cheese</td><td></td>

<td>gaa</td><td>ctt</td><td>atg</td><td>aat</td><td>aat</td><td>aat</td><td>tcc</td><td>agt</td><td>gag</td><td>cag</td><td>gtc</td><td>eta</td><td>tat</td><td>gtt</td><td>gen</td><td>ccc</td><td> 1414</td>

<td>Glu</td><td>Leu 395</td><td>Underworld</td><td>Asn</td><td>Asn</td><td>Asn</td><td>Cheese 400</td><td>Cheese</td><td>Glu</td><td>Gin</td><td>Val</td><td>Leu 405</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Pro</td><td></td>

<td>atg</td><td>att</td><td>aca</td><td>gag</td><td>ata</td><td>aaa</td><td>gaa</td><td>atc</td><td>ttc</td><td>atc</td><td>cca</td><td>gaa</td><td>cac</td><td>aag</td><td>cct</td><td>aca</td><td> 1462</td>

<td>Underworld 410</td><td>How much</td><td>Thr</td><td>Glu</td><td>How much</td><td>Lys 415</td><td>Glu</td><td>How much</td><td>Phe</td><td>How much</td><td>Pro 420</td><td>Glu</td><td>His</td><td>Lys</td><td>Pro</td><td>Thr 425</td><td></td>

<td>jacket</td><td>trays</td><td>aag</td><td>aag</td><td>gag</td><td>aat</td><td>aca</td><td>gga</td><td>ccc</td><td>ctg</td><td>gag</td><td>aca</td><td>aga</td><td>jacket</td><td>trays</td><td>ccg</td><td> 1510</td>

<td>Asp</td><td>Tyr</td><td>Lys</td><td>Lys</td><td>Glu 430</td><td>Asn</td><td>Thr</td><td>Gly</td><td>Pro</td><td>Leu 435</td><td>Glu</td><td>Thr</td><td>Arg</td><td>Asp</td><td>Tyr 440</td><td>Pro</td><td></td>

<td>all</td><td>aac</td><td>tcg</td><td>eta</td><td>ttc</td><td>jacket</td><td>aat</td><td>act</td><td>aca</td><td>gtt</td><td>gta</td><td>tat</td><td>att</td><td>cct</td><td>gen</td><td>ctc</td><td> 1558</td>

<td>Gin</td><td>Asn</td><td>Cheese</td><td>Leu 445</td><td>Phe</td><td>Asp</td><td>Asn</td><td>Thr</td><td>Thr 450</td><td>Val</td><td>Val</td><td>Tyr</td><td>How much</td><td>Pro 455</td><td>Asp</td><td>Leu</td><td></td>

<td>aac</td><td>act</td><td>gga</td><td>tat</td><td>aaa</td><td>ccc</td><td>all</td><td>att</td><td>tca</td><td>aat</td><td>ttt</td><td>ctg</td><td>cct</td><td>gag</td><td>gga</td><td>agc</td><td> 1606</td>

<td>Asn</td><td>Thr</td><td>Gly 460</td><td>Tyr</td><td>Lys</td><td>Pro</td><td>Gin</td><td>How much 465</td><td>Cheese</td><td>Asn</td><td>Phe</td><td>Leu</td><td>Pro 470</td><td>Glu</td><td>Gly</td><td>Cheese</td><td></td>

<td>cat</td><td>ctc</td><td>agc</td><td>aat</td><td>aat</td><td>aat</td><td>gaa</td><td>att</td><td>act</td><td>tcc</td><td>tta</td><td>aca</td><td>ctt</td><td>aaa</td><td>cca</td><td>cca</td><td> 1654</td>

<td>His</td><td>Leu 475</td><td>Cheese</td><td>Asn</td><td>Asn</td><td>Asn</td><td>Glu 480</td><td>How much</td><td>Thr</td><td>Cheese</td><td>Leu</td><td>Thr 485</td><td>Leu</td><td>Lys</td><td>Pro</td><td>Pro</td><td></td>

<td>gtt</td><td>gen</td><td>tcc</td><td>tta</td><td>jacket</td><td>tca</td><td>gga</td><td>aat</td><td>aat</td><td>ccc</td><td>agg</td><td>tta</td><td>all</td><td>aag</td><td>cat</td><td>cct</td><td> 1702</td>

<td>Val 490</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>Asp</td><td>Cheese 495</td><td>Gly</td><td>Asn</td><td>Asn</td><td>Pro</td><td>Arg 500</td><td>Leu</td><td>Gin</td><td>Lys</td><td>His</td><td>Pro 505</td><td></td>

<td>aat</td><td>ttt</td><td>gct</td><td>ttt</td><td>tet</td><td>gtt</td><td>tca</td><td>agt</td><td>gtg</td><td>aat</td><td>tca</td><td>eta</td><td>agc</td><td>aac</td><td>aca</td><td>ata</td><td> 1750</td>

<td>Asn</td><td>Phe</td><td>Ala</td><td>Phe</td><td>Cheese 510</td><td>Val</td><td>Cheese</td><td>Cheese</td><td>Val</td><td>Asn 515</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Asn</td><td>Thr 520</td><td>How much</td><td></td>

<td>ttt</td><td>ctt</td><td>gga</td><td>gaa</td><td>tta</td><td>agc</td><td>ctc</td><td>ata</td><td>tta</td><td>aat</td><td>all</td><td>gga</td><td>gaa</td><td>tgc</td><td>agt</td><td>tet</td><td> 1798</td>

<td>Phe</td><td>Leu</td><td>Gly</td><td>Glu 525</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>How much</td><td>Leu 530</td><td>Asn</td><td>Gin</td><td>Gly</td><td>Glu</td><td>Cys 535</td><td>Cheese</td><td>Cheese</td><td></td>

<td>cct</td><td>jacket</td><td>ata</td><td>all</td><td>aac</td><td>tca</td><td>gta</td><td>gag</td><td>gag</td><td>gaa</td><td>acc</td><td>acc</td><td>atg</td><td>ctt</td><td>ttg</td><td>gaa</td><td> 1846</td>

PL 209 128 B1

<td colspan="2">Pro Asp</td><td>How much 540</td><td colspan="2">Gin Asn</td><td>Cheese</td><td>Val</td><td colspan="2">Glu Glu 545</td><td colspan="3">Glu Thr Thr</td><td>Underworld 550</td><td>Leu</td><td>Leu</td><td colspan="2">Glu</td>

<td>aat</td><td>gen</td><td>tea</td><td>ccc</td><td>agt</td><td>gaa</td><td>act</td><td>att</td><td>cca</td><td>gaa</td><td>cag</td><td>acc</td><td>ctg</td><td>ctt</td><td>cct</td><td>gen</td><td> 1894</td>

<td>Asn</td><td>Asp</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Glu</td><td>Thr</td><td>How much</td><td>Pro</td><td>Glu</td><td>Gin</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Pro</td><td rowspan="2">Asp</td><td></td>

<td></td><td> 555</td><td></td><td></td><td></td><td></td><td> 560</td><td></td><td></td><td></td><td></td><td> 565</td><td></td><td></td><td></td><td></td>

<td>gaa</td><td>ttt</td><td>gtc</td><td>tcc</td><td>tgt</td><td>ttg</td><td>hhg</td><td>atc</td><td>gtg</td><td>aat</td><td>gag</td><td>gag</td><td>ttg</td><td>cca</td><td>tet</td><td>att</td><td> 1942</td>

<td>Glu</td><td>Phe</td><td>Val</td><td>Cheese</td><td>Cys</td><td>Leu</td><td>Gly</td><td>How much</td><td>Val</td><td>Asn</td><td>Glu</td><td>Glu</td><td>Leu</td><td>Pro</td><td>Cheese</td><td>How much</td><td></td>

<td> 570</td><td></td><td></td><td></td><td></td><td> 575</td><td></td><td></td><td></td><td></td><td> 580</td><td></td><td></td><td></td><td></td><td> 585</td><td></td>

<td>aat</td><td>act</td><td>tat</td><td>ttt</td><td>cca</td><td>all</td><td>aat</td><td>att</td><td>ttg</td><td>gaa</td><td>age</td><td>cac</td><td>ttc</td><td>aat</td><td>agg</td><td>att</td><td> 1990</td>

<td>Asn</td><td>Thr</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Gin</td><td>Asn</td><td>How much</td><td>Leu</td><td>Glu</td><td>Cheese</td><td>His</td><td>Phe</td><td>Asn</td><td>Arg</td><td>How much</td><td></td>

<td></td><td></td><td></td><td></td><td> 590</td><td></td><td></td><td></td><td></td><td> 595</td><td></td><td></td><td></td><td></td><td> 600</td><td></td><td></td>

<td>tea</td><td>ctc</td><td>ttg</td><td>gaa</td><td>aag</td><td colspan="8">tagagctgtg tggtcaaaat caatatgaga</td><td colspan="3">aagctgcctt</td><td> 2045</td>

<td>Cheese</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 605</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>gcaatctgaa</td><td>cttgggtttt</td><td>ccctgcaata</td><td>gaaattgaat</td><td>tctgcctctt</td><td>tttgaaaaaa</td><td> 2105</td>

<td>atgtattcac</td><td>atacaaatct</td><td>tcacatggac</td><td>acatgttttc</td><td>atttcccttg</td><td>gataaatacc</td><td> 2165</td>

<td>taggtagggg</td><td>attgctgggc</td><td>catatgataa</td><td>gcatatgttt</td><td>cagttctacc</td><td>aatcttgttt</td><td> 2225</td>

<td>ccagagtagt</td><td>gacatttctg</td><td>tgctcctacc</td><td>atcaccatgt</td><td>aagaattccc</td><td>gggagctcca</td><td> 2285</td>

<td>tgccttttta</td><td>attttagcca</td><td>ttcttctgcc</td><td>tmatttctta</td><td>aaattagaga</td><td>attaaggtcc</td><td> 2345</td>

<td>cgaaggtgga</td><td>acatgcttca</td><td>tggtcacaca</td><td>tacaggcaca</td><td>aaaacagcat</td><td>tatgtggacg</td><td> 2405</td>

<td>cctcatgtat</td><td>tttttataga</td><td>gtcaactatt</td><td>tcctctttat</td><td>tttccctcat</td><td>tgaaagatgc</td><td> 2465</td>

<td>aaaacagctc</td><td>tctattgtgt</td><td>acagaaaggg</td><td>taaataatgc</td><td>aaaatacctg</td><td>gtagtaaaat</td><td> 2525</td>

<td>aaatgctgaa</td><td>aattttcctt</td><td>taaaatagaa</td><td>teattaggee</td><td>aggcgtggtg</td><td>geteatgett</td><td> 2585</td>

<td>gtaatcccag</td><td>cactttggta</td><td>ggctgaggtr</td><td>ggtggatcac</td><td>ctgaggtcag</td><td>gagttegagt</td><td> 2645</td>

<td>ccagcctggc</td><td>caatatgctg</td><td>aaaccctgtc</td><td>tctactaaaa</td><td>ttacaaaaat</td><td>tagccggcca</td><td> 2705</td>

<td>tggtggcagg</td><td>tgcttgtaat</td><td>cccagctact</td><td>tgggaggctg</td><td>aggcaggaga</td><td>atcacttgaa</td><td> 2765</td>

<td>ccaggaaggc</td><td>agaggttgca</td><td>ctgagctgag</td><td>attgtgccac</td><td>tgcactccag</td><td>cctgggcaac</td><td> 2825</td>

aagagcaaaa ctctgtctgg aaaaaaaaaa aaaa 2859 &lt;210&gt; 2 &lt;211&gt; 629 &lt;212&gt; PRT &lt;213&gt; Unknown &lt;400&gt; 2

<td>Underworld</td><td>Asn</td><td>Xaa</td><td>Val -twenty</td><td>Thr</td><td>How much</td><td>Gin</td><td>Trp</td><td>Asp -15</td><td>Ala</td><td>Val</td><td>How much</td><td>Ala</td><td>Leu -10</td><td>Tyr</td><td>How much</td>

<td>Leu</td><td>Phe</td><td>Cheese -5</td><td>Trp</td><td>Cys</td><td>His</td><td>Gly -1</td><td>Gly 1</td><td>How much</td><td>Thr</td><td>Asn</td><td>How much 5</td><td>Asn</td><td>Cys</td><td>Cheese</td><td>Gly</td>

<td>His</td><td>How much</td><td>Trp</td><td>Val</td><td>Glu</td><td>Pro</td><td>Ala</td><td>Thr</td><td>How much</td><td>Phe</td><td>Lys</td><td>Underworld</td><td>Gly</td><td>Underworld</td><td>Asn</td><td>How much</td>

PL 209 128 B1

<td colspan="4"> 10</td><td colspan="5"> 15</td><td colspan="6"> 20</td><td> 25</td>

<td>Cheese</td><td>How much</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Ala</td><td>Ala</td><td>How much</td><td>Lys</td><td>Asn</td><td>Cys</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Lys</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td>

<td>His</td><td>Phe</td><td>Tyr</td><td>Lys</td><td>Asn</td><td>Gly</td><td>How much</td><td>Lys</td><td>Glu</td><td>Arg</td><td>Phe</td><td>Gin</td><td>How much</td><td>Thr</td><td>Arg</td><td>How much</td>

<td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td>

<td>Asn</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Ala</td><td>Arg</td><td>Leu</td><td>Trp</td><td>Tyr</td><td>Lys</td><td>Asn</td><td>Phe</td><td>Leu</td><td>Glu</td><td>Pro</td><td>His</td>

<td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td>

<td>Ala</td><td>Cheese</td><td>Underworld</td><td>Tyr</td><td>Cys</td><td>Thr</td><td>Ala</td><td>Glu</td><td>Cys</td><td>Pro</td><td>Lys</td><td>His</td><td>Phe</td><td>Gin</td><td>Glu</td><td>Thr</td>

<td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>How much</td><td>Cys</td><td>Gly</td><td>Lys</td><td>Asp</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Gly</td><td>Tyr</td><td>Pro</td><td>Pro</td><td>Asp</td><td>How much</td><td>Pro</td>

<td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td>

<td>Asp</td><td>Glu</td><td>Val</td><td>Thr</td><td>Cys</td><td>Val</td><td>How much</td><td>Tyr</td><td>Glu</td><td>Tyr</td><td>Cheese</td><td>Gly</td><td>Asn</td><td>Underworld</td><td>Thr</td><td>Cys</td>

<td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td>

<td>Thr</td><td>Trp</td><td>Asn</td><td>Ala</td><td>Xaa</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Tyr</td><td>How much</td><td>Asp</td><td>Thr</td><td>Lys</td><td>Tyr</td><td>Val</td><td>Val</td>

<td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td>

<td>His</td><td>Val</td><td>Lys</td><td>Cheese</td><td>Leu</td><td>Glu</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Glu</td><td>Gin</td><td>Gin</td><td>Tyr</td><td>Leu</td><td>Thr</td><td>Cheese</td>

<td></td><td></td><td> 14 0</td><td></td><td></td><td></td><td></td><td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td>

<td>Cheese</td><td>Tyr</td><td>How much</td><td>Asn</td><td>How much</td><td>Cheese</td><td>Thr</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Gly</td><td>Gly</td><td>Lys</td><td>Lys</td><td>Tyr</td>

<td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Val</td><td>Trp</td><td>Val</td><td>Gin</td><td>Ala</td><td>Ala</td><td>Asn</td><td>Ala</td><td>Leu</td><td>Gly</td><td>Underworld</td><td>Glu</td><td>Glu</td><td>Cheese</td><td>Lys</td>

170 175 180 185

Gin Leu Gin Ile His Leu Asp Asp Ile Val Ile Pro Ser Ala Ala Val 190 195 200

Ile Ser Arg Ala Glu Thr Ile Asn Ala Thr Val Pro Lys Thr Ile 205 210 215

Tyr Trp Asp Ser Gin Thr Thr Ile Glu Lys Val Ser Cys Glu Met Arg 220 225 230

Tyr Lys Ala Thr Thr Asn Gin Thr Trp Asn Val Lys Glu Phe Asp Thr 235 240 245

Asn Phe Thr Tyr Val Gin Gin Ser Glu Phe Tyr Leu Glu Pro Asn Ile

250 255 260 265

Lys Tyr Val Phe Gin Val Arg Cys Gin Glu Thr Gly Lys Arg Tyr Trp

270 275 280

Gin Pro Trp Ser Ser Pro Phe Phe His Lys Thr Pro Glu Thr Val Pro 285 290 295

Gin Val Thr Ser Lys Ala Phe Gin His Asp Thr Trp Asn Ser Gly Leu 300 305 310

Thr Val Ala Ser Ile Ser Thr Gly His Leu Thr Ser Asp Asn Arg Gly 315 320 325

Asp Ile Gly Leu Leu Leu Gly Met Ile Val Phe Ala Val Met Leu Ser 330 335 340 345

PL 209 128 B1

<td>How much</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>How much 350</td><td>Gly</td><td>How much</td><td>Phe</td><td>Asn</td><td>Arg 355</td><td>Cheese</td><td>Phe</td><td>Arg</td><td>Thr</td><td>Gly 360</td><td>How much</td>

<td>Lys</td><td>Arg</td><td>Arg</td><td>How much 365</td><td>Leu</td><td>Leu</td><td>Leu</td><td>How much</td><td>Pro 370</td><td>Lys</td><td>Trp</td><td>Leu</td><td>Tyr</td><td>Glu 375</td><td>Asp</td><td>How much</td>

<td>Pro</td><td>Asn</td><td>Underworld 380</td><td>Lys</td><td>Asn</td><td>Cheese</td><td>Asn</td><td>Val 385</td><td>Val</td><td>Lys</td><td>Underworld</td><td>Leu</td><td>Gin 390</td><td>Glu</td><td>Asn</td><td>Cheese</td>

<td>Glu</td><td>Leu 395</td><td>Underworld</td><td>Asn</td><td>Asn</td><td>Asn</td><td>Cheese 400</td><td>Cheese</td><td>Glu</td><td>Gin</td><td>Val</td><td>Leu 405</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Pro</td>

<td>Underworld 410</td><td>How much</td><td>Thr</td><td>Glu</td><td>How much</td><td>Lys 415</td><td>Glu</td><td>How much</td><td>Phe</td><td>How much</td><td>Pro 420</td><td>Glu</td><td>His</td><td>Lys</td><td>Pro</td><td>Thr 425</td>

<td>Asp</td><td>Tyr</td><td>Lys</td><td>Lys</td><td>Glu 430</td><td>Asn</td><td>Thr</td><td>Gly</td><td>Pro</td><td>Leu 435</td><td>Glu</td><td>Thr</td><td>Arg</td><td>Asp</td><td>Tyr 440</td><td>Pro</td>

<td>Gin</td><td>Asn</td><td>Cheese</td><td>Leu 445</td><td>Phe</td><td>Asp</td><td>Asn</td><td>Thr</td><td>Thr 450</td><td>Val</td><td>Val</td><td>Tyr</td><td>How much</td><td>Pro 455</td><td>Asp</td><td>Leu</td>

<td>Asn</td><td>Thr</td><td>Gly</td><td>Tyr</td><td>Lys</td><td>Pro</td><td>Gin</td><td>How much</td><td>Cheese</td><td>Asn</td><td>Phe</td><td>Leu</td><td>Pro</td><td>Glu</td><td>Gly</td><td>Cheese</td>

460 465 470

His Leu Ser Asn Asn Asn Glu Ile Thr Ser Leu Thr Leu Lys Pro Pro 475 480 485

Val Asp Ser Leu Asp Ser Gly Asn Asn Pro Arg Leu Gin Lys His Pro

490 495 500 505

Asn Phe Ala Phe Cheese Val Cheese Cheese Val Asn Ser Leu Cheese Asn Thr Ile

510 515 520

Phe Leu Gly Glu Leu Cheese Leu Ile Leu Asn Gin Gly Glu Cys Cheese Cheese 525 530 535

Pro Asp Ile Gin Asn Ser Val Glu Glu Glu Thr Thr Met Leu Leu Glu 540 545 550

Asn Asp Ser Pro Ser Glu Thr Ile Pro Glu Gin Thr Leu Leu Pro Asp 555 560 565

Glu Phe Val Cheese Cys Leu Gly Ile Val Asn Glu Glu Leu Pro Cheese Ile

570 575 580 585

Asn Thr Tyr Phe Pro Gin Asn Ile Leu Glu Ser His Phe Asn Arg Ile

590 595 600

Leu Leu Glu Lys 605 &lt;210&gt; 3 &lt;211&gt; 1887 &lt;212&gt; DNA &lt;213&gt; reverse translation &lt;220&gt;

&lt;221&gt; any_trait &lt;222&gt; (1) .. (1887)

PL 209 128 B1 &lt;223&gt; η may be a, c, g, or t &lt;400&gt; 3

<td>atgaaycayg</td><td>tnacnathca</td><td>rtgggaygcn</td><td>gtnathgcny</td><td>tntayathyt</td><td>nttywsntgg</td><td> 60</td>

<td>tgycayggng</td><td>gnathacnaa</td><td>yathaaytgy</td><td>wsnggncaya</td><td>thtgggtnga</td><td>rccngcnacn</td><td> 120</td>

<td>athttyaara</td><td>tgggnatgaa</td><td>yathwsnath</td><td>taytgycarg</td><td>cngcnathaa</td><td>raaytgycar</td><td> 180</td>

<td>ccnmgnaary</td><td>tncayttyta</td><td>yaaraayggn</td><td>athaargarm</td><td>gnttycarat</td><td>hacnmgnath</td><td> 240</td>

<td>aayaaracna</td><td>cngcnmgnyt</td><td>ntggtayaar</td><td>aayttyytng</td><td>arccncaygc</td><td>nwsnatgtay</td><td> 300</td>

<td>tgyacngcng</td><td>artgyccnaa</td><td>rcayttycar</td><td>garacnytna</td><td>thtgyggnaa</td><td>rgayathwsn</td><td> 360</td>

<td>wsnggntayc</td><td>cnccngayat</td><td>hccngaygar</td><td>gtnacntgyg</td><td>tnathtayga</td><td>rtaywsnggn</td><td> 420</td>

<td>aayatgacnt</td><td>gyacntggaa</td><td>ygcnmgnaar</td><td>ytnacntaya</td><td>thgayacnaa</td><td>rtaygtngtn</td><td> 480</td>

<td>caygtnaarw</td><td>snytngarac</td><td>ngargargar</td><td>carcartayy</td><td>tnacnwsnws</td><td>ntayathaay</td><td> 540</td>

<td>athwsnacng</td><td>aywsnytnca</td><td>rggnggnaar</td><td>aartayytng</td><td>tntgggtnca</td><td>rgcngcnaay</td><td> 600</td>

<td>gcnytnggna</td><td>tggargarws</td><td>naarcarytn</td><td>carathcayy</td><td>tngaygayat</td><td>hgtnathccn</td><td> 660</td>

<td>wsngcngcng</td><td>tnathwsnmg</td><td>ngcngaracn</td><td>athaaygcna</td><td>cngtnccnaa</td><td>racnathath</td><td> 720</td>

<td>taytgggayw</td><td>sncaracnac</td><td>nathgaraar</td><td>gtnwsntgyg</td><td>aratgmgnta</td><td>yaargcnacn</td><td> 780</td>

<td>acnaaycara</td><td>cntggaaygt</td><td>naargartty</td><td>gayacnaayt</td><td>tyacntaygt</td><td>ncarcarwsn</td><td> 840</td>

<td>garttytayy</td><td>tngarccnaa</td><td>yathaartay</td><td>gtnttycarg</td><td>tnmgntgyca</td><td>rgaracnggn</td><td> 900</td>

<td>aarmgntayt</td><td>ggcarccntg</td><td>gwsnwsnccn</td><td>ttyttycaya</td><td>aracnccnga</td><td>racngtnccn</td><td> 960</td>

<td>cargtnacnw</td><td>snaargcntt</td><td>ycarcaygay</td><td>acntggaayw</td><td>snggnytnac</td><td>ngtngcnwsn</td><td> 1020</td>

<td>athwsnacng</td><td>gncayytnac</td><td>nwsngayaay</td><td>mgnggngaya</td><td>thggnytnyt</td><td>nytnggnatg</td><td> 1080</td>

<td>athgtnttyg</td><td>cngtnatgyt</td><td>nwsnathytn</td><td>wsnytnathg</td><td>gnathttyaa</td><td>ymgnwsntty</td><td> 1140</td>

<td>mgn.acn.ggna</td><td>thaarmgnmg</td><td>nathytnytn</td><td>ytnathccna</td><td>artggytnta</td><td>ygargayath</td><td> 1200</td>

<td>ccnaayatga</td><td>araaywsnaa</td><td>ygtngtnaar</td><td>atgytncarg</td><td>araaywsnga</td><td>rynatgaay</td><td> 1260</td>

<td>aayaaywsnw</td><td>sngarcargt</td><td>nytntaygtn</td><td>gayccnatga</td><td>thacngarat</td><td>haargarath</td><td> 1320</td>

<td>ttyathccng</td><td>arcayaarcc</td><td>nacngaytay</td><td>aaraargara</td><td>ayacnggncc</td><td>nytngaracn</td><td> 1380</td>

<td>mgngaytayc</td><td>cncaraayws</td><td>nytnttygay</td><td>aayacnacng</td><td>tngtntayat</td><td>hccngayytn</td><td> 1440</td>

<td>aayacnggnt</td><td>ayaarccnca</td><td>rathwsnaay</td><td>ttyytnccng</td><td>arggnwsnca</td><td>yytnwsnaay</td><td> 1500</td>

<td>aayaaygara</td><td>thacnwsnyt</td><td>nacnytnaar</td><td>ccnccngtng</td><td>aywsnytnga</td><td>ywsnggnaay</td><td> 1560</td>

<td>aayccnmgny</td><td>tncaraarca</td><td>yccnaaytty</td><td>gcnttywsng</td><td>tnwsnwsngt</td><td>naaywsnytn</td><td> 1620</td>

<td>wsnaayacna</td><td>thttyytngg</td><td>ngarytnwsn</td><td>ytnathytna</td><td>aycarggnga</td><td>rtgywsnwsn</td><td> 1680</td>

<td>ccngayathc</td><td>araaywsngt</td><td>ngargargar</td><td>acnacnatgy</td><td>tnytngaraa</td><td>ygaywsnccn</td><td> 1740</td>

PL 209 128 B1 wsngaracna thccngarca racnytnytn ccngaygart tygtnwsntg yytnggnath 1800 gtnaaygarg arytnccnws nathaayacn tayttyccnc araayathyt ngarwsncay 1860 ttyaaymgna thwsnytnyt &lt;213287&gt; ngara &lt;Tanyaaymgna thwsnytnyt &lt;213&gt; &lt;Ngara&gt; &lt;213&gt; &lt;Ngara&gt;

&lt;223&gt; Description of unknown organism: primate; presumably homo sapiens

<td colspan="5"> &lt;400&gt; 4</td><td rowspan="2">Val</td><td rowspan="2">Val</td><td rowspan="2">Gin 10</td><td rowspan="2">Ala</td><td rowspan="2">Leu</td><td rowspan="2">Phe</td><td rowspan="2">How much</td><td rowspan="2">Phe 15</td><td rowspan="2">Leu</td>

<td>Underworld 1</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Gin Thr Trp 5</td>

<td>Thr</td><td>Thr</td><td>Glu</td><td>Cheese twenty</td><td>Thr Gly Glu</td><td>Leu</td><td>Leu 25</td><td>Asp</td><td>Pro</td><td>Cys</td><td>Gly</td><td>Tyr thirty</td><td>How much</td><td>Cheese</td>

<td>Pro</td><td>Glu</td><td>Cheese 35</td><td>Pro</td><td>Val Val Gin</td><td>Leu 40</td><td>His</td><td>Cheese</td><td>Asn</td><td>Phe</td><td>Thr 45</td><td>Ala</td><td>Val</td><td>Cys</td>

<td>Val</td><td>Leu 50</td><td>Lys</td><td>Glu</td><td>Lys Cys Met 55</td><td>Asp</td><td>Tyr</td><td>Phe</td><td>His</td><td>Val 60</td><td>Asn</td><td>Ala</td><td>Asn</td><td>Tyr</td>

<td>How much 65</td><td>Val</td><td>Trp</td><td>Lys</td><td>Thr Asn His 70</td><td>Phe</td><td>Thr</td><td>How much</td><td>Pro 75</td><td>Lys</td><td>Glu</td><td>Gin</td><td>Tyr</td><td>Thr 80</td>

<td>How much</td><td>How much</td><td>Asn</td><td>Arg</td><td>Thr Ala Ser 85</td><td>Cheese</td><td>Val</td><td>Thr 90</td><td>Phe</td><td>Thr</td><td>Asp</td><td>How much</td><td>Ala 95</td><td>Cheese</td>

<td>Leu</td><td>Asn</td><td>How much</td><td>Gin 100</td><td>Leu Thr Cys</td><td>Asn</td><td>How much 105</td><td>Leu</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Gin 110</td><td>Leu</td><td>Glu</td>

<td>Gin</td><td>Asn</td><td>Val 115</td><td>Tyr</td><td>Gly Ile Thr</td><td>How much 120</td><td>How much</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Pro 125</td><td>Pro</td><td>Glu</td><td>Lys</td>

<td>Pro</td><td>Lys 130</td><td>Asn</td><td>Leu</td><td>Cys Ile cheese 135</td><td>Val</td><td>Asn</td><td>Glu</td><td>Gly</td><td>Lys 14 0</td><td>Lys</td><td>Underworld</td><td>Arg</td><td>Cys</td>

<td>Glu 145</td><td>Trp</td><td>Asp</td><td>Gly</td><td>Gly Arg Glu 150</td><td>Thr</td><td>His</td><td>Leu</td><td>Glu 155</td><td>Thr</td><td>Asn</td><td>Phe</td><td>Thr</td><td>Leu 160</td>

<td>Lys</td><td>Cheese</td><td>Glu</td><td>Trp</td><td>Ala Thr His 165</td><td>Lys</td><td>Phe</td><td>Ala 170</td><td>Asp</td><td>Cys</td><td>Lys</td><td>Ala</td><td>Lys 175</td><td>Arg</td>

<td>Asp</td><td>Thr</td><td>Pro</td><td>Thr 180</td><td>Cys Thr</td><td>Val</td><td>Asp 185</td><td>Tyr</td><td>Cheese</td><td>Thr</td><td>Val</td><td>Tyr 190</td><td>Phe</td><td>Val</td>

<td>Asn</td><td>How much</td><td>Glu 195</td><td>Val</td><td>Trp Val Glu</td><td>Ala 200</td><td>Glu</td><td>Asn</td><td>Ala</td><td>Leu</td><td>Gly 205</td><td>Lys</td><td>Val</td><td>Thr</td>

<td>Cheese</td><td>Asp 210</td><td>His</td><td>How much</td><td>Asn Phe Asp 215</td><td>Pro</td><td>Val</td><td>Tyr</td><td>Lys</td><td>Val 220</td><td>Lys</td><td>Pro</td><td>Asn</td><td>Pro</td>

<td>Pro</td><td>His</td><td>Asn</td><td>Leu</td><td>Val Ile cheese</td><td>Asn</td><td>Cheese</td><td>Glu</td><td>Glu</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>How much</td><td>Leu</td>

225 230 235 240

PL 209 128 B1

Lys Leu Thr Trp Thr Asn Pro Ser 245

Tyr Asn Ile Gin Tyr Arg Thr Lys 260

Pro Pro Glu Asp Thr Ala Ser Thr 275 280

Leu Lys Pro Phe Thr Glu Tyr Val 290 295

Asp Gly Lys Gly Tyr Trp Ser Asp 305 310

Thr Tyr Glu Asp Arg Pro Ser Lys 325

Asp Pro Ser His Thr Gin Gly Tyr 340

Thr Leu Pro Pro Phe Glu Ala Asn 355 360

Thr Leu Thr Arg Trp Lys Ser His 370 375

Thr Lys Leu Thr Val Asn Leu Thr 385 390

Thr Val Arg Asn Leu Val Gly Lys 405

Pro Ala Cys Asp Phe Gin Ala Thr 420

Phe Pro Lys Asp Asn Met Leu Trp 435 440

Val Lys Lys Tyr Ile Leu Glu cheese 450 455

Pro Cys Ile Thr Asp Trp Gin Gin 465 470

Tyr Leu Arg Gly Asn Leu Ala Glu 485

Thr Pro Val Tyr Ala Asp Gly Pro 500

Tyr Leu Lys Gin Ala Pro Pro Ser 515 520

Ile Lys Cheese Val Ile Ile Leu Lys 250 255

Asp Ala Ser Thr Trp Ser Gin Ile 265 270

Arg Ser Ser Phe Thr Val Gin Asp 285

Phe Arg Ile Arg Cys Met Lys Glu 300

Trp Cheese Glu Glu Ala Cheese Gly Ile 315 320

Ala Pro Ser Phe Trp Tyr Lys Ile 330 335

Arg Thr Val Gin Leu Val Trp Lys 345 350

Gly Lys Ile Leu Asp Tyr Glu Val 365

Leu Gin Asn Tyr Thr Val Asn Ala 380

Asn Asp Arg Tyr Leu Ala Thr Leu 395 400

Asp Ala Ala Val Leu Thr Ile 410 415

His Pro Val Met Asp Leu Lys Ala 425 430

Val Glu Trp Thr Thr Pro Arg Glu 445

Trp Cys Val Leu Ser Asp Lys Ala 460

Glu Asp Gly Thr Val His Arg Thr 475 480

Lys Cys Tyr Leu Ile Thr Val 490 495

Gly Ser Pro Glu Ser Ile Lys Ala 505 510

Lys Gly Pro Thr Val Arg Thr Lys 525

Lys Val Gly Lys Asn Glu Ala Val 530 535

Asp Val Gin Asn Gly Phe Ile Arg 545 550

Leu Glu Trp Asp Gin Leu Pro Val 540

Asn Tyr Thr Ile Phe Tyr Arg Thr 555 560

PL 209 128 B1

<td>How much</td><td>How much</td><td>Gly</td><td>Asn</td><td>Glu 565</td><td>Thr</td><td>Ala</td><td>Val</td><td>Asn</td><td>Val 570</td><td>Asp</td><td>Cheese</td><td>Cheese</td><td>His</td><td>Thr 575</td><td>Glu</td>

<td>Tyr</td><td>Thr</td><td>Leu</td><td>Cheese 580</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cheese</td><td>Asp 585</td><td>Thr</td><td>Leu</td><td>Tyr</td><td>Underworld</td><td>Val 590</td><td>Arg</td><td>Underworld</td>

<td>Ala</td><td>Ala</td><td>Tyr 595</td><td>Thr</td><td>Asp</td><td>Glu</td><td>Gly</td><td>Gly 600</td><td>Lys</td><td>Asp</td><td>Gly</td><td>Pro</td><td>Glu 605</td><td>Phe</td><td>Thr</td><td>Phe</td>

<td>Thr</td><td>Thr 610</td><td>Pro</td><td>Lys</td><td>Phe</td><td>Ala</td><td>Gin 615</td><td>Gly</td><td>Glu</td><td>How much</td><td>Glu</td><td>Ala 620</td><td>How much</td><td>Val</td><td>Val</td><td>Pro</td>

<td>Val 625</td><td>Cys</td><td>Leu</td><td>Ala</td><td>Phe</td><td>Leu 630</td><td>Leu</td><td>Thr</td><td>Thr</td><td>Leu</td><td>Leu 635</td><td>Gly</td><td>Val</td><td>Leu</td><td>Phe</td><td>Cys 64 0</td>

<td>Phe</td><td>Asn</td><td>Lys</td><td>Arg</td><td>Asp 645</td><td>Leu</td><td>How much</td><td>Lys</td><td>Lys</td><td>His 650</td><td>How much</td><td>Trp</td><td>Pro</td><td>Asn</td><td>Val 655</td><td>Pro</td>

<td>Asp</td><td>Pro</td><td>Cheese</td><td>Lys 660</td><td>Cheese</td><td>His</td><td>How much</td><td>Ala</td><td>Gin 665</td><td>Trp</td><td>Cheese</td><td>Pro</td><td>His</td><td>Thr 670</td><td>Pro</td><td>Pro</td>

<td>Arg</td><td>His</td><td>Asn 675</td><td>Phe</td><td>Asn</td><td>Cheese</td><td>Lys</td><td>Asp 680</td><td>Gin</td><td>Underworld</td><td>Tyr</td><td>Cheese</td><td>Asp 685</td><td>Gly</td><td>Asn</td><td>Phe</td>

<td>Thr</td><td>Asp 690</td><td>Val</td><td>Cheese</td><td>Val</td><td>Val</td><td>Glu 695</td><td>How much</td><td>Glu</td><td>Ala</td><td>Asn</td><td>Asp 700</td><td>Lys</td><td>Lys</td><td>Pro</td><td>Phe</td>

<td>Pro 705</td><td>Glu</td><td>Asp</td><td>Leu</td><td>Lys</td><td>Cheese 710</td><td>Leu</td><td>Asp</td><td>Leu</td><td>Phe</td><td>Lys 715</td><td>Lys</td><td>Glu</td><td>Lys</td><td>How much</td><td>Asn 720</td>

<td>Thr</td><td>Glu</td><td>Gly</td><td>His</td><td>Cheese 725</td><td>Cheese</td><td>Gly</td><td>How much</td><td>Gly</td><td>Gly 730</td><td>Cheese</td><td>Cheese</td><td>Cys</td><td>Underworld</td><td>Cheese 735</td><td>Cheese</td>

<td>Cheese</td><td>Arg</td><td>Pro</td><td>Cheese 740</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Asp 745</td><td>Glu</td><td>Asn</td><td>Glu</td><td>Cheese</td><td>Cheese 750</td><td>Gin</td><td>Asn</td>

<td>Thr</td><td>Cheese</td><td>Cheese 755</td><td>Thr</td><td>Val</td><td>Gin</td><td>Tyr</td><td>Cheese 760</td><td>Thr</td><td>Val</td><td>Val</td><td>His</td><td>Cheese 765</td><td>Gly</td><td>Tyr</td><td>Arg</td>

<td>His</td><td>Gin</td><td>Val</td><td>Pro</td><td>Cheese</td><td>Val</td><td>Gin</td><td>Val</td><td>Phe</td><td>Cheese</td><td>Arg</td><td>Cheese</td><td>Glu</td><td>Cheese</td><td>Thr</td><td>Gin</td>

770 775 780

Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gin Leu Val Asp

785 790 795 800

His Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gin Gin Tyr Phe Lys

805 810 815

Gin Asn Cys Ser Gin His Glu Ser Ser Pro Asp Ile Ser His Phe Glu 820 825 830

Arg Cheese Lys Gin Val Cheese Cheese Val Asn Glu Glu Asp Phe Val Arg Leu 835 840 845

<img file="PL209128B1_D0001.tif" />

Gin

Gly

880

PL 209 128 B1

<td colspan="5">Thr Glu Gly Gin Val</td><td rowspan="2">Glu</td><td colspan="4" rowspan="2">Arg Phe Glu Thr 890</td><td rowspan="2">Val</td><td colspan="3" rowspan="2">Gly Met Glu Ala 895</td><td rowspan="2">Ala</td>

<td></td><td colspan="4"> 885</td>

<td>Thr</td><td>Asp</td><td>Glu</td><td>Gly</td><td>Underworld</td><td>Pro</td><td>Lys</td><td>Cheese</td><td>Tyr</td><td>Leu</td><td>Pro</td><td>Gin Thr</td><td>Val</td><td>Arg</td><td>Gin</td>

<td></td><td></td><td></td><td> 900</td><td></td><td></td><td></td><td></td><td> 905</td><td></td><td></td><td></td><td> 910</td><td></td><td></td>

<td>Gly</td><td>Gly</td><td>Tyr</td><td>Underworld</td><td>Pro</td><td>Gin</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

915 &lt;210&gt; 5 &lt;211&gt; 862 &lt;212&gt; PRT &lt;213&gt; Unknown &lt;220&gt;

&lt;223&gt; Description of unknown organism: primate; presumably homo sapiens

<td colspan="3"> &lt;400&gt; 5</td><td rowspan="2">Thr</td><td rowspan="2">Phe 5</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td><td rowspan="2">Cys</td><td rowspan="2">Cheese</td><td rowspan="2">Leu 10</td><td rowspan="2">Ala</td><td rowspan="2">Phe</td><td rowspan="2">Underworld</td><td rowspan="2">Phe</td><td rowspan="2">How much 15</td><td rowspan="2">How much</td>

<td>Underworld 1</td><td>Ala</td><td>His</td>

<td>Thr</td><td>Trp</td><td>Leu</td><td>Leu twenty</td><td>How much</td><td>Lys</td><td>Ala</td><td>Lys</td><td>How much 25</td><td>Asp</td><td>Ala</td><td>Cys</td><td>Lys</td><td>Arg thirty</td><td>Gly</td><td>Asp</td>

<td>Val</td><td>Thr</td><td>Val 35</td><td>Lys</td><td>Pro</td><td>Cheese</td><td>His</td><td>Val 40</td><td>How much</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Cheese 45</td><td>Thr</td><td>Val</td><td>Asn</td>

<td>How much</td><td>Thr 50</td><td>Cys</td><td>Cheese</td><td>Leu</td><td>Lys</td><td>Pro 55</td><td>Arg</td><td>Gin</td><td>Gly</td><td>Cys</td><td>Phe 60</td><td>His</td><td>Tyr</td><td>Cheese</td><td>Arg</td>

<td>Arg 65</td><td>Asn</td><td>Lys</td><td>Leu</td><td>How much</td><td>Leu 70</td><td>Tyr</td><td>Lys</td><td>Phe</td><td>Asp</td><td>Arg 75</td><td>Arg</td><td>How much</td><td>Asn</td><td>Phe</td><td>His 80</td>

<td>His</td><td>Gly</td><td>His</td><td>Cheese</td><td>Leu 85</td><td>Asn</td><td>Cheese</td><td>Gin</td><td>Val</td><td>Thr 90</td><td>Gly</td><td>Leu</td><td>Pro</td><td>Leu</td><td>Gly 95</td><td>Thr</td>

<td>Thr</td><td>Leu</td><td>Phe</td><td>Val 100</td><td>Cys</td><td>Lys</td><td>Leu</td><td>Ala</td><td>Cys 105</td><td>How much</td><td>Asn</td><td>Cheese</td><td>Asp</td><td>Glu 110</td><td>How much</td><td>Gin</td>

<td>How much</td><td>Cys</td><td>Gly 115</td><td>Ala</td><td>Glu</td><td>How much</td><td>Phe</td><td>Val 120</td><td>Gly</td><td>Val</td><td>Ala</td><td>Pro</td><td>Glu 125</td><td>Gin</td><td>Pro</td><td>Gin</td>

<td>Asn</td><td>Leu 130</td><td>Cheese</td><td>Cys</td><td>How much</td><td>Gin</td><td>Lys 135</td><td>Gly</td><td>Glu</td><td>Gin</td><td>Gly</td><td>Thr 140</td><td>Val</td><td>Ala</td><td>Cys</td><td>Thr</td>

<td>Trp 145</td><td>Glu</td><td>Arg</td><td>Gly</td><td>Arg</td><td>Asp 150</td><td>Thr</td><td>His</td><td>Leu</td><td>Tyr</td><td>Thr 155</td><td>Glu</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Gin 160</td>

<td>Leu</td><td>Cheese</td><td>Gly</td><td>Pro</td><td>Lys 165</td><td>Asn</td><td>Leu</td><td>Thr</td><td>Trp</td><td>Gin 170</td><td>Lys</td><td>Gin</td><td>Cys</td><td>Lys</td><td>Asp 175</td><td>How much</td>

<td>Tyr</td><td>Cys</td><td>Asp</td><td>Tyr 180</td><td>Leu</td><td>Asp</td><td>Phe</td><td>Gly</td><td>How much 185</td><td>Asn</td><td>Leu</td><td>Thr</td><td>Pro</td><td>Glu 190</td><td>Cheese</td><td>Pro</td>

<td>Glu</td><td>Cheese</td><td>Asn 195</td><td>Phe</td><td>Thr</td><td>Ala</td><td>Lys</td><td>Val 200</td><td>Thr</td><td>Ala</td><td>Val</td><td>Asn</td><td>Cheese 205</td><td>Leu</td><td>Gly</td><td>Cheese</td>

<td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Pro</td><td>Cheese</td><td>Thr</td><td>Phe</td><td>Thr</td><td>Phe</td><td>Leu</td><td>Asp</td><td>How much</td><td>Val</td><td>Arg</td><td>Pro</td>

PL 209 128 B1

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td>

<td>Leu 225</td><td>Pro</td><td>Pro</td><td>Trp</td><td>Asp</td><td>How much 230</td><td>Arg</td><td>How much</td>

<td>Arg</td><td>Cys</td><td>Thr</td><td>Leu</td><td>Tyr 245</td><td>Trp</td><td>Arg</td><td>Asp</td>

<td>Leu</td><td>Arg</td><td>Tyr</td><td>Arg 260</td><td>Pro</td><td>Cheese</td><td>Asn</td><td>Cheese</td>

<td>Thr</td><td>Lys</td><td>Ala 275</td><td>Lys</td><td>Gly</td><td>Arg</td><td>His</td><td>Asp 280</td>

<td>Glu</td><td>Tyr 290</td><td>Glu</td><td>Phe</td><td>Gin</td><td>How much</td><td>Cheese 295</td><td>Cheese</td>

<td>Trp 305</td><td>Cheese</td><td>Asp</td><td>Trp</td><td>Cheese</td><td>Glu 310</td><td>Cheese</td><td>Leu</td>

<td>Pro</td><td>Thr</td><td>Gly</td><td>Underworld</td><td>Leu 325</td><td>Asp</td><td>Val</td><td>Trp</td>

<td>Cheese</td><td>Arg</td><td>Gin</td><td>Gin 340</td><td>How much</td><td>Cheese</td><td>Leu</td><td>Phe</td>

<td>Ala</td><td>Arg</td><td>Gly 355</td><td>Lys</td><td>How much</td><td>Leu</td><td>His</td><td>Tyr 360</td>

<td>Gly</td><td>Gly 370</td><td>Lys</td><td>Ala</td><td>Underworld</td><td>Thr</td><td>Gin 375</td><td>Asn</td>

<td>Thr 385</td><td>Val</td><td>How much</td><td>Pro</td><td>Arg</td><td>Thr 390</td><td>Gly</td><td>Asn</td>

<td>Asn</td><td>Cheese</td><td>Lys</td><td>Gly</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Pro</td>

405

Cys Glu Ala Gly Leu Leu Ala Pro 420

Gly Met Asp Asn Ile Leu Val Thr 435 440

Ala Val Gin Glu Tyr Val Val Cheese 450 455

Gly Asp Thr Gin Val Pro Leu Asn 465 470

Val Ser Ala Leu Ile Cheese Glu Asn 485

Ile Arg Val Tyr Ala Leu Ser Gly 500

Leu Gly Asn Ser Lys His Lys Ala 515 520

Ala Ile Thr Glu Glu Lys Gly Ser 530 535

<td></td><td> 220</td>

<td>Lys Phe Gin 235</td><td>Lys Ala Ser Val Ser 240</td>

<td>Glu Gly Leu 250</td><td>Val Leu Leu Asn Arg 255</td>

<td>Arg Leu Trp 265</td><td>Asn Met Val Asn Val 270</td>

<td>Leu Leu Asp</td><td>Leu Lys Pro Phe Thr 285</td>

<td>Lys Leu His</td><td>Leu Tyr Lys Gly Ser 300</td>

<td>Arg Ala Gin 315</td><td>Thr Pro Glu Glu Glu 320</td>

<td>Tyr Met Lys 330</td><td>Arg His Ile Asp Tyr 335</td>

<td>Trp Lys Asn 345</td><td>Leu Cheese Val Cheese Glu 350</td>

<td>Gin Val Thr</td><td>Leu Gin Glu Leu Thr 365</td>

<td>How Much Thr Gly</td><td>His Thr Ser Trp Thr 380</td>

<td>Trp Ala Val 395</td><td>Ala Val Ser Ala Ala 400</td>

<td>Thr Arg Ile 410</td><td>Asn Ile Met Asn Leu 415</td>

<td>Arg Gin Val 425</td><td>Ala Asn Cheese Glu 430</td>

<td>Trp Gin Pro</td><td>Pro Arg Lys Asp Pro 445</td>

<td>Glu Trp Arg</td><td>Glu Leu His Pro Gly 460</td>

<td>Trp Leu Arg 475</td><td>Arg Pro Tyr Asn cheese 480</td>

<td>How Much Lys Cheese 490</td><td>Tyr Ile Cys Tyr Glu 495</td>

<td>Asp Gin Gly 505</td><td>Gly Cys Cheese Ile 510</td>

<td>Pro Leu Ser</td><td>Gly Pro His Ile Asn 525</td>

<td>How many Leu Ile</td><td>Trp Asn Cheese Ile</td>

540

PL 209 128 B1

<td>Pro 545</td><td>Val</td><td>Gin</td><td colspan="3">Glu Gin Met 550</td><td colspan="2">Gly Cys</td><td>Leu</td><td>Leu</td><td>His 555</td><td colspan="2">Tire Arg</td><td>How much</td><td>Tyr</td><td>Trp 560</td>

<td>Lys</td><td>Glu</td><td>Arg</td><td>Asp</td><td>Cheese</td><td>Asn</td><td>Cheese</td><td>Gin</td><td>Pro</td><td>Gin</td><td>Leu</td><td>'Cys</td><td>Glu</td><td>How much</td><td>Pro</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 565</td><td></td><td></td><td></td><td></td><td> 570</td><td></td><td></td><td></td><td></td><td> 575</td><td></td>

<td>Arg</td><td>Val</td><td>Cheese</td><td>Gin</td><td>Asn</td><td>Cheese</td><td>His</td><td>Pro</td><td>How much</td><td>Asn</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Val</td>

<td></td><td></td><td></td><td> 580</td><td></td><td></td><td></td><td></td><td> 585</td><td></td><td></td><td></td><td></td><td> 590</td><td></td><td></td>

<td>Thr</td><td>Tyr</td><td>Val</td><td>Leu</td><td>Trp</td><td>Underworld</td><td>Thr</td><td>Ala</td><td>Leu</td><td>Thr</td><td>Ala</td><td>Ala</td><td>Gly</td><td>Glu</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td> 595</td><td></td><td></td><td></td><td></td><td> 600</td><td></td><td></td><td></td><td></td><td> 605</td><td></td><td></td><td></td>

<td>His</td><td>Gly</td><td>Asn</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Phe</td><td>Cys</td><td>Leu</td><td>Gin</td><td>Gly</td><td>Lys</td><td>Ala</td><td>Asn</td><td>Trp</td><td>Underworld</td>

<td></td><td> 610</td><td></td><td></td><td></td><td></td><td> 615</td><td></td><td></td><td></td><td></td><td> 620</td><td></td><td></td><td></td><td></td>

<td>Ala</td><td>Phe</td><td>Val</td><td>Ala</td><td>Pro</td><td>Cheese</td><td>How much</td><td>Cys</td><td>How much</td><td>Ala</td><td>How much</td><td>How much</td><td>Underworld</td><td>Val</td><td>Gly</td><td>How much</td>

<td> 625</td><td></td><td></td><td></td><td></td><td> 630</td><td></td><td></td><td></td><td></td><td> 635</td><td></td><td></td><td></td><td></td><td> 640</td>

<td>Phe</td><td>Cheese</td><td>Thr</td><td>His</td><td>Tyr</td><td>Phe</td><td>Gin</td><td>Gin</td><td>Lys</td><td>Val</td><td>Phe</td><td>Val</td><td>Leu</td><td>Leu</td><td>Ala</td><td>Ala</td>

<td></td><td></td><td></td><td></td><td> 645</td><td></td><td></td><td></td><td></td><td> 650</td><td></td><td></td><td></td><td></td><td> 655</td><td></td>

<td>Leu</td><td>Arg</td><td>Pro</td><td>Gin</td><td>Trp</td><td>Cys</td><td>Cheese</td><td>Arg</td><td>Glu</td><td>How much</td><td>Pro</td><td>Asp</td><td>Pro</td><td>Ala</td><td>Asn</td><td>Cheese</td>

<td></td><td></td><td></td><td> 660</td><td></td><td></td><td></td><td></td><td> 665</td><td></td><td></td><td></td><td></td><td> 670</td><td></td><td></td>

<td>Thr</td><td>Cys</td><td>Ala</td><td>Lys</td><td>Lys</td><td>Tyr</td><td>Pro</td><td>How much</td><td>Ala</td><td>Glu</td><td>Glu</td><td>Lys</td><td>Thr</td><td>Gin</td><td>Leu</td><td>Pro</td>

<td></td><td></td><td> 675</td><td></td><td></td><td></td><td></td><td> 680</td><td></td><td></td><td></td><td></td><td> 685</td><td></td><td></td><td></td>

<td>Leu</td><td>Asp</td><td>Arg</td><td>Leu</td><td>Leu</td><td>How much</td><td>Asp</td><td>Trp</td><td>Pro</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Asp</td><td>Pro</td><td>Glu</td><td>Pro</td>

<td></td><td> 690</td><td></td><td></td><td></td><td></td><td> 695</td><td></td><td></td><td></td><td></td><td> 700</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Val</td><td>How much</td><td>Cheese</td><td>Glu</td><td>Val</td><td>Leu</td><td>His</td><td>Gin</td><td>Val</td><td>Thr</td><td>Pro</td><td>Val</td><td>Phe</td><td>Arg</td><td>His</td>

<td> 705</td><td></td><td></td><td></td><td></td><td> 710</td><td></td><td></td><td></td><td></td><td> 715</td><td></td><td></td><td></td><td></td><td> 720</td>

<td>Pro</td><td>Pro</td><td>Cys</td><td>Cheese</td><td>Asn</td><td>Trp</td><td>Pro</td><td>Gin</td><td>Arg</td><td>Glu</td><td>Lys</td><td>Gly</td><td>How much</td><td>Gin</td><td>Gly</td><td>His</td>

<td></td><td></td><td></td><td></td><td> 725</td><td></td><td></td><td></td><td></td><td> 730</td><td></td><td></td><td></td><td></td><td> 735</td><td></td>

<td>Gin</td><td>Ala</td><td>Cheese</td><td>Glu</td><td>Lys</td><td>Asp</td><td>Underworld</td><td>Underworld</td><td>His</td><td>Cheese</td><td>Ala</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>Pro</td><td>Pro</td>

<td></td><td></td><td></td><td> 740</td><td></td><td></td><td></td><td></td><td> 745</td><td></td><td></td><td></td><td></td><td> 750</td><td></td><td></td>

<td>Pro</td><td>Arg</td><td>Ala</td><td>Leu</td><td>Gin</td><td>Ala</td><td>Glu</td><td>Cheese</td><td>Arg</td><td>Gin</td><td>Leu</td><td>Val</td><td>Asp</td><td>Leu</td><td>Tyr</td><td>Lys</td>

<td></td><td></td><td> 755</td><td></td><td></td><td></td><td></td><td> 760</td><td></td><td></td><td></td><td></td><td> 765</td><td></td><td></td><td></td>

<td>Val</td><td>Leu</td><td>Glu</td><td>Cheese</td><td>Arg</td><td>Gly</td><td>Cheese</td><td>Asp</td><td>Pro</td><td>Lys</td><td>Pro</td><td>Glu</td><td>Asn</td><td>Pro</td><td>Ala</td><td>Cys</td>

<td></td><td> 770</td><td></td><td></td><td></td><td></td><td> 775</td><td></td><td></td><td></td><td></td><td> 780</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Trp</td><td>Thr</td><td>Val</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Gly</td><td>Asp</td><td>Leu</td><td>Pro</td><td>Thr</td><td>His</td><td>Asp</td><td>Gly</td><td>Tyr</td>

<td> 785</td><td></td><td></td><td></td><td></td><td> 790</td><td></td><td></td><td></td><td></td><td> 795</td><td></td><td></td><td></td><td></td><td> 800</td>

<td>Leu</td><td>Pro</td><td>Cheese</td><td>Asn</td><td>How much</td><td>Asp</td><td>Asp</td><td>Leu</td><td>Pro</td><td>Cheese</td><td>His</td><td>Glu</td><td>Ala</td><td>Pro</td><td>Leu</td><td>Ala</td>

<td></td><td></td><td></td><td></td><td> 805</td><td></td><td></td><td></td><td></td><td> 810</td><td></td><td></td><td></td><td></td><td> 815</td><td></td>

<td rowspan="2">Asp</td><td>Cheese</td><td>Leu</td><td>Glu</td><td>Glu</td><td>Leu</td><td>Glu</td><td>Pro</td><td>Gin</td><td>His</td><td>How much</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Val</td><td>Phe</td>

<td></td><td></td><td> 820</td><td></td><td></td><td></td><td></td><td> 825</td><td></td><td></td><td></td><td></td><td> 830</td><td></td><td></td>

<td>Pro</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>His</td><td>Pro</td><td>Leu</td><td>Thr</td><td>Phe</td><td>Cheese</td><td>Cys</td><td>Gly</td><td>Asp</td><td>Lys</td><td>Leu</td>

<td></td><td></td><td> 835</td><td></td><td></td><td></td><td></td><td> 840</td><td></td><td></td><td></td><td></td><td> 845</td><td></td><td></td><td></td>

<td>Thr</td><td>Leu</td><td>Asp</td><td>Gin</td><td>Leu</td><td>Lys</td><td>Underworld</td><td>Arg</td><td>Cys</td><td>Asp</td><td>Cheese</td><td>Leu</td><td>Underworld</td><td>Leu</td><td></td><td></td>

850 855 860

PL 209 128 B1