MXPA01011264A

MXPA01011264A - Novel chimpanzee erythropoietin (chepo) polypeptides and nucleic acids encoding the same.

Info

Publication number: MXPA01011264A
Application number: MXPA01011264A
Authority: MX
Inventors: Frederic Desauvage
Original assignee: Genentech Inc
Priority date: 1999-05-07
Filing date: 2000-05-05
Publication date: 2002-07-02
Also published as: JP3660880B2; WO2000068376B1; JP2002543784A; EP1177285A1; WO2000068376A8; AU766507B2; AU4704700A; IL145785A0; WO2000068376A1

Abstract

The present invention is directed to novel chimpanzee erythropoietin polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

Description

NEW POLYPES CHIMPANEE ERYTHROPOYETINE PTIDS (CHEPO) AND NUCLEIC ACIDS THAT CODE THE SAME FIELD OF THE INVENTION The present invention relates generally to the identification and isolation of novel chympanc erythropoietin polypeptides, nucleic acid molecules encoding those polypeptides and to the recombinant production of those polypeptides. BACKGROUND OF THE INVENTION Erythropoiesis, the production of red blood cells, occurs throughout the duration of human life to counteract cell destruction. Erythropoiesis 15 is a precisely controlled physiological mechanism that allows a sufficient number of red blood cells to be available in the blood for proper oxygenation of tissues, but not so many so that cells could impede circulation. The formation of 20 red blood cells occur in the spinal cord and this is the control of the hormone, erythropoietin. The erythropoieme, an acidic glycoprotein is of a molecular weight of approximately 34,000 daltons, can REF 133352 be presented in three forms: alpha, beta, and so on. The slightly different alpha and beta forms in the carbohydrate components have the same potency, biological activity and molecular weight. The asialo form is an alpha or beta form with the terminal carbohydrate (sialic acid) removed. Erythropoietin is present in very low concentrations in the plasma when the body is in a healthy state where tissues receive sufficient oxygenation from the existing number of erythrocytes. This low normal concentration is sufficient to stimulate the replacement of red blood cells which are normally lost through aging. The amount of erythropoietin in the circulation increases under hypoxic conditions when the transport of oxygen is reduced by the blood cells in the circulation. Hypoxia can be caused by the loss of large amounts of blood by hemorrhage, destruction of red blood cells by over-exposure to radiation, reduction in oxygen inhalation due to high altitudes or prolonged loss of consciousness, or various forms of anemia. In response to tissues suffering from hypoxic stress, erythropoietin will increase the production of red blood cells by stimulating the conversion of primitive precursor cells into the bone marrow within the proeritroblasts that subsequently mature, synthesizing hemoglobin and releasing into the circulation as red blood cells. When the number of red blood cells in the circulation is greater than that required for normal tissue oxygen requirements, erythropoietin in the circulation is decreased. Because erythropoietin is essential in the process of red blood cell formation, the hormone has a useful and potential application, both in the diagnosis and in the treatment of blood disorders, characterized by low production or defective red blood cells. See, in general, Pennathur-Das, et al., Blood 63 (5): 1168-71 (1984) and Haddy, Am. Jour. Ped. Hematol. Oncol., 4: 191-196 (1982) which relates to erythropoietin in the possible therapies for cellular diseases of depranocytosis, and Eschbach et al., J. Clin. Invest. 74 (2): 434-441 (1984), describes a therapeutic regimen for uraemic sheep based on the in vivo response to infusions of plasma rich in erythropoietin and proposes a dosage of 10 U EOP / kg per day for 15-40 days as an antidote to anemia of the associated type with chronic renal failure. See also, Krane, Henry Ford Hosp. Med. J., 31 (3): 177-181 (1983). The identification and characterization of a new erythropoietin polypeptide derived from the chimpanzee, designated here as CHEPO, is described herein.

BRIEF DESCRIPTION OF THE INVENTION A cDNA clone having homology with the nucleic acid encoding human erythropoietin encoding a new chimpanzee erythropoietin polypeptide has been identified, designated in the present application as "CHEPO". In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a CHEPO polypeptide. In one aspect, the isolated nucleic acid molecule comprises the nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, isolate at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% of nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% identity 10 of nucleic acid sequence, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% sequence identity of 15 nucleic acid, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% acid sequence identity 20 nucleic acid, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with (a) a DNA molecule encoding a CHEPO polypeptide having the amino acid residue sequence from about 1 or so approximately 28 to about 193, inclusive, of Figure 3 (SEQ ID NOS: 2 and 5), or (b) the complement of the DNA molecule of (a). In another aspect, the isolated nucleic acid molecule comprises (a) a nucleotide sequence encoding a CHEPO polypeptide having the amino acid residue sequence from about 1 or approximately 28 to about 193, inclusive, of Figure 3 ( SEC ID NOS: 2 and 5), or (b) the complement of the nucleotide sequence of (a). In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% identity of ac sequence nucleic acid, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93 % nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity , alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with (a) a DNA molecule having the nucleotide sequence from about 1 or from about 82 to about 579, inclusive, of Figure 2 (SEQ ID NO: 3), or (b) the complement of the DNA molecule of (a). In another aspect, the isolated nucleic acid molecule comprises (a) the nucleotide sequence from about 1 or approximately 82 to about 579, inclusive, of Figure 2 (SEQ ID NO: 3), or (b) the complement of the nucleotide sequence of (a). In another aspect, the invention relates to an isolated nucleic acid molecule encoding an active CHEPO polypeptide as defined below comprising a nucleotide sequence that hybridizes to the complement of the nucleic acid sequence encoding amino acids 1 or roughly 28 to about 193, inclusive, of Figure 3 (SEQ ID NOS: 2 and 5). Preferably, hybridization occurs under severe conditions of hybridization and washing. In still another aspect, the invention relates to a isolated nucleic acid molecule encoding an active CHEPO polypeptide as defined below comprising a nucleotide sequence that hybridizes to the complement of the nucleic acid sequence encoding amino acids 1 or approximately 82 to approximately 579, inclusive, of the Figure 2 (SEQ ID NO: 3). Preferably, hybridization occurs under severe conditions of hybridization and washing. In a further aspect, the invention relates to an isolated nucleic acid molecule that is produced by hybridizing a test DNA molecule under severe conditions with (a) a DNA molecule encoding a CHEPO polypeptide having the sequence of the residues of amino acids from about 1 or approximately 28 to about 193, inclusive, of Figure 3 (SEQ ID NOS: 2 and 5), or (b) the complement of the DNA molecule of (a), and, if the DNA test molecule has at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least approximately 83% nucleic acid sequence identity, ...? ¡? AA? TÍAA ± LJ? alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% identity of nucleic acid sequence, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively to less about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% sequence identity of ac nucleic acid, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with (a) or (b), and isolating the test DNA molecule. In another aspect, the invention relates to an isolated nucleic acid molecule comprising (a) a nucleotide sequence encoding a polypeptide registry of at least about 80% positive, at least at least about 81% positive, alternatively at least about 82% positive, alternately at least about 83% positive, alternately at least about 84% positive, alternately at least about 85% positive, alternately at least about 86% positive, alternately at least about 87% positive, alternately at least about 88% positive, alternately at least about 89% positive, alternating at least about 90% positive, alternating at least about 91% positive, alternating at least about 92% positive, alternating at least about 93% positive, alternating at least about 94% positive, alternately at least approximately 95% positive, alternatively at least approximately 96% positive, alternatively at least approximately 97% positive, alternatively at least about 98% positive, alternatively at least about 99% positive 5 when compared to the amino acid sequence of about residues 1 or roughly 28 to about 193, inclusive, of Figure 3 (SEQ ID NOS) : 2 and 5), or (b) the complement of the nucleotide sequence of (a). In a specific aspect, the invention provides an isolated nucleic acid molecule comprising the DNA encoding a CHEPO polypeptide without the N-terminal signal sequence and / or the initiating methionine, or is complementary to such a coding nucleic acid molecule . Signal peptide 15 has been tentatively identified as extending from about position 1 of the amino acid to about position 27 of the amino acid in the sequence of Figure 3 (SEQ ID NOS: 2 and 5). It is noted, however, that the C-terminal boundary of the signal peptide may vary, but most likely is not greater than about 5 amino acids on one side of the C-terminal boundary of the signal peptide as initially identified, where the C-terminal limit of the peptide of signal can be identified consistently with the criteria commonly employed in the art to identify that type of amino acid sequence element (eg, Nielsen et al., Prot. Eng. 10: 1-6 (1997) and von Heinje et al. ., Nucí, Acids, Res. 14: 4683-4690 (1986)). Furthermore, it is also recognized that, in some cases, the cleavage of a signal sequence from a secreted polypeptide is not completely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding them, are contemplated by the present invention. As such, for the purposes of the present application, the signal peptide of the CHEPO polypeptide shown in Figure 3 (SEQ ID NOS: 2 and 5) extends from amino acids 1 to X of Figure 3 (SEQ ID. NOS: 2 and 5), where X is any amino acid from 23 to 32 of Figure 3 (SEQ ID NOS: 2 and 5). Therefore, the mature forms of the CHEPO polypeptide that are encompassed by the present invention, include those comprising amino acids X to 193 of Figure 3 (SEQ ID NOS: 2 and 5), wherein X is any amino acid from 23 to 32 of Figure 3 (SEQ ID NOS: 2 and 5) and variants thereof as described below. Nucleic acid molecules are also contemplated, * fej. * faith *, JJtiÜ < , .J, Mt, .JÉ jjfcjá¿Uk. isolated, which encode these polypeptides. Another embodiment is directed to fragments of a sequence encoding the CHEPO polypeptide, which may find use as, for example, hybridization probes or to encode fragments of a CHEPO polypeptide which may optionally encode a polypeptide comprising a binding site for a anti-CHEPO antibody. Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length. length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternatively at least about 200 nucleotides in length, alternatively at least about 250 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternatively at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in the ngitude, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length, alternatively at least approximately 1000 nucleotides in length, wherein in this context the term approximately means the length of the reference nucleotide sequence plus or minus 10% of that length to which reference is made. In a preferred embodiment, the fragment of the nucleotide sequence is derived from any coding region of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1). It is noted that the new fragments of a sequence encoding the nucleotide CHEPO polypeptide can be determined in a customary manner by aligning the nucleotide sequence encoding the CHEPO polypeptide with other known nucleotide sequences using any of a number of programs for alignment of well known sequences, and determining that the fragment (s) of the nucleotide sequence encoding the CHEPO polypeptide are new. All such nucleotide sequences encoding the CHEPO polypeptide are contemplated herein and can be determined with or without undue experimentation. Also contemplated are fragments of the CHEPO polypeptide encoded by these fragments of the nucleotide molecule, preferably those fragments of the CHEPO polypeptide comprising a binding site for an anti-CHEPO antibody. In another embodiment, the invention provides a vector comprising a nucleotide sequence encoding the CHEPO or its variants. The vector can comprise any of the isolated nucleic acid molecules, identified hereinabove. A host cell comprising such a vector is also provided. By way of example, the host cells can be CHO cells, E. coli, or yeasts. A process for producing the CHEPO polypeptides is further provided and comprises culturing host cells under conditions suitable for the expression of CHEPO and recovering CHEPO from the cell culture. In another embodiment, the invention provides the isolated CHEPO polypeptide, encoded by any of the isolated nucleic acid sequences, subsequently identified. In a specific aspect, the invention provides the CHEPO polypeptide of the native, isolated sequence, which in certain embodiments, includes an amino acid sequence comprising approximately residues 1 or approximately 28 to approximately 193 of Figure 3 (SEQ. ID NOS: 2 and 5).

, J? ^? IAA? M '' ^^ í'íííJ ^ > í ^^^^ i? In another aspect, the invention relates to an isolated CHEPO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity , alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% sequence identity from amino acids, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% identity of amino acid sequence, alternatively at least about 98% amino acid sequence identity, alternatively at least about 99% amino acid sequence identity with the sequence of the amino acid residues from about 1 or from about 28 to about 193, inclusive , of Figure 3 (SEQ ID NOS: 2 and 5).

In a further aspect, the invention relates to an isolated CHEPO polypeptide comprising an amino acid sequence register encoding at least about 80% positives, alternatively at least about 81% positives, alternatively at least about 82% positives, alternatively at least about 83% positive, alternately at least about 84% positive, alternating at least about 85% positive, alternating at least about 86% positive, alternating at least about 87% positive, alternating at least about 88% positive, alternating at least about 89 % positive, alternately at least about 90% positive, alternately at least about 91% positive, alternating at least about 92% positive, alternating at least about 93% positive, alternatively at least about 94% positive, alternating at least about 95% positive, alternating at least about 96% positive, alternating at least about 97% positive, alternating at least about 98% positive, alternating at least about 99% positive when compares with the amino acid sequence of approximately residues 1 or approximately 28 to approximately 193, inclusive, of Figure 3 (SEQ ID NOS: 2 and 5). In a specific aspect, the invention provides an isolated CHEPO polypeptide without the N-terminal signal sequence and / or the start methionine and is encoded by a nucleotide sequence encoding such an amino acid sequence as described herein above. Processes are also described here to produce them, in ** - wherein those processes comprise culturing a host cell comprising a vector comprising the appropriate nucleic acid molecule, under conditions suitable for the expression of the CHEPO polypeptide and recovering the CHEPO from the cell culture. In yet another aspect, the invention relates to an isolated CHEPO polypeptide, which comprises the amino acid residue sequence from about 1 or approximately 28 to about 193, inclusive, of Figure 3 (SEQ ID NOS: 2). and 5), or a fragment thereof that is biologically active or sufficient to provide a binding site for an anti-CHEPO antibody, wherein the identification of CHEPO polypeptide fragments that possess biological activity or provide a binding site for an anti-CHEPO antibody can be completed in a usual manner using techniques that are well known in the art. Preferably, the CHEPO fragment retains a biological, qualitative, activity of a native CHEPO polypeptide. In yet a further aspect, the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under severe conditions with (a) a DNA molecule encoding a CHEPO polypeptide that has the sequence of amino acid residues from about 1 or approximately 28 to about 193, inclusive, of Figure 3 (SEQ ID NOS: 2 and 5), or (b) the complement of the DNA molecule of (FIG. a), and if the test DNA molecule has at least about 80% identity of the nucleic acid sequence, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% identity of nucleic acid sequence, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least approximately 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alt ernatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% identity of nucleic acid sequence and alternatively at least about 99% nucleic acid sequence identity with (a) or (b), (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for the expression of a polypeptide, and (iii) recovering the polypeptide from the cell culture. In another embodiment, the invention provides chimeric molecules comprising a CHEPO polypeptide fused to a heterologous polypeptide or amino acid sequence, wherein the CHEPO polypeptide can comprise any CHEPO polypeptide, variant or fragment thereof. as described here above. An example of such a chimeric molecule comprises a CHEPO polypeptide fused to an epitope tag sequence or an Fc region of an immunoglobulin. In another modality, the invention provides an antibody as defined below that specifically binds to a CHEPO polypeptide as described herein above. Optionally, the antibody is monoclonal antibody, an antibody fragment or a single chain antibody. In yet another embodiment, the invention relates to the agonists and antagonists of a native CHEPO polypeptide as defined below. In a particular embodiment, the agonist or antagonist is a CHEPO antibody or a small molecule. In a further embodiment, the invention relates to a method for identifying agonists or antagonists of a CHEPO polypeptide comprising contacting a CHEPO polypeptide with a candidate molecule and monitoring a biological activity mediated by said CHEPO polypeptide. Preferably, the CHEPO polypeptide is a native CHEPO polypeptide. Still in a further embodiment, the invention is refers to a composition of importance comprising a CHEPO polypeptide, or an agonist or antagonist of a CHEPO polypeptide as defined herein, or an anti-CHEPO antibody, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier. Another embodiment of the present invention is directed to the use of a CHEPO polypeptide, or an agonist or antagonist thereof as described herein, or an anti-CHEPO antibody, for the preparation of a medicament useful in the treatment of a condition that is sensitive to the CHEPO polypeptide, an agonist or antagonist thereof or an anti-CHEPO antibody. Yet another embodiment of the present invention is directed to CHEPO polypeptides having altered glycosylation patterns in one or more regions of the polypeptide when compared to the native sequence CHEPO polypeptide, preferably in the region enclosing and / or including position 84 of the amino acid in the amino acid sequence CHEPO shown in Figure 3 (SEQ ID NOS: 2 and 5). In various embodiments, polypeptides of the CHEPO variant are prepared using well-known techniques so as to create an N- or O-linked glycosylation site at or near position 84 of the amino acid in the CHEPO polypeptide sequence. For example, CHEPO polypeptides contemplated by the present invention include those wherein (a) amino acids 81-84 of the CHEPO amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5) (ie, et-Glu-Val -Arg; SEQ ID NO: 6) are replaced by the amino acid sequence Asn-X-Ser-X (SEQ ID NO: 7) or Asn-X-Thr-X (SEQ ID NO: 8) ), where X is any amino acid except Pro; (b) amino acids 82-85 of the CHEPO amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5) (ie, Glu-Val-Arg-Gln; SEQ ID NO: 9) they are replaced by the amino acid sequence Asn-X-Ser-X (SEQ ID NO: 7) or Asn-X-Thr-X (SEQ ID NO: 8), where X is any amino acid except Pro; (c) amino acids 83-86 of the CHEPO amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5) (ie, Val-Arg-Gln-Gln, SEQ ID NO: 10) they are replaced by the amino acid sequence Asn-X-Ser-X (SEQ ID NO: 7) or Asn-X-Thr-X (SEQ ID NO: 8), where X is any amino acid except Pro; or (d) amino acids 84-87 of the CHEPO amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5) (ie, Arg-Gln-Gln-Ala; SEQ ID NO: 11 ) are replaced by the amino acid sequence Asn-X-Ser-X (SEQ ID NO: 7) or Asn-X-Thr-X (SEQ ID NO: 8), where X is any amino acid except Pro , therefore, an N-glycosylation site is created in those positions. The nucleic acids encoding these variant polypeptides are also contemplated herein as are the vectors and the host cells comprising those nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1C show a nucleotide sequence (SEQ ID NO: 1) of an isolated genomic DNA molecule containing a nucleotide sequence (nucleotides 134-146, 667-812, 1071). -1157, 1760-1939 and 2074-2226, exclusive of others) that codes for CHEPO of native sequence. Also present in the genomic sequence are the locations of the start codon, exons and introns as well as the amino acid sequence (SEQ ID NO: 2) encoded by the coding sequence of SEQ. ID. NO: 1. Figure 2 shows the cDNA sequence of the CHEPO molecule (SEQ ID NO: 3) and the amino acid sequence encoded therein (SEQ ID NO: 2). Figure 3 shows a comparison of the amino acid sequence of human (human) eptropoietin (SEQ ID NO: 4) and that of chimpanzee erythropoietin (chepo) described herein, wherein the amino acid designated X at position 142 of the The amino acid of the CHEPO sequence is either glutamine (SEQ ID NO: 2) or lysine (SEQ ID NO: 5).

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES I. Definitions The terms "CHEPO polypeptide", "CHEPO protein" and "CHEPO" as used herein encompass the native sequence CHEPO and the CHEPO polypeptide variants (which are further defined herein). CHEPO polypeptides can be isolated from a variety of sources, such as human or other source tissue types, or can be prepared by recombinant and / or synthetic methods. A "native sequence CHEPO" comprises a polypeptide having the same amino acid sequence as a CHEPO derived from nature. Such native sequence CHEPO can be isolated from nature or can be produced by recombinant and / or synthetic means. The term "native sequence CHEPO" specifically encompasses secreted or truncated forms that occur naturally (eg, an extracellular domain sequence), Sa ^ j .- -a ^ a-JJO &irifca variant s that occur naturally (example, alternately spliced s) and allelic variants that occur naturally from CHEPO. In one embodiment of the invention, the native sequence CHEPO is a mature CHEPO or full-length native sequence, comprising amino acids 1 to 193 of Figure 3 (SEQ ID NOS: 2 and 5). However, while the CHEPO polypeptides described in Figure 3 (SEQ ID NOS: 2 and 5) are shown to start with the methionine residues designated here as position 1 of the amino acid, it is conceivable and possible that other residues of methionine, located either upstream or downstream of position 1 of the amino acid in Figure 3 (SEQ ID NOS: 2 and 5) can be used as the starting amino acid residue the CHEPO polypeptide. The "CHEPO variant polypeptide" means an active CHEPO polypeptide as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 28 to 193 of the CHEPO polypeptide shown in Figure 3 (SEQ ID NOS: 2 and 5), (b) X to 193 of the CHEPO polypeptide shown in Figure 3 (SEQ ID NOS: 2 and 5), where X is any residue of amino acids - ** "$ ev from 23 to 32 of Figure 3 (SEQ ID NOS: 2 and 5) or (c) another fragment specifically derived from the amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5) Such polypeptides of the CHEPO variant include, example, CHEPO polypeptides wherein one or more amino acid residues are aggregated, or deleted, at the N- or C-terminal, as well as within one or more internal domains, of the sequence of Figure 3 (SEQ ID NOS: 2 and 5) Ordinarily, a polypeptide of the CHEPO variant will have at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, at least at least apr approximately 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least approximately 89% < w -NSS 31 amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively less about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity with (a) residues 1 or about 28 up to 193 of the CHEPO polypeptide shown in Figure 3 (SEQ. ID. NOS: 2 and 5), (b) X to 193 of the CHEPO polypeptide shown in Figure 3 (SEQ ID NOS: 2 and 5), where X is any amino acid residue from 23 to 32 of Figure 3 ( SEQ ID NOS: 2 and 5) or (c) another fragment specifically derived from the amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5).

The polypeptides of the CHEPO variant do not encompass the native CHEPO polypeptide sequence. Ordinarily, polypeptides of the CHEPO variant are at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length. amino acids of length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or more. The "percentage (%) of the amino acid sequence identity" with respect to the CHEPO polypeptide sequences, identified herein, is defined as the percentage of the amino acid residues in a candidate sequence which is identical with the amino acid residues in a CHEPO sequence, after aligning the sequences and entering the spaces, if necessary, to achieve the maximum percentage of sequence identity, and without considering any conservative substitution as part of the sequence identity . The alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved in various ways that are within the skills in the art, for example, by using a publicly available computer program such as the BLAST, BLAST-2 programs. ALIGN, ALIGN-2 or Megalign (DNASTAR). Those skilled in the art can determine the appropriate parameters for measuring the alignment, which includes any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared. For the purposes of the present, however,% of the amino acid sequence identity values were obtained as described below using the computer program for comparison of ALIGN-2 sequences, wherein the complete source code for the ALIGN program -2 is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authorized by Genentech, Inc. and the source code shown in Table 1 below was presented with user documentation in the Copyright Office of the United States of America , Washington DC, 20559, where it was registered under the Copyright Registry of the United States of America No. TXU510087. The ALIGN-2 program is available to the public through Genentech, Inc., South of San Francisco, California or can be compiled from the source code provided in Table 1. The ALIGN-2 program must be compiled to be used in the UNIX operating system, preferably UNIX V4.0D digital. All sequence comparison parameters are adjusted by the ALIGN-2 program and do not vary. For the purposes of the present,% amino acid sequence identity of an amino acid sequence A given to, with, or against a given amino acid sequence B (which may alternatively be combined as a given amino acid sequence A having or comprising a certain% sequence identity of amino acids a, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / And where X is the number of amino acid residues recorded as identical matches by the ALIGN-2 sequence alignment program in which the alignment program of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that when the length of the amino acid sequence A is not equal to the length of the amino acid sequence B,% amino acid sequence identity from A to B will not be equal to% amino acid sequence identity from B to A. As examples of% of amino acid sequence identity calculations, the following Tables 2 and 3 demonstrate how to calculate the% amino acid sequence identity of the amino acid sequence designated Protein Comparison to the designated amino acid sequence PRO. Unless otherwise specified, all values of% amino acid sequence identity used herein are obtained as described above using the computer program for comparison of ALIGN-2 sequences. However,% amino acid sequence identity can also be determined using the NCBI-BLAST2 sequence comparison program (Altshul et al., Nucleic Acids Res.

B = fe ^ fea ^ - A 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, MD. The NCBI-BLAST2 uses several search parameters, where all search parameters are adjusted as default values that include, for example, unmasked = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15 / 5, multiple step e value = 0.01, constant for multiple steps = 25, decrease for the alignment of the final spacing = 25 and score matrix = BLOSUM62. In situations where the NCBI-BLAST2 is used for amino acid sequence comparisons, the% amino acid sequence identity of an amino acid sequence A given to, with, or against a given amino acid sequence B (which can be alternatively naming as a given amino acid sequence A having or comprising a certain% sequence identity of amino acids a, with, or against a given amino acid sequence B) is calculated as follows: 100 times the X / Y fraction where X is the number of amino acid residues recorded as identical matches by the NCBI-BLAST2 sequence alignment program in which the alignment program of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that when the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B through A. The "CHEPO variant polynucleotide" or nucleic acid sequence of the CHEPO variant means a nucleic acid molecule that encodes an active CHEPO polypeptide as defined below and which has at least about 80% sequence identity of nucleic acid with either (a) a nucleic acid sequence encoding residues 1 or about 28 to 193 of the CHEPO polypeptide shown in Figure 3 (S) EC, ID NO: 2 and 5), (b) a nucleic acid sequence encoding residues X to 193 of the CHEPO polypeptide shown in Figure 3 (SEQ. ID. NOS: 2 and 5), where X is any amino acid residue from 23 to 32 of Figure 3 (SEQ ID NOS: 2 and 5) or (c) a nucleic acid sequence encoding another fragment specifically derived from the amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5). Ordinarily, a polynucleotide of the CHEPO variant will have at least about 80% nucleic acid sequence identity, alternatively at least * about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity , alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least approximate 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% sequence identity of nucleic acid, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% amino acid sequence identity with either (a) a nucleic acid sequence encoding residues 1 or about 28 to 193 of the CHEPO polypeptide shown in Figure 3 (SEQ ID NOS: 2 and 5), (b) a nucleic acid sequence encoding residues X to 193 of the CHEPO polypeptide shown in Figure 3 (SEQ ID NOS: 2 and 5), wherein X is any amino acid residue from 23 to 32 of Figure 3 (SEQ. ID Nos: 2 and 5) or (c) a nucleic acid sequence encoding another fragment specifically derived from the amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5). The variants of the CHEPO polynucleotide do not encompass the native CHEPO nucleotide sequence. Ordinarily, the polynucleotides of the CHEPO variant are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length , alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more. The "percent (%) identity of the nucleic acid sequence" with respect to the nucleic acid sequences encoding the CHEPO polypeptide, identified here, it is defined as the percentage of nucleotides in a candidate sequence that is identical to the nucleotides in the nucleic acid sequence encoding the CHEPO polypeptide, after aligning the sequences and entering the spaces, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining the percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for example, by using a publicly available computer program such as the BLAST, BLAST- 2 ALIGN, ALIGN-2 or Megalign (DNASTAR). Those skilled in the art can determine the appropriate parameters for measuring the alignment, which includes any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared. For the purposes of the present, however, the nucleic acid sequence identity% values were obtained as described below using the ALIGN-2 sequence comparison computer program, where the complete source code for the ALIGN program -2 is provided in Table 1. The computer program for sequence comparison ALIGN-2 was authorized by Genentech, Inc. and the source code shown in Table 1 has been presented with user documentation in the Copyright Office of the United States of America, Washington DC, 20559, where it was registered under the Copyright Registry of the United States of America No. TXU510087. The ALIGN-2 program is available to the public through Genentech, Inc., South of San Francisco, California or can be compiled from the source code provided in Table 1. The ALIGN-2 program must be compiled to be used in the UNIX operating system, preferably UNIX V4.0D digital. All sequence comparison parameters are adjusted by the ALIGN-2 program and do not vary. For purposes of the present, the% nucleic acid sequence identity of a C nucleic acid sequence given to, with, or against a given D nucleic acid sequence (which may alternatively be referred to as a nucleic acid sequence) C given that it has or comprises a certain nucleic acid sequence identity identity a, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the W / Z fraction where W is the number of nucleotides recorded as identical matches by the program for ALIGN-2 sequence alignment in which the program alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that when the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, the% identity of nucleic acid sequence from C to D will not be equal to the% identity of nucleic acid sequence from D to C. As the % examples of the nucleic acid sequence identity calculations, Tables 4 and 5 below show how to calculate the% nucleic acid sequence identity of the nucleic acid sequence designated as DNA of Comparison to the designated nucleic acid sequence PRO-DNA Unless otherwise specified, all values of% sequence identity of the nucleic acid, used herein are obtained as described above using the computer program for comparison of ALIGN-2 sequences. However,% nucleic acid sequence identity can also be determined using the NCBI-BLAST2 sequence comparison program (Altshul et al., Nucleic Acids Res. 25: 3389-3402 (1997)). The comparison program NCBI-BLAST2 sequences can be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, MD. The NCBI-BLAST2 uses several search parameters, where all search parameters are adjusted as default values including, for example, unmasked = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-step e = 0.01, constant for multiple steps = 25, decrease for the alignment of the final spacing = 25 and score matrix = BLOSUM62. In situations where NCBI-BLAST2 is used for sequence comparisons, the% nucleic acid sequence identity of a C nucleic acid sequence given to, with, or against a given D nucleic acid sequence (which it may alternatively be named as a given C nucleic acid sequence having or comprising a certain nucleic acid sequence identity% a, with, or against a given D nucleic acid sequence) is calculated as follows: 100 times the W fraction / Z where W is the number of nucleotides registered as identical matches by the NCBI-BLAST2 sequence alignment program in which the alignment program of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that when the length of the nucleic acid sequence C does not is equal to the length of the nucleic acid sequence D, the% nucleic acid sequence identity of C to D will not be equal to the% nucleic acid sequence identity of D up to C. In other embodiments, the polynucleotides of the CHEPO variant, are nucleic acid molecules that encode an active CHEPO polypeptide and that are capable of hybridizing, preferably under harsh washing and hybridization conditions, to nucleotide sequences encoding the full-length CHEPO polypeptide, as shown in the Figure 3 (SEQ ID NOS: 2 and 5). The polypeptides of the CHEPO variant can be those that are encoded by a polynucleotide of the CHEPO variant. The term "positives", in the context of the amino acid sequence identity comparisons performed as described above, includes the amino acid residues in the compared sequences that are not only identical, but also those that have similar properties. The amino acid residues that register a positive value at a residue of amino acids of interest are those that are either identical to the amino acid residues of interest or are a preferred substitution (as defined in Table 6 below) of the amino acid residues. of interest. For purposes of the present, the value of the% of positives of a given amino acid sequence A, with, or against a given amino acid sequence B (which may alternatively be referred to as a given amino acid sequence A having or comprising a certain% of positives a, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues that register a positive value as defined above by the ALIGN-2 sequence alignment program, in which the alignment program of A and B, and where Y is the total number of the amino acid residues in B. It will be appreciated that when the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% positive from A to B will not be equal to the% positive of B up to TO.

"Isolated", when used herein, describes various polypeptides described herein, means that the polypeptide has been identified and separated and / or recovered from a component of its natural environment. Preferably, the isolated polypeptide is free of association with all the components with which it is naturally associated. The contaminating components of their natural environment are materials that would typically interfere with the diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequencer, or (2) homogenizing by SDS- PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, stained with silver. The isolated polypeptide includes the polypeptide itself within the recombinant cells, since at least one component of the natural environment of CHEPO will not be present. Ordinarily, however, the isolated polypeptide will be prepared by at least one purification step. An "isolated" nucleic acid molecule that encodes A CHEPO polypeptide is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid encoding CHEPO. Preferably, the isolated nucleic acid is free of association with all the components with which it is naturally associated. A nucleic acid molecule that encodes CHEPO is different from one that is in the form or as it is found in nature. The isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecules encoding CHEPO as found in natural cells. However, an isolated nucleic acid molecule encoding a CHEPO polypeptide includes the nucleic acid molecules encoding CHEPO, contained in cells that ordinarily express CHEPO where, for example, the nucleic acid molecule is a chromosomal location different from that of natural cells. The term "control sequences" refers to the DNA sequences necessary for the expression of a coding sequence operably linked in a particular host organism. The control sequences which are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers. The nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader that is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences that are linked are contiguous, and, in the case of a secretory or secretory leader, contiguous and in a reading phase. However, breeders do not have to be contiguous. The linkage is carried out by ligation at convenient restriction sites. If such sites do not exist, oligonucleotide adapters or linkers MtJtjdlUtorfartuJ & É-. synthetic, are used in accordance with conventional practice. The term "antibody" is used in the broadest sense and specifically covers, for example, the unique anti-CHEPO monoclonal antibodies (including the agonist, the antagonist, and the neutralizing antibodies), the anti-CHEPO antibody compositions with polyepitopic specificity, single-chain anti-CHEPO antibodies, and fragments of anti-CHEPO antibodies (see below) The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of homogeneous antibodies substantially, i.e., the individual antibodies comprising the population, are identical except for possible naturally occurring mutations that may be present in smaller amounts. The "stringency or severity" of the hybridization reactions is easily determined by someone with ordinary skill in the art, and in general is an empirical calculation that depends on the length of the probe, the wash temperature, and the salt concentration. In general, longer probes require higher temperatures to carry a formation of acid molecules hybrid nuclei, adequate, while shorter probes need lower temperatures. Hybridization in general depends on the ability of the denatured DNA to re-form hybridized nucleic acid molecules when complementary strands are present in an environment below their melting temperature. The larger the desired degree of homology between the probe sequence and the hybridizable one, the higher the temperature that can be used. As a result, it follows that relatively high temperatures will tend to make the reaction conditions more severe, while lower temperatures will make them less severe. For additional details and explanation of the severity of hybridization reactions, see Ausubel et al. Current Protocols m Molecular Biology, Wiley Interscience Publishers, (1995). 'Severe or severe conditions' or * conditions of high stringency or severity, "as defined herein, may be identified as those that: (1) employ high temperature and low ionic strength for washing, eg, sodium chloride 0.015 M / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C, (2) employ during denaturation a denaturing agent, such as formamide, for example, 50% formamide (v / v) with albumin of 0.1% bovine serum / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm (50 g / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C, washed at 42 ° C in 0.2 x SSC (sodium chloride / sodium citrate) and formamide 50% at 55 ° C, followed by a high severity wash consisting of 0.1 x SSC containing EDTA at 55 ° C. * Moderately stringent or severe conditions "can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of a washing solution and hybridization conditions. (eg, temperature, ionic strength, and% SDS) less severe than those described above An example of moderately severe conditions is an overnight incubation at 37 ° C in a solution comprising: 20% formamide , SSC 5 x (150 mM NaCl, trisodium citrate 15 mM), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured salmon sperm DNA, followed by washing the filters in SSC 1 x about 37-50 ° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc., as necessary to accommodate factors such as probe length and the like. The term "labeled epitope" as used herein refers to a chimeric polypeptide comprising a CHEPO polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, however short it does not interfere with the activity of the polypeptide to which it is to be fused. The label polypeptide is preferably also moderately unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues). -. iM ^^ dm ^^^ a? kí? Má ^ ^ á? é * - lÍ í - -a ^ t ^ - & feaá¿-a £, ri¿lü As used in the present, the term ' immunoadhesin "designates antibody-like molecules that combine the binding specificity of a heterologous protein (an 'adhesin') with the effector functions of the immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is different from the recognition of the antigens and the binding site of an antibody (i.e., it is 'heterologous'), and a sequence of the domain Immunoglobulin constant The part of the adhesin of an immunsadhesin molecule is typically a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand.The sequence of the immunoglobulin constant domain in the immunoadhesin is can be obtained from any immunoglobulin, such as subtypes IgG-1, IgG-2, IgG-3, or IgG-4, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. activity "for the purpose of the present refers to the form (s) of CHEPO that retain the biological and / or immunological activity of native CHEPO or that occurs naturally, where biological activity refers to a function biol logic (either inhibitory or stimulatory) caused by a native CHEPO or that occurs naturally, different from the ability to induce the production of an antibody against an antigenic epitope possessed by a native CHEPO or that occurs naturally and a "Immunological activity" refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally occurring CHEPO. Preferred biological activities include, for example, the ability to regulate the production of red blood cells, to bind receptors on the surface of compromised progenitor cells of the spinal cord and / or other hematopoietic tissues and / or induce proliferation and / or terminal maturation of erythroid cells. The term "antagonist" is used in a broad sense, and includes any molecule that partially or totally blocks, inhibits, or neutralizes the biological activity of a native CHEPO polypeptide described herein In a similar manner, the term "agonist" is used in a broad sense and includes any molecule that mimics a biological activity of a native CHEPO polypeptide described herein. Suitable agonist or antagonist molecules specifically include the antibodies of the agonist or antagonist or the antibody fragments, fragments or variants of the amino acid sequence of the native CHEPO polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists or antagonists of a CHEPO polypeptide may comprise contacting a CHEPO polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the CHEPO polypeptide. 'Treatment' refers to both therapeutic and prophylactic treatment or preventive measures, where the objective is to prevent or delay (decrease) the target condition or pathological condition.Those who need treatment include those who already have the condition as well as to those who are prone to have the condition or those where the condition is to be prevented The "chronic" administration refers to the administration of the agent or agents in a continuous manner as opposed to an acute mode, so as to maintain the effect Initial therapeutic (activity) for an extended period of time. Intermittent administration is the treatment that not only occurs consecutively without interruption but is cyclic in nature.

"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport animals, or pet animals, such as dogs, cats, cows, horses, sheep, pigs , goats, rabbits etc. Preferably, the mammal here is a human. Administration "in combination with" one or more therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. "Carrier" as used herein includes carriers, excipients, or pharmaceutically acceptable stabilizers that are not toxic to the cells or to the mammal that will be exposed thereto at the doses and concentrations employed. Often the physiologically acceptable carrier is a buffered solution of aqueous pH. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid; the low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or aa akAa.iaiij lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDT.H; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN, polyethylene glycol (PEG), and PLURCNICS. The "antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or the variable region of the intact antibody. Examples of the fragments of the antibody .ncluyer the fragments Fab, Fab1, F? Ab ') 2 / and Fv; the diabodies; Linear antibodies? 2apata et al., Protein Eng. 8 (10): 1057- .062

[2995]); the df- single chain antibodies; and the multispecific antibodies formed in the fragments of antibodies. The digestion of papain and the anti- cancer produces two fragments of binding of identical antigens, "beloved fragments" Fab, poo binds with a unique anion binding site, and a residual fragment "Fc", a designation that is rejectable. The ability to crystallize easily The L pepsin treatment generates an F (ab '*' fragment that has two antigen-attracting sites and is capable of binding cr: z dcmenre to the antigen.

The "Fv" is a minimal antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of a variable domain of heavy chain and one of light chain, in adjustment with a non-covalent association. It is in this configuration that there are three CDRs of each variable domain interaction that define a binding site for the antigen on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprises only three CDRs specific for an antigen) that has the ability to recognize and bind antigens, albeit with a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragments differ from the Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain that includes one or more cysteines from the antibody binding region. Fab'-SH is the designation herein for the Fab 'in which the cysteine residue (s) of the constant domains contain a free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments which have articulation cysteines between them. Other chemical couplings of the antibody fragments are also known. The "light chains" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two distinct types, clearly called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into * subclasses (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The "Single chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the sFv to form the structure a 'na fülHfr- tit * í¿? M desired for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). The term "diabodies" refers to fragments of small antibodies with two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain ( VH - VL). Using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Nati Acad. Sci. USA, 90: 6444-6448 (1993). An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of its natural environment are materials that would interfere with therapeutic and diagnostic uses for the antibody, and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the antibody will be purified (1) to be greater than 95% by weight of the antibody as determined by the Lowry method, and more preferably more than 99% by weight, (2) to a sufficient degree to obtain the less 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequencer, or (3) homogenizing by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. The isolated antibody includes the antibody in situ within the recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, the isolated antibody will be prepared by at least one purification step. The word "label" when used herein refers to a detectable compound or composition that is directly conjugated to the antibody to generate a "labeled" antibody. The label may be detectable by itself (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze the chemical alteration of a compound or composition of the substrate that is detectable. By "solid phase" a nonaqueous matrix is indicated to the which antibody of the present invention can adhere. Examples of the solid phases encompassed herein include those formed partially or completely of glass (for example, glass with controlled pores), polysaccharides (for example, agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase may comprise the well of a test plate; in others it is a purification column (for example, an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in US Patent No. 4,275,149. A "liposome" is a small vesicle composed of various types of lipids, phospholipids and / or surfactants that are useful for delivering a drug (such as a CHEPO polypeptide or an antibody thereof) to a mammal. The components of the liposome are arranged in a common manner in a bilayer formation, similar to the lipid arrangement of the biological membranes. A small molecule is defined herein that has a molecular weight below about 500 Daltons.

I ^ ^ be ^ lbi} ? Mí ^ AíAméJ? Ss-Má & »**? Z ?? * "Il tA ..

Table 1 / * * * CC increased from 12 to 15 * Z is average of EQ * B is average of ND * corresponds with the stop is _M, stop-stop = 0, J (unknown) correspondence = 0 * / #defin? R _M -8 / * value of a correspondence or match with an arrest v * / int _day [26] [26] =. { / * ABCDEFGHIJKLMNOPQRSTU VWXYZ * / l * A * l 2, 0, -2, 0, 0, -4, 1, -1, -1, 0, -1, -2, -1, 0, _M, 1, 0, -2, 1.1.0, 0, -6, 0, -3, 0.}. , / * B * / 0, 3, -4, 3, 2, -5, 0, 1, -2, 0, 0, -3, -2, 2, _M, -1, 1, 0, 0, 0, 0, -2, -5, 0, -3, 1.}. , / * C * / í-2, -4, 15, -5, -5, -4, -3, -3, -2, 0, -5, -6, -5, -4, _M, - 3, -5, -4, 0, -2, 0, -2, -8, 0, 0, -5} , D * / 0, 3, -5, 4, 3, -6, 1, 1, -2, 0, 0, -4, -3, 2, _M, -1, 2, -1, 0, 0 , 0, -2, -7, 0, -4, 2.}. , / * £ * / 0, 2, -5, 3, 4, -5, 0, 1, -2, 0, 0, -3, -2, 1, _M, -1, 2, -1, 0 , 0, 0, -2, -7, 0, -4, 3.}. , I * p * / -4, -5, -4, -6, -5, 9, -5, -2, 1, 0, -5, 2, 0, -4, _M, -5, -5 , -4, -3, -3, 0, -1, 0, 0, 7, -5} , / * G * / 1, 0, -3, 1, 0, -5, 5, -2, -3, 0, -2, -4, -3, 0, _M, -l, -l, - 3, 1, 0, 0, -1, -7, 0, -5, 0.}. , / * H [-1, 1, -3, 1, 1, -2, -2, 6, -2, 0, 0, -2, -2, 2, _M, 0, 3, 2, -1 , -1, 0, -2, -3, 0, 0, 2.}. , / * j * / -1, -2, -2, -2, -2, 1, -3, -2, 5, 0, -2, 2, 2, -2, _M, -2, -2 -2, -l, 0, 0, 4, -5, 0, -1, -2} , 1 * 1 * 1 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0.}. , / * K * / -1, 0, -5, 0, 0, -5, -2, 0, -2, 0, 5, -3, 0, 1, _M, -1, 1, 3, 0 , 0, 0, -2, -3, 0, -4, 0.}. , l * L * l (-2, -3, -6, -4, -3, 2, -4, -2, 2, 0, -3, 6, 4, -3, _M, -3, - 2, -3, -3, -l, 0, 2, -2, 0, -1, -2.}., / * M * / -1, -2, -5, -3, -2, 0 , -3, -2, 2, 0, 0, 4, 6, -2, _M, -2, -l, 0, -2, -1, 0, 2, -4, 0, -2, -1 .}., / * N * / 0, 2, -4, 2, 1, -4, 0, 2, -2, 0, 1, -3, -2, 2, _M, - 1, 1, 0 , 1, 0, 0, -2, -4, 0, -2, 1.}., 1 * 0 * 1 _M, _M, _M, _M, _M, _M < _M, _M, _M, _M) _M , _M, _M, _M, 0, _M, _M, _M, _M, _M, _M, _M, _M! _M, _M, _M} I l *? * L 1, -1, -3, -1, -1, -5, -1, 0, -2, 0, -1, -3, -2, - 1, _M, 6, 0 , 0, 1, 0, 0, -1, -6, 0, -5, 0.}. , / * Q * / 0, 1, -5,2,2, -5, -1,3, -2,0, 1, -2, -1, 1, _M, 0.4, 1, -1 , -1.0, -2, -5.0, -4.3} , / * R * / -2, 0, -4, -1, -1, -4, -3, 2, -2, 0, 3, -3, 0, 0, _M, 0, 1, 6, 0, -1, 0, -2, 2, 0, -4, 0.}. , / * S * / 1, 0, 0, 0, 0, -3, 1, -1, -1, 0, 0, -3, -2, 1, _M, 1, -1, 0, 2, 1, 0, -1, -2, 0, -3, 0.}. , / * j * / 1, 0, -2, 0, 0, -3, 0, -1, 0, 0, 0, -1, -1, 0, _M, 0, -1, -1, 1 , 3, 0, 0, -5, 0, -3, 0.}. , / * U * / 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0.}. , / * v * / 0, -2, -2, -2, -2, -1, -1, -2, 4, 0, -2, 2, 2, -2, _M, -l, -2 -2, -l, 0, 0, 4, -6, 0, -2, -2} , / * w * / -6, -5, -8, -7, -7, 0, -7, -3, -5, 0, -3, -2, -4, -4, _M, -6 , -5, 2, -2, -5, 0, -6.17, 0, 0, -6} ,? * x *? 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0.}. , /* Y */ . { -3, -3, 0, -4, -4, 7, -5, 0, -1, 0, -4, -l, -2, -2, _M, -5, -4, -4, - 3, -3, 0, -2, 0, 0.10, -4} ,? * z *? . { 0, 1, -5, 2, 3, -5, 0, 2, -2, 0, 0, -2, -1, 1, _M, 0, 3, 0, 0, 0, 0, -2, -6, 0, -4, 4.}. } , ^ ^^^ ei ^^^^^^ - r ^^^^^^^^^^^^^^^^ - - - - - frat.1. i- 'Table 1 (cont.) * / # ¡Nclude < stdio.h > #include < ctype.h > #define MAXJMP 16 / * hops max. in a diag * / ^ define MAXSEP 24 / * does not continue to penalize spaces larger than this * / # Define JMPS 1024 / * max jumps. on a trajectory * / #define MX 4 / * save if there are at least MX-1 bases since the last jump * / ^ define DMAT 3 / * value of bases with correspondence * / # define DMIS 0 / * penalty for bases that do not correspond * / ^ define DINSO 8 / * penalty for a space * / #def? nir D1NS1 1 / * penalty for base * / # define PINSO 8 / * penalty for space * / #def? nir PINS1 4 / * penalty for waste * / short jmp structure [MAXJMP]; / * jump size (neg for dely) * / short not labeled xfMAXJMP]; / * base no. of jump of sec. x * / / * limits sec. to 2? 16 -l * / structure diag int score; / * score on the last jump * / long deviation; / * deviation from the previous block * / short isalto; / * index of current jumps * / salt structure. jp; / * jump list * / estruct tray { int espac; / * number of guide spaces * / short n [JMPS]; / * jump size (separation) * / int x [JMPS]; / * salt loe (last item before space) * / car * or file; / * output file name * / car * namex [2]; / * names of sec: getsecsQ * / car * prog; / * name prog for mens. of error * / car * secx [2]; / secs getsecs () * / int dmax; / * diag better: nw () * / int d axO; / * final diag * / int adn; / * adjust if adn: main () * / int sep. of extremes: / * adjust if extreme sep * / int sepx, sepy; / * total separations in secs * / int lenO, lenl; / * lens sec. * / int nsepx, nsepy; / * total size of separations * / int s ax; / * max score: nvv () * / int * xbm; / * bitmap for corresp. * / long desv .; / * current deviation in jump file * / struct diag * dx; / * diagonal retentions * / estruct tray pp [2J; / * tray. retentions for secs * / 10 car * calloc (), * mallot car * getsec (). * e cali fifteen •twenty Table 1 (cont.J / * Needleman-Wunsch alignment program * use: progs archivol file2 * where archivol and file2 are two DNA or two protein sequences. * The sequences may be in the upper or lower portion or may contain ambiguity * Any line that begins with ';', '> 'or' < 'ignored Max file length. is 65535 (limited by short x not labeled in the salt structure) A sequence with 1/3 or more of its ACGTU elements is considered to be DNA Output is in the file "align.out" * The program can create a tmp file in / tmp to hold info on backup. * Original version developed below BSD 4.3 in a vax 8650 * / #include "nw.h" #include "day.h" static _dbval [26] =. { 1, 14.2.13,0,0,4, 1 1, 0,0, 12,0,3, 15,0,0,0,5,6,8,8,7,9,0, 10,0 static _pbval [26] =. { 1. 2 | (1 «('D, -' A ')) | (1« (' N'-? ')), 4, 8, 16, 32, 64, 128, 256, OxFFFFFFF, 1 «10 , 1 «1 1, 1« 12, 1 «13. 1 «14. 1 «15, 1« 16, 1 «17, 1« 18, 1 «19. 1 «20. 1 «2 I, 1« 22, 1 «23, 1« 24, 1 «25 | (1« (,, -, A,)) | (1 «(, Q'-? ')) principal (ac, av) principal int ac; car * av [] { prog = av [0J; yes (ac! = 3). { fimprimirf (stderr, "use:% s archivol file 2 \ n", prog); fimppmirf (stderr, "where file I and file 2 are two DNA sequences or two proteins? n"); f? mprimirf (stderr, "Sequences can be in the upper or lower portion"); fimprimirf (stderr, "Any line that begins with '.' or '<' is ignored \ n"); fimprimirf (stderr, "Output is in the file \" align.out \ "\ n"); sahda (l); namex [0] = av [l]; namex [l] = av [2]; secx [0] = getsec (namex [0], & lenO); secx [l] = getsec (namex [l], & lenl); xbm = (adn)? _dbval: .pbval; end separations = 0; / * 1 to penalize end separations * / file or = "align.out"; / * output file * / nw (), / * fill in the matrix, get possible jumps * / leersalt (); / * adjust real jumps * / printO; / * print alignment, * / clean (O); / * do not link any tmp file * / Table 1 (cont.) / * Perform alignment, return to best score: principal () * adn: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983 * pro: PAM 250 values * When the scores are equal they prefer no correspondence to any space, prefer * a new space to extend a space in course, and prefer a space in secx * to a separation in sec and. * / nw () nw . { car * p ?, py, / secs and ptrs * / int * ndely, * dely; / * keep dely lane * / int ndelx, delx; / * keep delx lane * / int * tmp; / * to swap row, row * / int mis; / * score for each type * / int insO, insl; / * insertion of penalties * / register id; / * diagonal index * / register ij; / * jump index * / register * col0, * col l; / * record for last row, current * / register xx, yy, / * index in secs. * / dx = (struct diag *) g_calloc ("get diags", lenO + lenl + 1, size of (struct diag)); ndely = (int *) g_calloc ("get ndely", len l + 1, size of (int)); dely = (int *) g_calloc ("get dely", len 1 + 1, size of (? nt)); colO = (int *) g_calloc ("get colO", lenl + I, size of (int)) coi l = (int *) g_calloc ("get col l", lenl + 1, size of (? nt)); insO = (adn) 9 DINSO: PINSO; ins l = (adn)? DINS l. PINS 1; smax = - 10000; yes (end separations). { for (col0 [0] = dely [0] = -insO, yy = 1, yy < = len 1; yy ++). { colOfyy] = delyfyy] = col0 [yy-l] - insl; ndely [yy] = yy; J col0 [0] = 0, / * Waterman Bull Math Biol 84 * / J then for (yy = 1, yy < = len I, yy ++) dely [yy] = -insO, / * fill in correspondence matpz * / for (px = secx [0], xx = 1, xx < = IenO, px ++, xx ++). { / * start first entry in col * / si (end separations). { if (xx = 1) col l [0] = delx = - (? nsO +? nsl), then col 1 [0] = delx = col0 [0] - ins l, ndelx = xx,} so . { col l [0] = 0, delx = -insO, ndelx = 0.

Table 1 (cont.) ..nw for (py = secx [l], yy = 1; yy < = len 1; py ++, yy ++). { mis = colO [yy- l]; if (adn) my + = (xbm [* px-, A,] & xbm [* py-? '])? DMAT: DMIS; then my + = _day [* px-'A '] [* py-'A']; / * penalty for update for on sec x, * favor new envelope of the in progress * ignore M.AXSEP if end separations weigh more * / if (end separations || ndelyfyy] <MAXSEP). { yes (col0 [yy] - insO > = delyfyy]). { 10 delyfyy] = coIOfyy] - (insO + insl); ndelyfyy] = 1; } so . { delyfyy] - = insl; ndely [yy] ++; then yes (coIOfyy] - (insO + insl) > = delyfyy]). { delyfyy] = coIOfyy] - (insO + insl); 15 ndelyfyy] = 1; } then ndely [yy] ++; / * penalty for updating for the in sec and; * favor the new envelope of the in progress * / if (end separations || ndelx <MAXSEP). { 20 yes (col 1 [yy- 1] - insO > = delx). { delx = col 1 [yy- 1] - (insO + ins 1); ndelx = 1; } so . { delx - = insl; ndelx ++; } then yes (col 1 [yy- 1] - (insO + ins 1) > = delx). { delx = col 1 [yy- 1] - (ins + insl), ndelx = 1; } then ndelx ++; J / * collect the maximum record, we are favoring * missing about any of the and delx about dely * / 10 fifteen •twenty l * n Table 1 (cont.) .nw id = xx - yy + len 1 - 1; if (my > = delx & mis > = dely [yy]) col 1 [yy] = mis; then yes (delx > = delyfyy]). { col 1 [yy] = delx; ij = dx [id] .isalt; yes (dx [id] .jp.n [0] & (! adn || (ndelx > = MAXJMP & &xx> dx [id] .jp.x [ij] + MX) | | mis &gd; dx [id] .puntaje + DINSO)). { dx [id] .isalt ++; yes (++ ij> = MAXJMP). { escpbirsalt (id); ij = dx [id] .isalt = 0; dx [id], deviate = deviate; deviate + = size of (estruct salt) + size of (deviate); } dx [id] jp.n [ij] = ndelx; dx [id] .jp.x [ij] = xx; dx [id] score = delx; so . { col l [yy] = dely [yy]; ij = dx [id] .isalt; yes (dx [id] jp.n [0] & (! adn || (ndelyfyy] > = MAXJMP & &xx > dx [id] .jp.x [ij] + MX) | | my > dx [id] score + DINSO)). { dx [id] .isalt ++; yes (++ ij> = MAXJMP). { escribsalts (id); ij = dx [id] isalt = 0; dx [? d]. divert = divert; divert + = size of (struct jmp) + size of (bypass), dx [? d] .jp.n [? j] = -ndely [yy]; dx [id] .jp x [ij] = xx; dx [id]. score = dely [yy]; yes (xx = lenO & &y &< lenl). { / * last col * / si (end separations) collfyy] - = insO + insl * (lenl-y), if (col 1 [yy] > smax). { smax = coll [yy]; d ax = id; } } yes (end separations &&xx < lenO) coll [yy-l] - = ins0 + insl * (len0-xx); yes (coll [yy-l] > smax). { smax = coll [yy-l]; dmax = id; tmp = colO; colO = coll; coll = tmp; > (empty) hbre ((car *) ndely); (empty) free ((car *) dely) (empty) free ((car *) col0) (empty) free ((car *) coll) Table 1 (cont.) * imppmirO - only the routine visible outside this module * * static: * obtenmat () - backup of the traces of the best way, accounting or counting of correspondences: print () * pr_al? near () - alignment of print from what is described in the p []: print () * dum? block () - memory dump block-dump a block of lines with numbers, stars: pr_alinear () * nums () - put out a line from the number: memory dump block () * line () - place a line outside (name, [num], sec, [num]). memory dump block () * stars () - - put a line of stars: memory dump block () * removename () - remove any path and prefix from a sec name * / «Include" nw.h " «Define ESPAC 3« define P JNEA 256 / * maximum output line * / «define P ESPAC 3 / * space between name or number and sec * / external _day [26] [26]; int olen; / * reset the output line length * / FILE * fx; / * output file * / print () print int Ix, ly, first spacing, last spacing; / * overlap * / if ((fx = f ab? erto (or file, "w")) = 0). { fimprimir (stderr, "% s: can not write% s \ n", prog, or file); clean (l); } fimprim? rf (fx, "< first sequence-% s (length =% d) \ n", namex [0], lenO); fimprimirf (ßa, "< second sequence:% s (length =% d) \ n", namex [l], lenl); olen = 60; lx = lenO. ly = len l, first spacing = last spacing = 0, if (dmax < lenl - 1). { / * guide spacing in x * / pp [0]. spacing = first spacing = len l - dmax - 1; ly - = pp [0] spac, > then yes (dmax > lenl - 1). { / * sep guide in y * / pp [l]. spac = first spacing = dmax - (len l - 1); lx - = pp [l] spac; } yes (dmaxO < lenO - 1). { / * sep tail at x * / last spacing = lenO - dmaxO -1; Ix - = last spacing; } then yes (dmaxO > lenO - 1). { / * spacing of overlap in y * / last spacing = dmaxO - (lenO - 1); ly - = last spacing; } getmat (lx, ly, ppmer spacing, last spacing), pr_al? near (), Table 1 (cont.) / * * Trailing trace of the best path, count of correspondences * / static getmat (lx, l, first spacing, last spacing) getmat int lx. ly, / * "core" (less end spacings) * / int first spacing, last spacing, / * overlap of the guide tail * /. { int nm, lO, 11, tamO, tam 1, car salt? dax [32], double pet, record nO, record car * p0, * pl, / * obtain the total correspondences, score * /? 0 =? l = tam0 = taml = 0, pO = secx [0] + pp [l] spac, pl = secxfl] + pp [0] spac. n0 = pp [l] spac + 1, n 1 = pp [0] spac + 1, nm = 0, then (* p0 & * pl). { yes (tamO). { pl ++, n l ++, tamO-. then yes (ta l). { p0 ++, n0 ++, taml - then if (xbm [* pO-'A '] & xbm [* pl-?']) nm ++; yes (? 0 ++ = = pp [0] .x [? O]) tamO = pp [0] .n [iO ++]; if (nl ++ = = pp [l] .x [il]) taml = pp [l] .n [il ++]; p0 ++; pl ++; J / * pet homology: * if the end separations are pened, the base is the shortest sec * then, destroy the protrusions and take a shorter core * / si (end separations) 10 Ix = (len0 <lenl) ? len0: lenl; then lx = (lx < ly) 9 lx: ly; pct = 100, * (double) nm / (double) lx; fimprimirf (fx, "\ n"); fimprirmifffx, "<% d corresponds to% s in an overlap of% d:% .similarity of 2f percent \ n", nm, (nm = = l)? "": "is", lx, pet); fifteen •twenty ÍA, -jS.i »jJÍ-aa ... a-M £ ^" ". : li. 80 Table 1 (cont.) f? mprimirf (fx, "< separation in a first sequence:% d", sepx), ... getmat if (sepx). { (empty) simprimirf (outputx, "(% d% s% s)", nsepx, (adn)? "base": "residual", (nsepx = 1)? "": "s") fimprimirf (fx, " % s ", outputx); fimprimirf (fx, ", separations in a second sequence:% d", sepy); yes (sepy) { (empty) simpr? (sal? dax, "(% d% s% s)", nsepy, (adn)? "base": "residual", (nsepy = = 1)? "": "s"); f? mprim? rf (fx, "% s", outputx); } if (adn) fimirrimirf (fx, "\ n < score:% d (correspondence =% d, no correspondence =% d, separation penalty =% d +% d per base) \ n", smax, DMAT, DMIS , D1NS0, DINS 1); then fimprimirf (fx, "\ n < score:% d (Dayhoff matrix PAM 250, separation penalty =% d +% d per residue) \ n" smax, PINSO, PINS 1); if (separation of ends) fimprimirf (fx, '^ separations of pened ends, left end separations.% d% s% s, right end separations'% d% s% s \ n ", first spacing, (adn) ? "base": "residue", (first spacing = = 1)? "": "s", last spacing, (adn)? "base": "residue", (last spacing = = 1)? "": "s"), then fimprimirf (fx, "< extreme separations not pened \ n"); static nm; / * correspondences in the kernel - to verify * / static lmax; / * file name lengths deleted * / static Ü [2]; / * jump index for a path * / static nc [2]; / * number at the beginning of the current line * / static or [2], / * current element number - to form the spacing * / static tam [2]; static car * ps [2]; / * ptr to the current element * / static car * po [2]; / * ptr to the car slot of the next output * / static output car [2] [P_LINEA]; / * exit line * / static car star [P_LINEA]; / * adjustment using starsQ * / / * * print nment of what is described in the path structure [] * / static pr_ear () pr to n int nn; / * car count * / int more; register i; for (i = 0, lmax = 0, i <2; i ++). { nn = name removed (namex [i]); if (nn> lmax) lmax = nn; nc [i] = 1; ni [i] = l; tam [i] = ij [i] = 0; ps [i] = secxfi]; po [i] = outputfi]; Table 1 (cont.) for (nn = nm = 0, plus = 1; more;). { ... pr to align for (i - more = 0; i <2; i ++). { / * * Do we have more of this sequence? * / if (! ps [i]) continue; more-H-; yes (pp [i] spac). { / * guide space * / * po [i] ++ = ''; pp [i] espac-; J then yes (tam [i]). { / * in a space * / * po [i] ++ = '-'; tam [i] -, J then. { / * we are placing a sec element * / * po [?] = * ps [i]; yes (it is lower (* ps [?])) ps [i] = toupper (* ps [i]); po [?] ++, ps [i] ++, / * * Are we in the next space for this sec? * / si (ni [i] = = pp [i] .x [ij [i]]). { / * * we need to join all the separations * in this location * / tam [i] = pp [i] .n [ij [i] ++]; then (ni [i] = = pp [i] x [ij [i]]) tamfi] + = pp [i] .n [ij [i] ++]; ni [i] ++,} J if (++ nn = olen || 'more & amp; nn). { memory dump block (); for (i = 0; i < 2; i ++) po [i] = out [i]; nn = 0; / * * dump a block of lines, which include numbers, stars: pr_alinear () * / static dump dump block () memory dump block register i; for (i = 0, i <2, i ++) * po [i] «= '\ 0'; . J. .-- -É & hm? Mkr. -jaé t. iá,.? *? ¿, * ,. -r, d ¿^ * ^ ik ^ .tk¿ * * A.At, ^ ¿iA.-...:! I. - 84 Table 1 (cont.) ... memory dump block (empty) putcCW, fx); for (i = 0; i <2; i ++). { if (* output [i] & (* output [i]! = "|| * (po [i])! = '')) { si (i = = 0) nums (i); if (i = = 0 & * output [l]) stars (); put line (i); yes (i = = 0 & * outputfl]) fimprimirf (fx, star); yes (i) = = l) nums (i); } / *: put out a number line: memory dump block Q * / static nums (ix) nums int ix; / * index in and outf] the sec line that is maintained * / nline line [P_LINEA]; to register '. j. register car * pn, px, py; for (pn = nline, i = 0, i <lmax + P_ESPAC; i ++, pn ++) * pn = ''; for (i = ncfix], py = outfix]; * py; py ++, pn ++). { if (* py = = "|| * py = = '-') * pn: then yes (% 10 = = O || (i = = 1 & ncfix]! = 1)). { j = (i <0)? -i. i; for (px = pn; j, j / = 10, px-) * px = j% 10 + '0'; if (i <0) * px = '-'- J then * pn ='? ++; J J * pn = '\ 0', ncfix] = i; for (pn = nline, * pn; pn ++) (empty) putc (* pn, fx), (empty) putc ('\ n', fx), put out a line (name, [num], sec, [num]) dump block of memona () * / static put line (? x) put int line Table 1 (cont.) put line int record car px; for (px = xfixname), i = 0; * px & * px! = ':'; px ++, i ++) (empty) putc (* px, fx); for (; i < Imax + P_ESPAC; i ++) (empty) putc ('', fx); / * this count from 1: * ni [] is the current element (from 1) * ncf] is the number at the beginning of the current line * / for (px = outputfix]; * px; px ++) (empty) put c (* px &0x7F, fx): (empty) putc ('\ n', fx); / * * put a line of stars (the secs always inside outfO], outfl]): block of memory dump () * / static starsQ stars int i, register car * p0, * pl, cx, * px; if (! * output [0] || (* output [0] = = "& * (po [0]) = =") I! * output [l] | l (* output [l] = = "& & * (po [l]) = =")) return; px = star; for (i = lmax + P_ESPAC: i; i-) * px ++ = ''; for (pO = $ ahda [0], pl = sal? da [l], * pO && pl, pO ++, pl ++). { yes (esalfa (* p0) & ampalfa (* pl)). { yes (xbm [* pO-'A '] & xbm [* pl-'A']). { cx = '*', nm ++,} then if ('adn & _day [* pO-'A'] [* p l-'A ']> 0) cx = ", then cx =", then cx = ", * px ++ = cx, * px ++ = V, * px = '\ 0', ia ^ jtog ^^ A ^ Table 1 (cont.) / * * separate the path or prefix of pn, return len: pr_alinear () * / static remove the name (pn) remove the name car * pn; / * file name (can be path) * /. { register "car * px, * py; py = 0; for (px = pn;, px, px ++) if (* px = '/') py = px + l; if (py) (empty) strcpy (pn, py); returns r (strlen (pn)); ~ «I s < mn & kmi &? &é & m Table 1 (cont.) * clean () - clean any file tmp * get sequence () - read on the sec, adjust adn, len, maxlen. * g_ca! loc () - calloc () with verification error * leersaltsO - get the good jumps, from the tmp file if necessary * escpbirsalts () - write an array full of jumps to a tmp file: nw () * / "Include" nw h "« include < sys / archivo.h > car * jname = "/ tmp / homgXXXXXX"; / * tmp file for jumps * / FILE * fj; int l? mpiar (); / * clean file tmp * / long search I (); / * * delete any tmp file if indicated l? mp? ar (?) clean int if (fj) (empty) unlinkedname j); sal? da (i), / * * read, return ptr to sec, set adn, len, maxlen * jump lines that start with ';', '< ', or' > 'sec in the upper or lower portion * / car * get sequence (file, len) get sequence car * arch? vo; /* filename */ int * Ien; / * len sec * / car line

[1024], * psec; register car * px, * py; int natgc, tlen; FILE * fp; if ((fp = manufacture (file, "r")) = 0). { f? mpressionf (stderr, "% s: can not be 11er% s \ n", prog, file), output (l); } tlen = natgc = 0; while (fobteners (line, 1024, fp)). { if ("line = ';' ||" line = '<' || * line = '>') continue; for (px = line; * px! = '\ n'; px ++) if (top (* px) || is lower (* px)) tlen ++; } if ((psec = malloc ((without signaling) (tlen + 6))) = 0). { fimprimirf (stderr, "% s: malloc () failed to get% d bits for% s \ n", prog, tlen + 6, file); exit (l),} psec [0] = psec [l] = psec [2] = psec [3] = '\ 0', Table 1 (cont.). Get sec py = psec + 4; * len = tlen; rebob? nar (fp); while (fobteners (line, 1024, fp)). { if (* line = = ';' || * line = = '<' || * line = = &') continue; for (px = line; * px '= V; px ++). { yes (top (* px)) * py ++ = * px; then if (lower (* px)) * py ++ = upper (* px); yes (index ("ATGCU", * (py-l))) natgc ++; } } * py ++ = '\ 0'; * py = '\ 0'; (empty) fierrar (fp); adn = natgc > (tlen / 3); return (pSec + 4); car * g_calloc (msg, nx, sz) g_caIIoc car * msj; / * program, calling routine * / int nx, sz, / * number and size of the elements * /. { car * px, * calloc (); if ((px = calloc ((unsignalize) nx, (unsignalize) sz)) = 0). { yes (* msj). { fimprimirf (stderr, "% s: g_calloc () failure% s (n =% d, sz =% d) \ n", prog, msj, nx, sz); exit (l). } returns r (px); get final hops of dx [] or tmp file, adjust ppf], restart dmax: principalQ * / Ieersalts () leersalts. { int fd = -l; int tam iO, íl; register U, xx: yes (fj). { (empty) fierrar (fj); if ((id = open jnombre, 0_RDSOALLY, 0)) < 0). { fimprimirf (stderr, "% s: can not open ()% s \ n", prog, jname); clean (l); } } for (i = iO = 11 - = 0, dmaxO - dmax, xx = lenO;; i ++). { then (I). { for (j = dx [dmax] isalt; j> = 0 & dx [dmax] jp.x [j] > = xx; j-) Table 1 (cont.) ... leersalts if (j <0 && dxfdmax] deviate && fj). { (empty) search (fd, dxfdmax]. divert, 0); (empty) read (fd, (car *) & dx [dmax] jp, size of (jump structure)); (empty) Ieer (fd, (car *) & dx [dmax]. deflect, size of (dx [dmax], deviate)); dx [dmax] .isalt = MAXJMP-1; } then finish; } yes (i> = SALTS). { f? mprimirf (stderr, "% s: too many separations in the alignment \ n", prog), clean (l); } yes (j> = 0). { tam = dx [dmax] .jp.n [j]; xx = dx [dmax] .jp.x [j]; dmax + = tam; yes tam < 0). { / * separation in the second sec * / pp [l] .n [il] = -tam; xx + = tam; / * id = xx - yy + Ienl - I * / pp [l] .x [? l] = xx-dmax + len l-1; sepy ++; nsepy - = tam; / * ignore MAXSEP when end separations are done * / tam = (-tam <MAXSEP || end separations) '-tam MAXSEP, il ++; } then yes (tam> 0). { / * separation in the first sec * / pp [0] n [i0] = tam, pp [0] .x [¡0] = xx, sepx ++; nsepx + = tam; * r * r r A. JJjt a ,. *, ij.i'tÍi, r, ¿j femjanaaH "-» * - ^ - »- 'norar MAXSEP when the end separations are done * / tam = (tam <MAXSEP || separation of ends)' ' tam MAXSEP, 0 ++; } then finish; / * invert the order of the jumps * / for (j = 0, i0 ~; j <0, j ++, i0 ~). { i = pp [0] n [j]; pp [0] .nfj] = pp [0] .n [i0]; pp [0] .n [i0] = i; i = pp [0] xfj]; pp [0] .x [j] = pp [0] .x [i0]; pp [0] .x [i0] = i; } for < j = 0, i l -; j < il; j ++, il-). { i = pp [l] .nfj], pp [l] -nD] = ppfl] nfil], pp [l] .nfil] = i; i = pp [l].? [j]; ppflj.xfj] = PPfll-xfil]; PP [1]? [Il] = i; yes (fd > = 0) (empty) close (fd); i (j). { (empty) unlinked (name j), fj = 0; divert = 0; } } Table 1 (cont.) / * * write a full jump deviation of the previous one (yes it does exist): nw () * / write jumps (ix) write breaks int ix; . { car * mktemp (); yes (! «). { yes (mktemp (name j) < 0). { fimprimirf (stderr, "% s: can not mktemp ()% s \ n", prog, name j); clean (l); } if ((fj = manufactureirname, "w")) = 0). { fimprimirf (stderr, "% s: can not write% s \ n", prog, name j); exit (l); (empty) fwrite ((car *) & dx [ix] .st, size (struct salt), 1, fj); (empty) fwrite ((car *) & dx [ix]. deviate, size of (dx [ix]. deviate), 1, fj); Table 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) Comparison protein XXXXXYYYYYYY (Length = 12 amino acids) % amino acid sequence identity = (the number of amino acid residues that correspond identically between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) = 5 divided by 15 = 33.3% Table 3 PRO XXXXXXXXXX (Length = 10 amino acids) Comparison protein XXXXXYYYYYYZZYZ (Length = 15 amino acids) 5% amino acid sequence identity = (the number of amino acid residues that correspond identically between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) = 5 divided by 10 = 50% fifteen 'twenty 'Mi ^? ^ A ^^^^^ iß ^^^^^^^ M ^? ^ Isi ^ Su i ^? ^ B ^? ^ M Table 4 PRO-DNA NNNNNNNNNNNNN (Length = 14 nucleotides) Comparison DNA NNNNNNLLLLLLLLL (Length = 16 nucleotides) % nucleic acid sequence identity = (the number of nucleotides running identically between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) = 6 divided by 14 = 42.9% Table 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity = (the number of nucleotides that correspond identically between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) = 4 divided by 12 = 33.3% ^^^^ tttt «* jj ^^ l ^ j ^^^^^? g? ^^^ «^^^^ j II. Compositions and Methods of the Invention A. Full-Length CHEPO Polypeptides The present invention provides novel identified and isolated nucleotide sequences encoding the polypeptides referred to in the present invention as CHEPO. In particular, the DNA encoding a CHEPO polypeptide has been identified and isolated, as described in more detail in the following Examples.

B. CHEPO Variants In addition to the full-length native sequence CHEPO polypeptides described herein, it is contemplated that CHEPO variants can be prepared. CHEPO variants can be prepared by introducing the appropriate nucleotide changes into the CHEPO DNA, and / or by synthesis of the desired CHEPO polypeptide. Those skilled in the art will appreciate that amino acid changes can alter the post-translational processes of CHEPO, such as changing the number or position of the glycosylation sites or altering the anchoring characteristics of the membrane. Variations in the CHEPO of native sequence, of full length, or in various domains of CHEPO described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations established, for example, in U.S. Patent No. 5,364,934. The variations may be a substitution, deletion or insertion of one or more codons encoding CHEPO resulting in a change in the amino acid sequence of CHEPO compared to native sequence CHEPO. Optionally, the variation is by substitution of at least one amino acid with any other amino acid in one or more of the CHEPO domains. The guide for determining which amino acid residue can be inserted, replaced or deleted without adversely affecting the desired activity can be found by comparing the sequence of CHEPO with that of the homolog of known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. The amino acid substitutions can result in the replacement of an amino acid with another amino acid having similar chemical and / or structural properties, such as the replacement of a leucine with a serine, i.e., the replacements of conservative amino acids. The inserts or ^^^^ ¿¿¡^ ^ ^ X ^^^^ i ^ i ^ jA ?? deletions may optionally be in the range of 1 to 5 amino acids. The allowed variation can be determined by insertions, deletions or substitutions that are made systematically of the amino acids in the sequence and by testing the resulting variants for the activity exhibited by the native, mature, or full-length sequence. Fragments of the CHEPO polypeptide are provided herein. Such fragments may be truncated at the N-terminal or C-terminal, or they may lack internal residues, for example, when compared to a native, full-length protein. Certain fragments lack the amino acid residues that are not essential for a desired biological activity for the CHEPO polypeptide. CHEPO fragments can be prepared by any number of conventional techniques. The fragments of the desired peptide can be chemically synthesized. An alternative method involves the generation of CHEPO fragments by enzymatic digestion, for example, by treating the protein with a known enzyme for the cleavage proteins at the sites defined by the particular amino acid residues, or by digesting the DNA with the enzymes of appropriate restriction and isolating the desired fragment. Yet another suitable technique involves the isolation and amplification of a DNA fragment encoding a desired polypeptide fragment by the polymerase chain reaction (PCR). Oligonucleotides that define the desired terminal end of the DNA fragment are used in the 5 'and 3' primers in the PCR. Preferably, the CHEPO polypeptide fragments share at least one biological and / or immunogenic activity with the native CHEPO polypeptide shown in Figure 3. 10 (SEQ ID NOS: 2 and 5). In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in activity Biological, then more substantial changes, called exemplary substitutions in Table 6, or as further described below with reference to the amino acid classes are introduced into the selected products. t ^ «* - j.j & ^^? ^ k ^? MA s, A? á ^ ¿^ A¡í.

Table 6 Residue Substitutions Substitutions Original Exemplary Preferred Wing (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) be Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; wing wing His (H) asn; gln; lys; arg arg He (I) leu; val; met; to; leu phe; norlisucin Leu) norlieucine; ile; val; ile met; to; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; to; leu tyr Pro (P) wing wing Ser (S) thr thr Thr (T) be Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; to be phe Val (V) ile; leu; met; phe; leu wing; norleucinai Substantial modifications in the function or immunological identity of the CHEPO polypeptide are complemented by the selection of substitutions that differ significantly in their effect of maintaining (a) the structure of the polypeptide backbone in the area of the substitution, eg, as a sheet or helical conformation, (b) the hydrophobicity load of the molecule at the target site, or (c) the density of the side chain. The naturally occurring residues are divided into groups based on properties of common side chains: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence the orientation of the chain: gly, pro; and (6) aromatics: trp, tyr, phe. Non-conservative substitutions will exchange a member of one of these classes for another class. Such substituted residues can also be introduced at the conservative substitution sites or, more preferably, at the remaining (non-conserved) sites. Variations can be made using methods known in the art such as PCR mutagenesis, alanine scanning, oligonucleotide-mediated mutagenesis (site-directed). Site-directed mutagenesis [Cárter et al., Nucí. Acids Res., 13: 4331 (1986); Zoller et al., Nucí. Acids Res., 10: 6487 (1987)], cassette or cartridge mutagenesis [Wells et al., Gene, 3_4: 315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)], or other known techniques can be performed on the cloned DNA to produce the DNA of the CHEPO variant. Analysis of the amino acids by scanning can also be used to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are the relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is typically a preferred scanning amino acid among this group because it removes the side chain beyond beta-carbon and is less susceptible to altering the main conformation of the variant chain [Cunningham and Wells, Science, 244: 1081 -1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. In addition, it is frequently found both in hidden and exposed positions [Creighton, The Proteins, (W.H. Freeman &Co. N.Y.); Chothia, J. Mol. Biol., 150: 1 (1976)]. If the alanine substitution does not generate the adequate amounts of variant, an isoteric amino acid can be used.

C. Modifications of CHEPO Covalent modifications of CHEPO are included within the scope of this invention. One type of covalent modification includes the amino acid residues targeted by the reaction of the CHEPO polypeptide with an organic derivatizing agent that is capable of reacting with the selected side chains or the N- or C-terminal residues of CHEPO. Derivatization with bifunctional agents is useful, for example, for the cross-linking of CHEPO to a water-insoluble support matrix or surface for use in the method for the purification of anti-CHEPO antibodies, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- ? TikMe¿i¡my & & amp; They are also dithiobis (succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3- [(p-azidophenyl) dithio] propioimidate. Other modifications include the deamidation of the glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, the hydroxylation of proline and lysine, the phosphorylation of the hydroxyl groups of the seryl or threonyl residues, the methylation of the a-amino groups of the lateral chains of lisma, arginine, and histidine [TE Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], the acetylation of the N-terminal amine, and the amidation of any C-terminal carboxylic group. Another type of covalent modification of the polypeptide CHEPO included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. "Altering the native glycosylation pattern" is indicated herein with the purpose of eliminating one or more portions of carbohydrates found in a CHEPO of native sequence (either eliminating the underlying glycosylation site or suppressing glycosylation by chemical and / or enzymatic means), and / or adding one or more glycosylation sites that are not present in the native sequence CHEPO. In addition, the phrase includes qualitative changes in the glycosylation of native proteins, which imply a change in the nature and proportions of various carbohydrate moieties present. In addition to the glycosylation sites for the CHEPO polypeptide, they can be completed by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the CHEPO of native sequence (for glycosylation sites linked to oxygen). The amino acid sequence of the CHEPO polypeptide can optionally be altered through changes at the DNA level, particularly by mutation of the DNA encoding the CHEPO polypeptide at the preselected bases such as the codons that are generated that will result in the desired amino acids. . Another means of increasing the number of carbohydrate moieties in the CHEPO polypeptide is by chemical or enzymatic coupling of the glycosides to the polypeptide. Such methods are described in the art, for example, in WO 87/05330 published September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev.

Biochem. , pp. 259-306 (1981). The removal of the carbohydrate moieties present in the CHEPO polypeptide can be carried out chemically or enzymatically or by mutational substitution of codons encoding the amino acid residues which serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin, et al., Arch. Biochem. Biophys., 259: 52 (1987) and by Edge et al., Anal. Biochem. , 118: 131 (1981). Enzymatic cleavage of the carbohydrate moieties in the polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol. , 138: 350 (1987). Another type of covalent modification of CHEPO comprises linking the CHEPO polypeptide to one of a variety of non-protein polymers, for example, polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The CHEPO of the present invention can also be modified in such a way as to form a chimeric molecule comprising a CHEPO fused to another, the polypeptide heterologous or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of CHEPO with a tag or tag polypeptide which provides an epitope to which the anti-tag antibody can be selectively linked. The brand epitope is usually placed in the amino- or carboxy-terminus of CHEPO. The presence of such labeled forms of the CHEPO epitope can be detected using an antibody against the tag polypeptide. Also, the provision of the epitope tag allows CHEPO to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various brand polypeptides and their respective antibodies are well known in the art. Examples include the labels polyhistidine (poly-his) or poly-histidine-glycine (poly-his-gly); the HA-tagged polypeptide flu and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8: 2159-2165 (1988)]; the c-myc tag and antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10 thereto [Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)]; and the glycoprotein D (gD) tags of Herpes Simplex virus and its antibody [Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)]. Other polypeptides from marga include the Flag peptide [Hopp et al., BioTechnology, 6: 1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255: 192-194 (1992)]; an α-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991)]; and the peptide tag of the T7 gene 10 protein [Lutz-Freyermuth et al., Proc. Nati Acad. Sci. USA, 8J7: 6393-6397 (1990)]. In an alternative embodiment, the chimeric molecule may comprise a fusion of CHEPO with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an immunoadhesin), such fusion could be the Fc region of an IgG molecule. Ig fusions preferably include the substitution of a soluble form (deleted or inactivated transmembrane domain) of a CHEPO polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the fusion of the immunoglobulin includes the joint, CH2 and CH3, or the joint, the CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see also U.S. Patent No. 5,428,130 issued June 27, 1995.

^ »X ... l. - Á¡ * Á.? M¿ * t? .. Á *: D. Preparation of CHEPO The description that follows mainly relates to the production of CHEPO by cultured cells transformed or transfected with a vector containing the desired CHEPO nucleic acid. Of course, it is contemplated that alternative methods, which are well known in the art, can be employed to prepare CHEPO. For example, the CHEPO sequence, or portions thereof, can be produced by direct peptide synthesis using solid phase techniques [see, for example, Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Soc, 8_5: 2149-2154 (1963)]. The synthesis of the protein in vi tro can be done using manual techniques or by automation. Automated synthesis can be complemented, for example, by using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. Various portions of the desired CHEPO can be chemically synthesized separately and combined using chemical or enzymatic methods to produce full-length CHEPO. .-áiit ?, 1. Isolation of the DNA that Codifies CHEPO The DNA that codes for CHEPO can be obtained from a cDNA library prepared from the tissue that is believed to possess the desired CHEPO mRNA and express it at a level detectable Accordingly, the human CHEPO DNA can be conveniently obtained from a cDNA library prepared from human tissue, as described in the Examples. The gene encoding CHEPO can also be obtained from a genomic library or by known synthetic methods (e.g., automated nucleic acid synthesis). The libraries can be selected with probes (such as antibodies to the desired CHEPO or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. The selection or separation of the cDNA or the genomic library with the selected probe can be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative method to isolate the gene that codes for CHEPO is to use the PCR methodology [Sambrook et al., Supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)]. The following Examples describe the techniques for selecting or separating a cDNA library. The sequences of oligonucleotides selected as probes should be of sufficient length and not of sufficient ambiguity to minimize false positives. The oligonucleotide is preferably labeled such that it can be detected after hybridization to the DNA in the library being selected. Labeling methods are well known in the art, and include the use of radiolabels such as ATP labeled with 32P, biotinylation or enzyme labeling. Hybridization conditions include moderate severity and high severity, and are provided in Sambrook et al., Supra. Sequences identified in such library selection methods can be compared and aligned with other known sequences deposited and available in public databases such as GenBank or other databases of private sequences. Sequence identity (at any nucleotide or amino acid level) within defined regions of the molecule or through the sequence The full-length library can be determined using methods known in the art and as described herein. The nucleic acid having the protein coding sequence can be obtained by separating the selected cDNA or the genomic libraries using the deduced amino acid sequence described herein for the first time, and, if necessary, using the extension methods of conventional primers as described in Sambrook et al., supra, to detect the precursors and process the mRNA intermediates that may not have been reverse transcribed into the cDNA. 2. Selection and Transformation of Host Cells Host cells are transfected or transformed with cloning or expression vectors described herein for the production and culture of CHEPO in modified conventional nutrient media such as those suitable for the induction of promoters, the selection of transformants, or the amplification of genes that encode the desired sequences. The culture conditions, such as the medium, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, the principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., Supra. The methods of eukaryotic cell transfection and prokaryotic cell transformation are known to technicians with ordinary skills, for example, CaCl2, CaP04, by means of liposomes and electroporation. Depending on the host cell used, the transformation is performed using standard techniques appropriate for such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., Supra, or electroporation is generally used for prokaryotes. The infection with Agrobacterium um tumefaciens is used for the transformation of certain plant cells, as described by Shaw et al., Gene, 23: 315 (1983) and by the publication WO 89/05859 published on June 29, 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 5: 2: 456-457 (1978) may be employed. The general aspects of Transformations of the host or host system of mammalian cells have been described in U.S. Patent No. 4,399,216. Transformations in yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Nati Acad. Sci. (USA), 76: 3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations, for example, polybrene, polyornithine, can also be used. For various mammalian cell transformation techniques, see Keown et al., Methods in Enzymoloqy, 185: 527-537 (1990) and Mansour et al., Nature, 336: 348-352 (1988). Host cells suitable for cloning or expressing the DNA in the vectors herein include prokaryotes, yeasts, or higher eukaryotic cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, eg, Enterobacteriaceae such as E. coli. Several strains of E. coli are publicly available, such as strain MM294 from E. coli K12 (ATCC 31,446); E. coli X1776 (ATCC 31,537); the strain E. Coli W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serra tia, e.g., Serra tia marcescans, and Shigella, as well as Bacilli. such as B. subtilis and B. li cheniformi s (for example, B. licheniformis 41P described in DD 266,710 published April 12, 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is a particularly preferred host or main host because it is a common host strain for fermentations of the recombinant DNA product. Preferably, the host cells secrete minimal amounts of proteolytic enzymes. For example, strain W3110 can be modified to effect a genetic mutation in genes encoding proteins endogenous to the host, with examples of such hosts including strain 1A2 of E. coli W3110, which has the complete tonA genotype; strain 9E4 of E. coli W3110, which has the complete genotype tonA ptr3; the strain 27C7 of E. coli W3110 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT ka; strain 37D6 of E. coli W3110, which has the genotype the? fcj, jB £ k * ??. »í *. * 3A..m -Ai *. »., Complete tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs lilvG kan; strain 40B4 of E. coli W3110, which is strain 37D6 with a degP elimination mutation not resistant to kanamycin; and an E. coli strain having the mutant periplasmic protease described in U.S. Patent No. 4,946,783 issued August 7, 1990. Alternatively, the methods of in vitro cloning, for example, PCR or other reactions of the nucleic acid polymerase, are suitable. In addition to prokaryotes, eukaryotic microbes such as fungi or filamentous yeasts are suitable for cloning or expression of hosts for the vectors encoding the CHEPO polypeptide. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140

[1981]; EP 139,383 published May 2, 1985); Kluyveromyces guests (North American Patent No. 4,943,529; Fleer et al. , Bio / Technology, 9: 968-975 (1991)) such as, for example, K. lactis (MW98-8C, CBS683, CBS4574, Louvencourt et al., J. Bacteriol., 737

[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045;, K. wi ckeramii (ATCC 24,178), K. wal tii (ATCC 56,500), K. faith tjffiéte 4 **? * < ii-r-: * t, iV.v¡it? ¡. drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8: 135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreejrishna et al., J. Basic Microbiol., 28: 265-278

[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Nati, Acad. Sci. USA, 76: 5259-5263

[1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published October 31, 1990); and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium (WO 91/00357 published January 10, 1991), and Aspergillus us as A. nidulans (Ballance et al., Biochem. Biophys., Res. Commun., 11: 284-289

[1983], Tilburn et al., Gene, 26: 205-221

[1983], Yelton et al., Proc. Nati. Acad Sci. USA, 81: 1470-1474

[1984]) and A. niger (Kelly and Hynes, EMBO J., -4: 475-479

[1985]). Methylotropic yeasts are suitable herein and include, but are not limited to yeasts capable of growing in methanol, selected from the genus consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Host cells suitable for the expression of glycosylated CHEPO are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 line, transformed by SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol., 3_6: 59 (1977)); chmo hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Nati, Acad. Sci. USA, 77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and the mouse mammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriate host cells is considered to be within the skills in the art. 3. Selection and Use of a Replicable Vector Nucleic acid (for example, cDNA or genomic DNA) which encodes a desired CHEPO can be inserted into a replicable vector for cloning (DNA amplification) or for expression. Several vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector with a variety of procedures. In general, the DNA is inserted into an appropriate restriction endonuclease site or sites using techniques known in the art. The vector components in general include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The construction of suitable vectors containing one or more of these components employs standard ligation techniques that are known to a skilled technician. CHEPO can be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the protein or mature polypeptide. In general, the signal sequence can be a component of the vector, or it can be a part of the DNA that codes for CHEPO that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, lpp, or the heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, for example, the yeast invertase leader, the alpha factor leader (which includes the leaders of the a factor of Saccharomyces and Kl uyveromyces, the last described in the US Patent No 5,010,182), or the leader of acid phosphatase, the leader of glucoamylase of C. albicans (EP 362,179 published April 4, 1990), or the signal described in WO 90/13646 published November 15, 1990. In mammalian cell expression, mammalian signal sequences can be used to direct the secretion of the protein, such as the signal sequences of the secreted polypeptides of the same or related species, as well as viral secretory leaders. Both the expression and cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication of plasmid pBR322 is suitable for most Gram-negative bacteria, 'the origin of plasmid 2μ is suitable for yeast, and several viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. The expression and cloning vectors will typically contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, eg, ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic differences, or (c) supply critical nutrients not available from the complex medium , for example, the gene encoding D-alanine racemase for Bacilli. An example of the selectable markers for mammalian cells are those that allow identification of the cells competent to take the nucleic acid encoding CHEPO, such as DHFR or thymidine kinase. An appropriate host cell when wild-type or wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Nati Acad. Sci.

USA, 77: 4216 (1980). A suitable selection gene for use in yeast is the trpl gene present in yeast plasmid YRp7 [Stinchcomb et al., Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980)]. The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)]. Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding CHEPO to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [de Boer et al., Proc. Nati Acad. Sci. USA, 80: 21-25 (1983)]. Promoters for use in bacterial systems will also contain a Shine-sequence Delgarno (S.D.) operably linked to the DNA encoding CHEPO. Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)] or other glycolytic enzymes [Hess et al. , J. Adv. Enzyme Res., 7: 149 (1968)]; Holland, Biochemistry, .17: 4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase , isomerase of triosefosfato, isomerasa of phosphoglucose, and glucokinase. Other promoters of yeasts, which are inducible promoters that have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocitochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the use of maltose and galactose. Suitable vectors and promoters for use in the expression of yeast are further described in EP 73,657.

Transcription of the CHEPO polypeptide of the vectors into mammalian host or host cells is controlled, for example, by promoters obtained from the genomes of the viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published July 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, poultry sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and Simian Virus 40 (SV40), from the promoters of heterologous mammals, for example, the promoter of actin or an immunoglobulin promoter, and calorific shock promoters, provided that such promoters are compatible with the host cell systems. The transcription of a DNA encoding CHEPO by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. The enhancers are cis-acting elements of DNA, usually approximately 10 to 300 bp, which act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, a eukaryotic cell virus enhancer will be used. Examples include the improver Ato taarittaaiS-fct SV40 on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancers. The enhancer can be spliced into the vector at the 5 'or 3' position to the CHEPO coding sequence, but preferably it is located at a 5 'site of the promoter. Expression vectors used in host, eukaryotic cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells of other multicellular organisms) will also contain sequences necessary for the termination of transcription and for the stabilization of mRNA. Such sequences are commonly available from the 5 'and occasionally 3' untranslated regions of the viral or eukaryotic cDNAs or DNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding CHEPO. Still other methods, vectors, and host cells suitable for adaptation to the synthesis of CHEPO in recombinant vertebrate cell cultures are described in Gething et al., Nature, 293: 620-625 (1981).; Mantei et al., Nature, 281: 40-46 (1979); EP 117,060; and EP 117,058.

. Detection of the Amplification / Expression Gene The amplification and / or expression of the gene can be measured in a sample directly, for example, by staining or conventional Southern blotting, spotting or Northern blotting to quantitate the transcription of the mRNA [Thomas, Proc. Nati Acad. Sci. USA, T7: 5201-5205 (1980)], spot spotting (DNA analysis), or hybridization in itself, using an appropriately labeled probe, are based on the sequences provided herein. Alternatively, antibodies can be employed, which recognize specific duplexes or duplexes, including DNA duplexes, RNA duplexes, and duplexes of DNA-RNA hybrids or duplexes or DNA-protein duplexes. The antibodies in turn can be labeled and assays can be carried out where the duplex or double binds to the surface, so that after the formation of the duplex on the surface, the presence of the antibody bound to the surface can be detected. duplex or double. The expression of the gene, alternatively, can be measured by immunological methods, such as immunohistochemical staining of cells or sections of tissues and assays of cell cultures or body fluids, to directly quantitate the expression of the gene product.

Antibodies useful for staining and / or immunohistochemical testing of sample fluids can be either monoclonal or polyclonal, and can be prepared in any mammal. Conveniently, the antibodies can be prepared against a CHEPO polypeptide of native sequence or against a synthetic peptide based on the DNA sequences provided herein or against the exogenous sequence fused to a DNA of the CHEPO polypeptide and encoding an epitope of the specific antibody. 5. Purification of the Polypeptide The CHEPO forms can be recovered from the culture medium or host cell lysates. If the membrane bound, it can be released from the membrane using a suitable detergent solution (eg, Triton-X 100) or by enzymatic cleavage. Cells used in the expression of CHEPO can be broken by various physical or chemical means, such as freezing cyclization, sonication, mechanical disruption, or cell lysate agents. It may be desired to purify CHEPO from recombinant cell proteins or polypeptides. The following procedures are exemplary of the jS¿fc > iü¿SSAitj, IÉaki.i suitable purification procedures: by fractionation in an ion exchange column; precipitation with ethanol; Reverse phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; precipitation with ammonium sulfate; gel filtration using, for example, Sephadex G-75; Sepharose columns of protein A to remove contaminants such as IgG; and chelating metal columns to link forms labeled with epitope of CHEPO. Various methods of protein purification can be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification steps selected will depend, for example, on the nature of the purification processes used and the particular CHEPO produced.

E. Uses for CHEPO The nucleotide sequences (or their complements) that encode CHEPO have several applications in the field of molecular biology, including uses as = »V < ¿. * Hybridization probes, in the mapping of chromosomes and genes, and in the generation of RNA and antisense DNA. The CHEPO nucleic acid will also be useful for the preparation of the CHEPO polypeptides by the recombinant techniques described herein. The full-length native sequence CHEPO cDNA (SEQ ID NO: 3), or portions thereof, can be used as hybridization probes for a cDNA library to isolate the full length CHEPO cDNA or 10 to further isolate other cDNAs (e.g., those naturally occurring variants, encoding CHEPO or CHEPO from other species) having a desired sequence identity to the native CHEPO sequence, described in Figure 2 (SEQ. ID NOS: 3). Optionally, the length 15 of the waves will be from about 20 to about 50 bases. Hybridization probes can be derived from at least partially the new regions of the SEC nucleotide sequence. ID. NO: 3, where those regions can be determined without experimentation Or from genomic sequences that include CHEPO promoters, enhancer elements and introns of native sequence. By way of example, a selection or separation method will comprise isolating the region of encoding the CHEPO gene that uses the known DNA sequence to synthesize a selected probe of approximately 40 bases. Hybridization probes can be labeled by a variety of labels, including radionucleotides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin / biotin coupling systems. The labeling probes have a sequence complementary to that of the CHEPO gene of the present invention and can be used to select libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries will hybridize the probe. Hybridization techniques are described in more detail in the following Examples. Any EST sequence described in the present application can be used similarly as probes, using the methods described herein. Other useful fragments of the CHEPO nucleic acids include the sense or antisense oligonucleotides comprising the single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to the target CHEPO mRNA (sense) or the DNA sequences of the CHEPO. CHEPO (antisense). The sense and antisense oligonucleotides, according to the present invention, they comprise a fragment of the coding region of the CHEPO DNA. Such a fragment generally comprises at least about 14 nucleotides, preferably about 14 to 30 nucleotides. The ability to derive a sense or antisense oligonucleotide, based on a DNA sequence encoding a given protein is described, for example, in Stein and Cohen (Cancer Res. 48: 2659, 1988) and van der Krol et al. (BioTechniques 6: 958, 1988). Binding of the sense or antisense oligonucleotides to the target nucleic acid sequences results in the formation of doubles or duplexes that block the transcription or translation of the target sequence by one of several means, including improved degradation of the doubles or duplex, the premature termination of transcription or translation, or by other means. The antisense oligonucleotides can therefore be used to block the expression of CHEPO proteins. Sense or antisense oligonucleotides further comprise oligonucleotides having modified sugar phosphodiester backbones (other than sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. . Such oligonucleotides with resistant sugar bonds are stable in vivo (i.e., capable of resisting enzymatic degradation), but retain the sequence specificity to be capable of binding to the target nucleotide sequences. Other examples of the sense and antisense oligonucleotides include those oligonucleotides that are covalently linked to the organic moieties, such as those described in WO 90/10048, and other portions that increase the affinity of the oligonucleotide for a target nucleic acid sequence, such as poly. - (L-lysine). further, intercalating agents, such as ellipticine, and alkylating agents or metal complexes can be attached to the sense and antisense oligonucleotides to modify the binding specificities of the sense or antisense oligonucleotide to the target nucleotide sequence. Sense and antisense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any method of gene transfer, including, for example, CaP0-mediated transfection of DNA, electroporation, or by using the transfer vectors of genes such as the Epstein-Barr virus.

In a preferred procedure, the sense and antisense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from MuLV), or double copy vectors, designated DCT5A, DCT5B and DCT5C (see WO 90/13641). . The sense and antisense oligonucleotides can also be introduced into a cell containing the target nucleotide sequence by the formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules, include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or blocks the entry of the sense or antisense oligonucleotide or its conjugated version in the cell. Alternatively, a sense or antisense oligonucleotide can be introduced into a cell containing the target nucleic acid sequence by forming an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex preferably dissociates within the cell by an endogenous lipase. The probes can also be used in PCR techniques to generate a set of sequences for the identification of closely related CHEPO coding sequences. The nucleotide sequences encoding a CHEPO can also be used to construct hybridization probes for the mapping of genes encoding that CHEPO and for the genetic analysis of individuals with genetic diseases. The nucleotide sequences provided herein can map to a chromosome and specific regions of a chromosome using known techniques, such as in-situ hybridization, binding analysis against known chromosomal markers, and selection of hybridization with libraries. When the coding sequences for CHEPO encodes a protein which binds to another protein (eg, where CHEPO is a receptor), CHEPO can be used in assays to identify other proteins or molecules involved in the binding interaction. By such methods, inhibitors of receptor / ligand linkage interaction can be identified. The proteins involved in such binding interactions can also be used to select peptides or inhibitors of small molecules or agonists of the binding interaction. Also, the CHEPO receptor can be used to isolate the ligand or ligands correlated. Selection assays can be designed to find leader or leader compounds that mimic the biological activity of a native CHEPO polypeptide or a receptor for CHEPO. Selection trials will include assays capable of highly specific selection of chemical libraries, making them particularly suitable for identifying candidates for small molecule drugs. The contemplated small molecules include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical separation or screening assays, immunoassays and cell-based assays, which are well characterized in the art. Nucleic acids that encode a CHEPO or its modified forms can also be used to generate either transgenic animals or "tagged" animals which, in turn, are useful in the development and selection of therapeutically useful reagents. A transgenic animal (e.g., a rat or mouse) is an animal that has cells that contain a transgene, said transgene was introduced into the animal or an ancestor of the animal in a prenatal stage, e.g., an embryonic stage. A transgene is a DNA that is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, the cDNA encoding a CHEPO can be used to clone the genomic DNA encoding CHEPO according to established techniques and the genomic sequences used to generate the transgenic animals that contain the cells expressing the DNA encoding CHEPO . Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells will be the target for incorporation of the CHEPO transgene with tissue-specific improvers. Transgenic animals that include a copy of a transgene encoding a CHEPO introduced into a germline of the animal at an embryonic stage can be used to examine the effect of increased expression of the DNA encoding CHEPO. Such animals can be used as test animals for reagents to confer protection from, for example, the pathological conditions associated with their overexpression. According to this facet of the invention, an animal is treated with the reagent and the incidence of the pathological condition is reduced, compared to untreated animals carrying the transgene, which would indicate a potential therapeutic intervention for the pathological condition. Alternatively, the non-human CHEPO homologs can be used to construct a "non-transgenic" animal of CHEPO that has a defective or altered gene that codes for CHEPO as a result of homologous recombination between the endogenous gene encoding CHEPO and genomic DNA altered that encodes CHEPO introduced into an embryonic stem cell of the animal. For example, the cDNA encoding a CHEPO can be used to clone the genomic DNA encoding CHEPO according to established techniques. A portion of the genomic DNA that encodes a CHEPO can be deleted or replaced with another gene, such as. a gene that encodes a selectable marker that can be used to monitor the integration. Typically, several kilobases of unaltered flanking DNA (both at the 5 'and 3' ends) are included in the vector [see for example, Thomas and Capecchi, Cell, 51: 503 (1987) for a description of homologous recombination vectors ] The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells are selected in which the introduced DNA has recombined homology with the endogenous DNA [see for example, Li et al. , Cell, 69: 915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see for example, Bradley, in Tera tocacinomas and Embryoni c Stem Cells: A Practical Approach, EJ Robertson, ed. . (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted in a suitable pseudopregnant female animal and the embryo is brought to term to create a "marked" animal. By harvesting the progeny, the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed more animals in which all the cells of the animal will contain the homologously recombined DNA. The labeled animals can be characterized, for example, by their ability to defend against certain pathological conditions and by developing pathological conditions due to the absence of the CHEPO polypeptide. The nucleic acid encoding the CHEPO polypeptides can also be used in gene therapy. In the applications of gene therapy, the genes are introduced into the cells to achieve the synthesis in vivo of an effective therapeutic gene product, for example for the replacement of a defective gene. "Genetic therapy" includes both conventional gene therapy wherein a permanent effect is achieved by a simple treatment, and administration of the genetic therapeutic agents, involving a single administration or repeated administration of therapeutically effective mRNA or DNA. RNAs or antisense DNAs can be used as therapeutic agents to block the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors despite their low intracellular concentrations caused by their restricted absorption by the cell membrane. (Zamecnik et al., Proc. Nati.

Acad. Sci. USA 8_3: 4143-4146

[1986]). The oligonucleotides can be modified to improve their absorption, for example, by replacing their negatively charged phosphodiester groups with non-charged groups. There are a variety of techniques available to introduce nucleic acids into viable cells. The techniques vary depending on whether the nucleic acid is transferred to cells cultured in vi tro, or in vivo in the cells of the intended host. Suitable techniques for the transfer of nucleic acid in mammalian cells include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the method of calcium phosphate precipitation, etc. Currently preferred in vivo gene transfer techniques include transfection with viral vectors (typically retroviral) and viral-coated liposome-mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210

[1993]). In some situations it is desirable to provide the nucleic acid source with an agent targeting the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. When they are used liposomes, proteins that bind to a cell surface membrane protein associated with endocytosis can be used to target and / or facilitate absorption, for example, capsid proteins or fragments of the same tropics for a type of particular cell, the antibodies for the proteins that undergo internalization in cycles, the proteins that point to the intracellular localization and improve the intracellular half-life. The technique of endocytosis mediated with the receptor is described, for example, by Wu et al. , J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al. , Proc. Nati Acad. Sci. USA 87, 3410-3414 (1990). For review of gene labeling and gene therapy protocols see Anderson et al. , Science 256, 808-813 (1992). The CHEPO polypeptides described herein can also be used as molecular weight markers for the purposes of electrophoresis of the protein and isolated nucleic acid sequences can be used to recombinantly express those markers. The nucleic acid molecules encoding the CHEPO polypeptides or fragments thereof described herein are useful for the identification of chromosomes. In this regard, there is a current need to identify new chromosome markers, since only some chromosome labeling reagents, based on current sequence data, are currently available. Each CHEPO nucleic acid molecule of the present invention can be used as a chromosome marker. The CHEPO polypeptides and nucleic acid molecules of the present invention can also be used to classify the type of tissue, wherein the CHEPO polypeptides of the present invention can be expressed differently in one tissue when compared to another. The CHEPO nucleic acid molecules will be used to generate probes for PCR, Northern analysis, Southern analysis and Western analysis. The CHEPO polypeptides described herein are also used as therapeutic agents. The CHEPO polypeptides of the present invention can be formed according to known methods for preparing the pharmaceutically useful compositions, whereby the CHEPO product thereof is combined in a mixture with the pharmaceutically acceptable carrier vehicle. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with the carriers, excipients or stabilizers.

Physiologically acceptable (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are not toxic to containers at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants that include ascorbic acid; the low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates that include glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; the counterions that form salts such as sodium; and / or non-ionic surfactants such as TWEEN ™, PLURONICS ™ or PEG. The formulations to be used in vivo must be sterile. This is easily completed by filtering through the sterile filtration membranes, before or after lyophilization and reconstitution. Therapeutic compositions herein are generally placed in a container having a sterile access port, for example, a bag or vial of intravenous solution having a stopper pierceable by a needle for hypodermic injection. The route of administration is in accordance with known methods, for example, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes, topical administration or by sustained release systems. The dosages and desired drug concentrations of the pharmaceutical compositions of the present invention may vary depending on the particular use visualized. The determination of the appropriate dosage or route of administration is well within the skills of an ordinary technician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. The escalation between species of the effective doses can be done following the principles determined by Mordenti, J. and Chappell, W. "The use of interspecies scaling in ? ± iaAUÁL.

Toxicokinetics "In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Pr New York 1989, pp. 42-96 When the in vivo administration of a CHEPO polypeptide or agonist or antagonist thereof is used, the amounts of normal dosages may vary from about 10 ng / kg to 100 mg / kg of the body weight of the mammal or more per day, preferably about 1 μg / kg / day to 10 mg / kg / day, depending on the route of Administration Dosage guidance and particular delivery methods are provided in the literature, see, for example, U.S. Patent Nos. 4,657,760, 5,206,344, or 5,225,212 It is anticipated that the different formulations will be effective for different treatment compounds and different conditions, that the objective of administration to the organ or tissue, for example, may need the supply in a different way from that to a different tissue or organ. if the administration of sustained release of a CHEPO is desired in a formulation with adequate release characteristics for the treatment of any condition or disorder that requires the administration of the CHEPO polypeptide, microencapsulation of the dHEPO polypeptide. The microencapsulation of recombinant proteins for sustained release has been succully performed with human growth hormone (rhGB), interferon- (rhIFN-), interleukin-2, and MN rgpl20. Johnson et al. , Nat. Med., 2: 795-799 (1996); Yasuda, Biomed. Ther. , 27: 1221-1223 (1993); Hora et al. , Bio / Technology, 8: 755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines using Polylactide Polyglycolide Microsphere Systems", in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Pr New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Patent No. 5,654,010. Sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to their biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids, can be rapidly eliminated within the human body. In addition, the degradability of this polymer can be adjusted from months to years depending on its weight and molecular composition. Lewis, "Controlled release of bioactive agents from lactide / glycolide polymer", in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41 This invention encompasses methods of selecting compounds to identify those that mimic the 5 CHEPO polypeptide (agonists) or avoid the effect of CHEPO polypeptide (antagonists). Selection tests for candidates for antagonist drugs are designed to identify compounds that bind or complex with the CHEPO polypeptides encoded by the genes 10 identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins. Such selection trials will include treatable assays for the high-throughput screening of chemical libraries, making them particularly 15 suitable for identifying candidates for small molecule drugs. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical selection assays, immunoassays, cell-based assays, which are well characterized in the art. All assays for antagonists are common because they require contacting the drug candidate 152 with a CHEPO polypeptide encoded by a nucleic acid identified herein under conditions and for a sufficient time to allow these two components to interact. In the link tests, the interaction is the bond and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the CHEPO polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, for example, on a microtiter plate, by covalent or non-covalent attachment. The non-covalent binding is usually completed by coating the solid surface with a solution of the CHEPO polypeptide and drying it. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the CHEPO polypeptide that is immobilized can be used to anchor it to a solid surface. The test is performed by adding the immobilized component, which can be labeled by a detectable label, to the immobilized component, for example, the coated surface containing the anchored component. When the reaction is completed, the components that did not react are removed, for example, by washing, and the complexes anchored on the surface are detected solid When the originally non-immobilized component carries a detectable label, detection of the immobilized label on the surface indicates that the complex occurred. When the originally non-immobilized component carries a label, the complex can be detected, for example, by using a labeled antibody that binds specifically to the immobilized complex. If the candidate compound interacts with but does not bind to a particular CHEPO polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by well-known methods for detecting protein-protein interactions. Such assays include traditional methods, such as, for example, crosslinking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored using the genetic system based on yeast, described by Fields and colleagues (Fields and Song, Nature (London), 340: 245-246 (1989); Chien et al., Proc. Nati, Acad. Sci. USA, 88: 9578-9582 (1991)) as described by Chevray and Nathans, Proc. Nati Acad. Sci. USA, 89: 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two domains modular physically discrete, one that acts as the domain that binds to DNA, and the other that functions as the domain of transcription-activation. The yeast expression system described in previous publications (generally referred to as the "double hybrid system"), takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA binding domain of the GAL4, and another, in which the activation proteins, candidates, are fused to the activation domain. The expression of a GALl-IacZ reporter gene under the control of a promoter activated with GAL4 depends on the reconstruction of GAL4 activity via the protein-protein interaction. The colonies containing the interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER ™) to identify protein-protein interactions between two proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to the protein domains of the map, involved in the interactions of the specific protein as well as the precise amino acid residues that are crucial for these interactions. Compounds that interfere with the interaction of a gene encoding a CHEPO polypeptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture containing the product of the gene and the intra- or extracellular component is prepared under conditions and for a time that allows the interaction and link of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is activated in the absence and in the presence of the test compound. In addition, a placebo can be added to the third reaction mixture, for servile as a positive control. The binding (complex formation) between the test compound and the intra- or extracellular compound present in the mixture is monitored as described above. The formation of a complex in the control reaction but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner. For an assay for the antagonists, the CHEPO polypeptide can be added to a cell together with the compound to be selected for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the CHEPO polypeptide indicates that ei compound is an antagonist of the CHEPO polypeptide. Alternatively, antagonists can be detected by combining the CHEPO polypeptide and a potential antagonist with the membrane-bound CHEPO polypeptide receptors or recombinant receptors under conditions appropriate for a competitive inhibition assay. The CHEPO polypeptide can be labeled, such as by means of radioactivity, such that the number of the CHEPO polypeptide molecules, bound to the receptor, can be 10 use to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those skilled in the art, for example, separation by screening the ligand and FACS classification. Coligan et 15 ai. , Current Protocols in Immun., 1 (2): Chapter 5 (1991). Preferably, expression cloning is employed wherein the polyadenylated RNA is prepared from a cell responsive to the CHEPO polypeptide and a cDNA library created from this RNA is divided into pools and 20 used to transfer COS cells or other cells that are not sensitive to the CHEPO polypeptide. Transfected cells that are grown on glass slides are exposed to the labeled CHEPO polypeptide. The CHEPO polypeptide can be labeled by a variety of means including the iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to auto-radiographic analysis. The positive sets are identified and the subsets are prepared and re-transfected using an interactive subset and re-selection process, which eventually produces a single clone encoding the putative receptor. As an alternative method for the identification of the receptor, the tagged CHEPO polypeptide can be linked by photoaffinity with the membrane or cell extract preparations expressing the receptor molecule. The crosslinked material is resolved by PAGE and exposed to an X-ray film. The tagged complex containing the receptor can be removed, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from the micro-sequencing could be used to design a set of degenerate oligonucleotide probes to select a cDNA library that identifies the gene encoding the putative receptor. In another assay for the antagonists, the cells of mammals or a preparation of the membrane that expresses the receptor that would be incubated with a labeled CHEPO polypeptide in the presence of the candidate compound. The ability of the compound to improve or block this interaction could then be measured. More specific examples of potential antagonists include an oligonucleotide that binds to immunoglobulin fusions with the CHEPO polypeptide, and, in particular, the antibodies include, without limitation, the polyclonal and monoclonal antibodies and the antibody fragments, single chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the CHEPO polypeptide that recognizes the receptor but has no effect, thereby competitively inhibiting the action of the CHEPO polypeptide. Another potential antagonist of the CHEPO polypeptide is an antisense RNA or DNA construct prepared using antisense technology, where, for example, an antisense RNA or DNA molecule acts to directly block the translating the mRNA by hybridizing to the target mRNA and preventing translation of the protein. Antisense technology can be used to control the expression of the gene through triple helix formation or antisense DNA or RNA, both methods are based on the binding of a polynucleotide to RNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence, which I codes for the mature CHEPO polypeptides herein, is used to design an antisense RNA oligonucleotide. 3 (0 or from approximately 10 to 40 base pairs of i I 1 length.A DNA oligonucleotide is designated to be 1 complementary to the region of the gene involved in the I transcription (triple helix - see Lee et al., Nucí.

'Res., 6: 3073 (1979); Cooney et al. , Science, 241: 456 I (1988); Dervan et al. , Science, 251: 1360 (1991)), which i i prevents transcription and production of the CHEPO polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks the translation of the mRNA molecule j into the CHEPO polypeptide (antisense - Okano, Neurochem., I (| _56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988) Oligonucleotides described above can be delivered to cells in such a way that RNA or DNA The antisense can be expressed in vivo to inhibit the production of the CHEPO polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site are preferred, for example, between approximately positions -10 and +10 of the nucleotide sequence of the target gene. Potential antagonists include small molecules that bind to the active site, the receptor binding site, or the growth factor or other relevant binding site of the CHEPO polypeptide, thereby blocking the normal biological activity of the CHEPO polypeptide. Examples of the small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic, organic or non-peptidyl inorganic compounds. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. The specific ribozyme cleavage sites within the potential RNA target can be identified by known techniques.

For further details see, for example, Rossi, Current Biology, 4: 469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997). Nucleic acid molecules in the triple helix formation used to inhibit transcription should be single stranded or composed of deoxynucleotides. The base composition of these oligonucleotides is designated such that triple helix formation is promoted via the Hoogsteen base pairing rules, which generally requires considerable elasticities of purines or pyrimidines in a single strand of a duplex. For more details see, for example, PCT publication No. WO 97/33551, supra. These small molecules can be identified by any one or more of the screening assays described above and / or by any other selection technique well known to those skilled in the art.

F. Anti-CHEPO Antibodies The present invention further provides anti-CHEPO antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies. 1. Polyclonal Antibodies Anti-CHEPO antibodies may comprise polyclonal antibodies. Methods for preparing polyclonal antibodies are well known to those skilled in the art. Polyclonal antibodies can be generated in mammals, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and / or will be injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent can include the CHEPO polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a known protein that will be immunogenic in the mammal to be immunized. Examples of such immunogenic proteins include but are not limited to key limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and the MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorinomycolate). The immunization protocol can be selected by someone skilled in the art without undue experimentation. 2. Monoclonal Antibodies Anti-CHEPO antibodies can alternatively be monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohier and Milstein, Nature, 256: 495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to generate lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vi tro. The immunizing agent will typically include the CHEPO polypeptide of interest or a fusion protein thereof. In general, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells if sources of non-human mammals are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) p. 59-103]. The immortalized cell lines are usually transformed mammalian cells, particularly rodent, bovine and human origin myeloma cells. Usually mouse or rat myeloma cell lines are employed. Hybridoma cells can be cultured in a culture medium that preferably contains one or more substances that inhibit the growth or survival of immortalized, unfused cells. For example, if the parental cells lack the hypoxanthine guanine phosphoribosyl transferase of the enzyme (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, inopterin, and thymidine ("HAT medium"), which substances avoid the growth of cells deficient in HGPRT. Preferred immortalized cell lines are those that efficiently fuse, and support stable high level expressions of the antibody by the cells that produce selected antibodies, and are sensitive to a medium such as the HAT medium. The most preferred immortalized cell lines are the murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Coliection, Manassas Virginia. Also described are mouse-human heteromyeloma and human myeloma cell lines for the production of human monoclonal antibodies [Kozbor, J. Immunol. , 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) p. 51-63]. The culture medium in which the hybridoma cells are grown can be tested for the presence of monoclonal antibodies directed against CHEPO. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as the radioimmunoassay (RIA) or the enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. , 107: 220 (1980). After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution and growth procedures by standard methods [Goding, supra]. The culture medium suitable for this purpose includes, for example, the of Eagle Modified with Dulbecco and the RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or the ascites fluid by conventional immunoglobulin purification methods such as, for example, chromatography of A-Sepharose, hydroxylapatite, gel electrophoresis, dialysis, or affinity chromatography. Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the invention can be easily isolated and sequenced using conventional methods (for example, using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed in the expression vectors, which are then transfected into the host cells such as simian COS cells, the cells of Chinese hamster ovary (CHO), or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of monoclonal antibodies in recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for the human heavy and light chain constant domains instead of the homologous murine sequences [U.S. Patent No. 4,816,467; Morrison et al., Supra] or by covalently binding to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be replaced by the constant domains of an antibody of the invention, or it can be replaced by the variable domains of a combination site of the antigen of an antibody of the invention to create a chimeric bivalent antibody. The antibodies can be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves the recombinant expression of modified heavy chains and immunoglobulin light chains. The heavy chain is truncated generally at any point in the Fc region Í ^^^^^ A? ^ Ü ^ - ^ I &? to avoid cross-linking or cross-linking of the heavy chain. Alternatively, the relevant cysteine residues are replaced with another amino acid residue or are eliminated to avoid cross-linking. The in vi tro methods are also suitable for the preparation of monovalent antibodies. Digestion of the antibodies produces fragments thereof, particularly, Fab fragments, which can be supplemented using routine techniques known in the art. 3. Human and Humanized Antibodies The anti-CHEPO antibodies of the invention may further comprise humanized antibodies or antibodies from humans. Humanized forms of non-human antibodies (eg, murine) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other antigen binding subsequences of the antibodies) which contain the minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which the residues of a complementary determinant region (CDR) of the container are replaced by residues of a CDR of a non-human species (donor antibody) such as the mouse, rat or rabbit that have the desired specificity, affinity and capacity. In some cases, the residues of the Fv structure of the human immunoglobulin are replaced by the corresponding non-human residues. The humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR nor in the sequences of the structure. In general, the humanized antibody will comprise substantially all or at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions of a consensus sequence of human immunoglobulin. The humanized antibody optimally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)]. Methods for the humanization of non-human antibodies are well known in the art. Generally, a humanized antibody has one or more residues of amino acids introduced therein from a source that is non-human. These non-human amino acid residues are often referred to as "imported" residues, which are typically taken from an "imported" variable domain. Humanization can be carried out essentially following the method of Winter et al. [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], replacing the CDR or CDRs sequences of the rodent with the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than one intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues of analogous sites in rodent antibodies. Human antibodies can also be produced using various techniques known in the art, including the phage sample libraries [Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol.

Biol., 22: 581 (1991)]. The techniques of Colé et al., And Boerner et al., Are also available for the preparation of human monoclonal antibodies [Colé et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147 (1): 86-95 (1991)]. Similarly, human antibodies can be made by introducing the human immunoglobulin site in the transgenic animals, for example, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. During the challenge, the production of human antibodies was observed, which closely resembles that observed in humans in all estimates, including gene restructuring, assembly, and antibody repertoire. This scope is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al. , Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al. , Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995). 4. Bispecific Antibodies Bispecific antibodies are monoclonal, humanized or preferably human antibodies, which have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for CHEPO, the other is one for any other antigen, and preferably for a cell surface protein or receptor or receptor subunit. Methods for preparing bispecific antibodies are well known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two heavy chain / light chain immunoglobulin pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305: 537-539 (1983 )]. Due to the randomization of heavy and light chains of immunoglobulin, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually carried out by the affinity chromatography steps. Similar procedures are described in WO 93/08829, published on May 13, 1993, and aa & feSif tj &ifcf aa, ..,. a | M * j. ^ tojaj. in Traunecker et al., EMBO J., 10: 3655-3659 (1991). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the joint regions, CH2 and CH3. It is preferred to have the first heavy chain constant region (CH1) containing the necessary site for the present light chain link in at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chains, are inserted into separate expression vectors, and co-transfected into a suitable host organism. For further details of the generation of bispecific antibodies' see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986). According to another method described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that were recovered from the recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of a constant domain of the .-, j, - .. **. ~ < í r i.J antibody. In this method, one or more side chains of amino acids, small, from the interface of the first antibody molecule are replaced with the longer side chains (for example, tyrosine or tryptophan). Compensating "cavities" of similar or identical size were created for the long chain or side chains at the interface of the second antibody molecule by replacing the long amino acid side chains with smaller ones (eg alanine or threonine). This provides a mechanism to increase the performance of the heterodimer over other undesired terminal products such as homodimers. Bispecific antibodies can be prepared as full-length antibodies or fragments of antibodies (for example bispecific antibodies F (ab ') 2). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using a chemical bond. Brennan et al., Science 229: 81 (1985) describes a method wherein the intact antibodies are proteolytically cleaved to generate the F (ab ') 2 fragments. These fragments are reduced in the presence of sodium arsenite, the agent that forms dithiol complexes to stabilize the neighboring dithiols and prevent the formation of intermolecular disulfide. The generated Fab 'fragments are then converted into thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted to Fab '-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of the enzymes. Fab 'fragments can be recovered directly from E. coli and chemically ablated to form bispecific antibodies. Shalaby et al. , J. Exp. Med. 175: 217-225 (1992) describes the production of an F (ab ') 2 molecule of the fully humanized bispecific antibody. Each Fab 'fragment is secreted separately from E. coli and subjected to chemical coupling in vi tro to form the bispecific antibody. The bispecific antibody thus formed is capable of binding to cells overexpressing the ErbB2 receptor and normal T cells, as well as causing the violent activity of human cytotoxic lymphocytes against the targets of the human breast tumor. Several techniques have been described to make and isolate the bispecific antibody fragments directly from the recombinant cell culture. For example, bispecific antibodies have been produced using leucine closures. Kostelny et al. , J. Immunol. 148 (5): 1547-1553 (1992). Peptides from the leucine lock of the Fos and Jun proteins are linked to the Fab 'portions of two different antibodies by genetic fusion. The antibody homodimers are reduced to the joint region to form the monomers and then re-oxidized to form the antibody heterodimers. This method can also be used for the production of the antibody heterodimers. The diabody technology described by Hollinger et al. , Proc. Nati Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism for the construction of the bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to the light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy has also been reported for the construction of bispecific antibody fragments by the use of single chain Fv dimers (sFv). See, Gruber et al. , J. Immunol. 152: 5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. , J. Immunol. 147: 60 (1991). Exemplary bispecific antibodies can bind to two different epitopes on a CHEPO polypeptide given herein. Alternatively, an arm of the anti-CHEPO polypeptide can be combined with an arm that binds to an activation molecule in a leukocyte such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc R), such as Fc Rl (CD64), Fc RII (CD32) and Fc RUI (CD16) in regard to local cell defense mechanisms for the cell expressing the particular CHEPO polypeptide . Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing a particular CHEPO polypeptide. These antibodies possess the CHEPO binding arm and an arm that binds to a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Other antibody The bispecific of interest binds to the CHEPO polypeptide and in addition binds to the tissue factor (TF). 5. Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies, for example, have been proposed as targets of cells of the immune system to unwanted cells [US Patent No. 4,676,980], and for the treatment of HIV infection [WO 91/00360].; WO 92/200373; EP 03089]. It is contemplated that the antibodies can be prepared in vi tro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by the formation of a thioether linkage. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those described, for example, in U.S. Patent No. 4,676,980. 6. Genetic Engineering of the Effector Function It is desirable to modify the antibody of the invention with respect to the function of the effect, to improve, for example, the effectiveness of the antibody in the treatment of cancer. For example, the cysteine residue (s) can be introduced into the Fc region, thereby allowing the formation of interchain chain disulfide bonding in this region. The homodimeric antibody thus generated may have the enhanced internalization capacity and / or increased cell death mediated by complement and antibody-dependent cellular cytotoxicity (ADCC). See Carón et al. , J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using the cross-linked heterobifunctional linkers described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody having the dual Fc regions can be engineered and therefore complement lysis and ADCC capabilities can be improved. See Stevenson et al. , Anti-Cancer Drug Design, 3: 219-230 (1989). 7. Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof). themselves), or a radioactive isotope (i.e., a radioconjugate). The chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. The enzymatically active toxins and fragments thereof that can be used include the diphtheria A chain, the unbound active fragments of the diphtheria toxin, the exotoxin A chain (from Pseudomonas aeruginosa), the ricin A chain, the chain of abrin A, the chain of modeccin A, alpha-sarcin, the proteins Aleuri tes fordii, the proteins diantinas, the proteins Phytolaca ameri cana (PAPI, PAPII, and PAP-S), the inhibitor of momordica charantia, curcina, crotina, inhibitor of sapaonaria officinalis, gelonin, mitogeline, restrictocin, phenomycin, enomycin, and trichothecenes.

A variety of radionuclides is available for the production of radioconjugated antibodies. The examples ^ ^ ^ J ^ ij ^^^ iM ^ include 212Bi, 131I, 131In, 90Y, and 186Re. The conjugates of the antibody and the cytotoxic agent are made using a variety of protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional byproducts of the imidoesters ( such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexandiamine), bis-diazonium by-products (such as bis- (p-diazonium benzoyl) -ethylenediamine), diisocyanates (such as 2,6-tolienium diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) . For example, a castorium immunotoxin can be prepared as described in Vitetta et al. , Science, 238: 1098 (1987). The 1-isothiocyanatobenzyl-3-methyldiethylene triaminopentaacetic acid labeled with carbon 14 (MX-DTPA) is an exemplary chelating agent for the conjugation of the radionuclide with the antibody. See WO94 / 11026. In another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for use in the pre-target of the tumor wherein the * ^ J ^^ J conjugate antibody-receptor is administered to the patient, followed by removal of an unbound conjugate from the circulation using a clarifying agent and then administration of the "ligand" (eg, avidin). ) that is conjugated with a cytotoxic agent (for example, a radionuclide). 8. Immunoliposomes The antibodies described herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al. , Proc. Nati Acad. Sci. USA, 82: 3688 (1985); Hwang et al. , Proc. Nati Acad. Sci. USA, 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are described in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and phosphatidylethanolamine derived with PEG (PEG-PE). The liposomes are extruded through the filters of defined pore size to produce the liposomes with the desired diameter. The Fab 'fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al. , J. Biol. Chem., 257: 286-288 (1982) via a disulfide exchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al. , J. National Cancer Inst., 81 (19): 1484 (1989). 9. Pharmaceutical Compositions of Antibodies Antibodies that specifically bind to a CHEPO polypeptide identified in the present invention, as well as to other molecules identified by the screening assays described hereinbefore, may be administered for the treatment of various conditions in the form of pharmaceutical compositions. If the CHEPO polypeptide is intracellular and the whole antibodies are used as inhibitors, internalization antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into the cells. When fragments of the antibody are used, the fragment is preferred -? Ifit? Ll? Ii | -airi? Í fai > - frr.-íí% .- smaller inhibitor that binds specifically to the domain of the target protein. For example, based on the variable region sequences of an antibody, peptide molecules can be designed to retain the ability to bind to the target protein sequence. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. See, for example, Marasco et al. , Proc. Nati Acad. Sci. USA, 90: 7889-7893 (1993). The formulation herein may also contain more than one active compound that is necessary for the particular indication to be treated, preferably those with complementary activities that do not adversely affect one another. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, growth inhibitory agent. Such molecules are suitably present in combination with amounts that are effective for the intended purpose. The active ingredients can also be entrapped in microcapsules prepared, for example, by particle gathering techniques or by interfacial polymerization, for example, hydroxymethylcellulose or Httü * - r'Jj? I. J * m £ t. * Íítt. * Á aia Jita Ji gelatin microcapsules and poly- (methylmetacylate) microcapsules, respectively, in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences, supra. The formulations that are to be used for in vivo administration must be sterile. This is easily completed by filtering through the sterile filtration membranes. Sustained-release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, whose matrices are in the form of shaped articles, for example, films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), L-glutamic acid copolymers, and ethyl-L-glutamate, non-degradable ethylene vinyl acetate, glycolic acid-degradable lactic acid copolymers such as LUPRON DEPOT ™ (injectable microspheres) composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) - 3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid glycolic acid are capable of releasing the molecules over a period of 100 days, certain hydrogels release proteins for shorter times. When the encapsulated antibodies remain in the body for a very long time, they can denature or aggregate as a result of exposure to moisture at 37 ° C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if it is discovered that the aggregation mechanism is an intermolecular SS bond formation through the thio-disulfide exchange, stabilization can be achieved by modifying the sulfhydryl residues, lyophilizing from the acid solutions, controlling the content of humidity, using the appropriate additives, and developing matrix compositions of the specific polymer. | ^^^ - ^^ t-j1'T? r'Ti'liTi? í? i? H * -k ^ & - ~ Mt-j¿ ^^ fct-. { - ^ J ^^^ & fc «a» - * fc - »« ^^ a¿i < G. Uses for Anti-CHEPO Antibodies The anti-CHEPO antibodies of the invention have various utilities. For example, anti-CHEPO antibodies can be used in diagnostic assays for a CHEPO, for example, for the detection of its expression in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art can be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in the diagnostic assays can be labeled with a detectable portion. The detectable portion should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable portion can be a radioisotope, such as 3H, 4C, 32P, 35S, or 125I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art can be employed to conjugate the antibody with the detectable portion, including those methods described by Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40: 219 (1981); and Nygren, J ^ Histochem. and Cytochem. , 30: 407 (1982). Anti-CHEPO antibodies are also useful for the affinity purification of CHEPO from the culture of recombinant cells or natural sources. In this process, antibodies against CHEPO are immobilized on a suitable support, such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody is then contacted with a sample containing the CHEPO to be purified, and subsequently the support is washed with a suitable solvent that will remove substantially all of the material in the sample except CHEPO, which binds to the antibody immobilized. Finally, the support is washed with another suitable solvent that will liberate the CHEPO from the antibody. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. All patents and references in the literature cited in the present specification are incorporated herein by reference in their entirety.

EXAMPLES Commercially available reagents referred to in the examples were used according to the manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, using the ATCC access numbers is the American Type Culture Collection, Manassas, VA.

EXAMPLE 1 Isolation of the Nucleic Acid Encoding CHEPO Genomic DNA was isolated from two chimpanzee cell lines (ATCC CRL 1609 and CRL 1857) using the Qiagen kit (cat # 10262) as recommended in the manufacturer's instructions. The chimpanzee Epo gene is then obtained in 3 fragments separated by PCR using 1 g of genomic DNA as standard and the following primer pairs: EPO.F: 5'-ACCGCGCCCCCTGGACAG-3 '(SEQ ID NO: 12) EPO .INT1.R: 5 '-CATCCACTTCTCCGGCCAAACTTCA-3' (SEQ ID NO: 13) EPO.INT1F: 5 '-TTTGGCCGGAGAAGTGGATGC-3' (SEQ ID NO: 14) EPO. INT4R: 5 '-TCACTCACTCACTCATTCATTCATTCATTCA-3' (SEQ ID NO: 15) EPO.INT4F: 5 '-GT GAATGAATGATTGAATGAATGAGTGA-3' (SEQ ID NO: 16) EPO.R: 5'-GCACTGGAGTGTCCATGGGACAG-3 '(SEQ ID NO: 17) Each PCR reaction contains 5 L of the lOx PCR stretcher (Perkin Elmer), 1 L of dNTP (20 mM), 1 g of genomic DNA, 1 L of each primer, 1 L of Taq polymerase (Clontech) and H20 to carry the total volume up to 50 L. The reaction is first denatured for 4 min. at 94 ° C then amplified for 40 cycles of 1 min. at 94 ° C, 1 min. at 65 ° C or 66 ° C then it is extended for 1 min. at 72 ° C. A final extension stage of 5 min. at 72 ° C. The reaction was then analyzed on agarose gel. The PCR product of 500 bp, 1200 bp, 750 bp was observed for each PCR product, respectively. The PCR reactions were then purified using a Wizard kit (Promega cat. # A7170) then sequenced directly. DNA sequencing of the PCR producers was done using an Applied Biosystems 377 DNA Sequencer (PE / Applied Biosystems, Foster City, CA). The chemistry used was Dye Terminator Cycle Sequencing with dRhodamine and the BIG DYE terminators (PE / Applied Biosystems, Foster City, CA). The sequence assembly and editing were done with the Sequencher computer program (Gene Codes, Ann. Arbor, MI). The 5 coding exons were identified by homology to the human erythropoietin sequence and assembled into a predicted full-length cDNA. The coding region of the CHEPO cDNA is 579 nucleotides in length (Figure 1) and encodes a predicted protein of 193 amino acids (Figure 3). There are 3 putative signal cleavage sites, predicted after amino acid residues 22, 24 and 27. According to the N-terminus of human Epo, the most recent one probably corresponds to the cleavage site of the chimpanzee Epo. The signal peptide of amino acid 27, hydrophobic, is followed by a mature protein of 166 amino acids in length, which contains 3 potential N-glycosylation sites. A polymorphism of the single nucleotide is present in the predicted sequence obtained from CRL1609 and changes the protein sequence at position 142 of the amino acid from a Q to a K. The alignment of the chimpanzee Epo protein with the human sequence indicates a change unique at position 84 of the amino acid (Figure 3).

EXAMPLE 2 Use of the CHEPO cDNA as a hybridization probe The following method describes the use of a nucleic acid sequence encoding a CHEPO as a hybridization probe. The DNA comprising the sequence encoding ie a mature or full length CHEPO (as shown in xa Figure 2, SEQ ID NO: 3) is used as a probe that selects homologous DNAs (such as those encoding variants). They are presented naturally, encoding the CHEPO) umano tissue cDNA libraries or in human tissue genomic libraries. Hybridization and washing of the filters that contain either the DNA of the library, are performed ba]? the following highly severe conditions. The hib? The radiolabel of the CHEPO-derived probe was radiolabelled to the filters in a 50% formamide solution, 5x SSC, 0.% SDS, 0.1% sodium pyrophosphate, 50 mM soaxo phosphate pH 6.8 , solution of Denhard 2x, and its dextran 10% at 42"C for 20 hours.The liter-liter was washed in an aqueous solution of SSC O.lx and SDS at 0.1 to 4 ° C. DNA that have a desired sequence identity with the DNA encoding the full-length native sequence CHEPO, it can then be identified using standard techniques known in the art.

EXAMPLE 3 Expression of CHEPO in E. coli This example illustrates the preparation of a non-glycosylated form of a CHEPO by recombinant expression in E. coli. The DNA sequence encoding CHEPO is initially amplified using the selected PCR primers. The primers should contain the restriction enzyme sites corresponding to the restriction enzyme sites in the selected expression vector. A variety of expression vectors can be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains the genes for resistance to ampicillin and tetracycline. The vector is digested with the restriction enzyme and dephosphorylated. The amplified PCR sequences are then ligated into the vector. The vector preferably will include sequences that code for an antibiotic resistance gene, a trp promoter, a leader polyhis (which includes the first six STII codons, the polyhis sequence, and the enterokinase cleavage site), the specific CHEPO coding region, the lambda transcriptional terminator, and an argU gene. The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., Supra. Transformants are identified by their ability to grow on LB plates and antibiotic-resistant colonies are thus selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing. The selected clones can be grown overnight in a liquid culture medium such as LB broth supplemented with antibiotics. The culture can be used subsequently throughout the night to inoculate a large-scale culture. The cells are then grown to a desired optical density, during which the expression promoter is driven. After culturing the cells for several more hours, the cells can be harvested by centrifugation. The pellets of cells obtained by centrifugation can be solubilized using several known agents in the technique, and the solubilized CHEPO protein can then be purified using a metal chelating column under conditions that allow a linkage outside the protein. CHEPO can be expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding CHEPO was initially amplified using the selected PCR primers. The primers will contain the restriction enzyme sites corresponding to the restriction enzyme sites in the selected expression vector, and other useful sequences that provide reliable and efficient translation initiation, rapid purification in a metal chelating column, and proteolytic elimination with enterokinase. The poly-His-labeled sequences amplified with PCR were then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA (tonA) Ion galE rpoHts (htpRts) clpP (lacIq) The transformants were first grown in LB containing 50 mg / ml carbenicillin at 30 ° C with agitation until an optical density or OD600 of 3-5 was reached.The cultures were then diluted from 50 to 100. times in a CRAP medium (prepared by mixing 3.57 g of (NH4) S04, 0.71 g of sodium citrate-2H20, 1.07 g of KCl, 5.36 g of Difco yeast extract, 5.36 g of SF hycase from Sheffield in 500 mL of water, as well as MPOS 110 mM, pH 7.3, glucose at 0.55% (w / v) and 7 mM MgSO4) and grown for about 20-30 hours at 30 ° C with shaking. Samples were removed to verify expression by SDS-PAGE analysis, and the bulk culture was centrifuged to pellet cells. The cell pellets were frozen until purification and refolded. The E. coli paste was resuspended from fermentations of 0.5 to 1 L (pellets of 6-10 g) in 10 volumes (w / v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Sulfite was added of solid sodium and tetrathionate of sodium to make the final concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred overnight at 4 ° C. This step results in a denatured protein with all the cysteine residues blocked by sulfitolization. The solution was centrifuged at 40,000 rpm in a Beckman Ultracentrifuge for 30 minutes. The supernatant was diluted with 3-5 volumes of the metal quenching column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through ¡A ..., .- * -.? .- i 0.22 micron filters for clarification. The clarified extract was loaded on a Qiagen Ni-NTA metal chelating column of 5 ml, equilibrated in the buffer of the metal chelate column. The column was washed with an additional buffer solution containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4 ° C. The protein concentration was estimated by its absorbance at 280 nm using the extinction coefficient calculated based on its amino acid sequence. The proteins were refolded by slow dilution of a sample in a freshly prepared refolding or redouble buffer, which consisted of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine, and EDTA 1 mM. The refolding volumes were chosen so that the final concentration of the protein was between 50 to 100 micrograms / ml. The refolding solution was gently stirred at 4 ° C for 12-36 hours. The refolding reaction was rapidly quenched by the addition of TFA to a final concentration of 0.4% (pH of about 3). Before a further purification of the protein, the solution was filtered through a 0.22 micron filter and I ^ &g? j ^^^^^^ jj ^ ^ ^ added acetonitrile to a final concentration of 2-10%. The refolding protein was subjected to chromatography on a Poros Rl / H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. The aliquots of the fractions with an absorbance of A280 were analyzed in SDS polyacrylamide gels and the fractions containing the homogeneous refolded protein were pooled. In general, appropriately redone species of most proteins were eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors coated from the interaction with the reverse phase resin. The species that formed aggregates usually elute at higher acetonitrile concentrations. In addition to solving the erroneously redoubled forms of the proteins from the desired form, the reverse phase stage also eliminates the endotoxin from the samples. The fractions containing the desired folded CHEPO polypeptide were conjugated and the acetonitrile was removed using a gentle stream of nitrogen directed to the solution. Proteins were formulated in 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by Dialysis or by gel filtration using G25 Superfine resins (eg, dialysis). Pharmacia) equilibrated in the formulation buffer and filtered sterilely.

EXAMPLE 4 Expression of CHEPO in Mammalian Cells This example illustrates the preparation of a potentially glycosylated form of CHEPO by recombinant expression in mammalian cells. The vector, pRK5 (see EP 307,247, published March 15, 1989), was used as the expression vector. Optionally, the DNA encoding CHEPO was ligated into pRK5 with the selected restriction enzymes to allow insertion of the CHEPO DNA using ligation methods such as those described in Sambrook et al., Supra. The resulting vector was called pRK5-CHEPO. In one embodiment, the selected host cells can be 293 cells. Human 293 cells (ATCC CCL 1573) were grown to confluence in tissue culture plates in a medium such as DMEM supplemented with fetal calf serum and optionally, with the components of nutrients and / or antibiotics. Approximately 10 μg of pRK5-CHEPO DNA was mixed with "^ j ^^ á ^ tójgi? ^ ^ ^^^^^ j ^^^^^ j ^^^^^^^^^ Jj? i? ^ ijj? ^ itt about 1 μg of the DNA encoding the gene of RNA VA [Thimmappaya et al., Cell, 31: 543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture was added, dropwise, 500 μL of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaP04, and a precipitate was allowed to form for 10 minutes at 25 ° C. The precipitate was suspended and added to the 293 cells and allowed to settle for approximately four hours at 37 ° C. The culture medium was aspirated and 2 L of 20% glycerol in 20% PBS was added for 30 seconds.The 293 cells were then washed with a serum-free medium, the fresh medium added and the cells were incubated for approximately 5 days.Approximately 24 hours after the transfections, the culture medium was removed and replaced with culture medium (alone) or with a culture medium containing 200 Ci / mL of 35S- cysteine and 200 Ci / mL of S-methionine. After 12 hours of incubation, the conditioned medium was collected, concentrated on a rotary filter, and loaded on 15% SDS gel. The processed gel can be dried and exposed to a film for a selected period of time to reveal the presence of the CHEPO polypeptide. The cultures that contain the cells i'ir. transfected can undergo further incubation (in a serum free medium) and the medium was tested in selected bioassays. In an alternative technique, CHEPO can be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Nati Acad. Sci., 12: 7575 (1981). The 293 cells are grown to a maximum density in a spinner flask and 700 μg of pRK5-CHEPO DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate was incubated in cell pellets for four hours. The cells are then treated with 20% glycerol for 90 seconds, washed with a tissue culture medium, and 5 μg / ml bovine insulin is re-introduced into the spinner flask containing the tissue culture medium. and 0.1 μg / ml of bovine transferrin. After about four days, the conditioned medium is centrifuged and filtered to remove the cells and debris. The sample containing the expressed CHEPO can then be concentrated and purified by any selected method, such as dialysis and / or column chromatography.

In another embodiment, CHEPO can be expressed in CHO cells. PRK5-CHEPO can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium is replaced with the culture medium (alone) or the medium containing a radiolabel such as 35S-methionine. After determining the presence of the CHEPO polypeptide, the culture medium can be replaced with a serum-free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed CHEPO can then be concentrated and purified by any selected method. CHEPO labeled with the epitope can also be expressed in host CHO cells. CHEPO can be subcloned from the vector pRK5. The subclone insert can be subjected to PCR to be fused in a structure with a selected epitope tag such as a poly-his tag in a Baculovirus expression vector. The CHEPO insert labeled with poly-his can then be subcloned into an SV40 drive vector containing a selection marker such as DHFR for the selection of clones stable Finally, the CHO cells can be transfected (as described above) with the SV40 drive vector. The labeling can be done, as described above, to verify the expression. The culture medium containing the CHIPO labeled with expressed poly-His can then be concentrated by any selected method, such as by affinity chromatography of Ni2 + -kelate. CHEPO can also be expressed in CHO and / or COS cells by the transient expression procedure or in CHO cells, or by another stable expression method. Stable expression in CHO cells was performed using the following procedure. The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g., extracellular domains) of the respective proteins are fused to an IgG1 constant region sequence containing the hinged portion, CH2. and the CH2 domains and / or is a poly-His tagged form. After amplification with PCR, the respective DNAs were subcloned into a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors were constructed to have compatible restriction sites 5 'and 3' of the DNA of interest to allow convenient transport of the cDNAs. The expression used of the vector in CHO cells is as described in Lucas et al., Nucí. Acids Res. 24: 9 1774-1779 (1996), and utilizes the SV40 early promoter / enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). The expression of DHFR allows selection for stable maintenance of the plasmid after transfection. Twelve micrograms of the desired plasmid DNA were introduced into approximately 10 million CHO cells using the commercially available transfection reagents Superfect® (Quiagen), Dosper® or Fugene® (Boehringer Mannheim). The cells were grown and described in Lucas et al., Supra. Approximately 3 x 10"7 cells were frozen in an ampoule for further growth and production as described below The ampoules containing the plasmid DNA were sealed by placing them in a water bath and mixed by a vortex motion. were placed through a pipette in a centrifuge tube containing 10 mLs of the medium and centrifuged at 1000 rpm for 5 minutes.The supernatant was aspirated and the cells were resuspended in 10 mL of the selective medium (0.2 μm of filtered PS20 with 0.2 μm of 5% diafiltered fetal bovine serum.) The cells were then aliquoted into a 100 mL centrifuge device containing 90 mL of the selective medium.After 1-2 days, the cells were transferred to a flask. of 250 mL filled with 150 mL of selective growth medium and incubated at 37 ° C. After another 2-3 days, the 250 mL, 500 mL and 2000 mL flasks were seeded with 3 x 10 cells / ml The cell medium was exchanged with a fresh medium by centrifugation and resuspension in the production medium. Although any suitable CHO medium can be employed, a production medium described in US Patent No. 5,122,469, issued June 16, 1992, was actually used. The 3L production flask was seeded with 1.2 x 10 cells / mL. On day 0, the pH number of the cells was determined. On day 1, samples were obtained from the flask and spraying with filtered air was started. On day 2, samples were obtained from the flask, the temperature was changed to 33 ° C, and 30 mL of 500 g / L of glucose and 0.6 mL of glucose were added. 10% antifoam (e.g., a 35% polydimethylsiloxane emulsion, Medical Grade Emulsion 365 from Dow Corning). Throughout the production, the pH was adjusted as necessary to stay at around 7.2. After 10 days, or until the viability was decreased below 70%, the cell culture was harvested by centrifugation and filtered through a 0.22 μm filter. The filtrate was either stored at 4 ° C or immediately loaded onto columns for purification. For the poly-His tagged constructs, the proteins were purified using a Ni-NTA column (Qiagen). Prior to purification, the imidazole was added to the conditioned medium to a concentration of 5 mM. The conditioned medium was pumped to a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml / min. at 4 ° C. After loading, the column was washed with an additional equilibration buffer and the protein was eluted with an equilibration buffer containing 0.25 M imidazole. The highly purified protein was subsequently desalted in a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a G25 Superfine column (Pharmacia) and stored at -80 ° C. The immunoadhesin constructs (containing Fc) were purified from the conditioned medium as follows. The conditioned medium was pumped into a 5 ml Protein A column (Pharmacia) which was equilibrated in 20 mM Na-phosphate buffer, pH 6.8. After loading, the column was washed extensively with an equilibrium buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by the collection of 1 ml fractions in tubes containing 275 μL of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted in storage buffer as described above for the labeled polyprotein proteins. His. The homogeneity was assessed by SDS polyacrylamide gels and by sequencing the N-terminal amino acids by Edman degradation.

EXAMPLE 5 Expression of CHEPO in Yeasts The following method describes the recombinant expression of CHEPO in yeasts. First, yeast expression vectors are constructed for the production or intracellular secretion of CHEPO from the ADH2 / GAPDH promoter. The DNA encoding CHEPO and a promoter are inserted into the appropriate restriction enzyme sites in the selected plasmid to direct the intracellular expression of CHEPO. For secretion, DNA encoding the CHEPO can clone the selected plasmid, together with DNA encoding the ADH2 / GAPDH promoter, a signal peptide native CHEPO or other signal peptide mammal, or for example a factor yeast alpha or a signal sequence / secretory leader of the invertase, and linker sequences (if needed) for the expression of CHEPO. Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in the selected fermentation medium. Transformed yeast supernatants can be analyzed by precipitation with 10% tpcloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue staining. The recombinant CHEPO can subsequently be isolated and purified by removing yeast cells from the fermentation medium by centrifugation and then concentrating the medium using the selected cartridge filters. The concentrate containing the CHEPO can be further purified using the selected column chromatography resins.

EXAMPLE 6 Expression of CHEPO in Insect Cells Infected with Baculovirus The following method describes the recombinant expression of CHEPO in insect cells infected with Baculovirus. The coding sequence for CHEPO is fused in the 5 'direction of an epitope tag contained within a baculovirus expression vector. Such epitope tags include the poly-his tags and the immunoglobuyin tags (such as the Fc regions of IgG). A variety of plasmids are used, including plasmids derived from commercially available plasmids such as pVI.1393 (Novagen). Briefly, the sequence cocifica ai CHEPO or the desired portion of the coding sequence CHEPO as the sequence encoding the exfracelular domain ana transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with the primers complementary to the 5 'and 3' regions. The 5 'primer can incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subciled in the expression vector. Recombinant baculovirus is generated by co-transfecting the above plasmid and DNA EaculoGold ™ (Pharmingen) into Spodoptera virus frugiperda ( "Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28 ° C, the released viruses are harvested and used for subsequent amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Bacuiovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994). The poly-his labeled CHEPO can then be purified, for example, by affinity chromatography of Ni2 + -kelate as follows. Extracts are prepared from Sf9 cells infected with the recombinant virus as described by Rupert et al., Nature, 362: 175-179 (1993). Briefly, the Sf9 cells are ? To ^^ | t ^^ ^^^^^ TEIà washed, resuspended in sonication buffer (25 L Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; NP-40 0.1 %; KCl 0.4 M), and were subjected to sound twice for 20 seconds on ice. The sonicates are clarified by centrifugation and the supernatant is diluted 50 times in charge buffer (50 M phosphate)., 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. The Ni2 + -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 L of charge buffer. The extract of the cells, filtrate is loaded on the column at 0.5 mL per minute. The column is washed to a A2go baseline with charge buffer, at which point the collection of the fraction begins. Next, the column is washed with a secondary washing buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 6.0), which is eluted with a non-specific binding protein. After reaching the baseline A280 again, the column is developed with a gradient of Imidazole from 0 to 500 mM in the secondary wash buffer. Fractions of one mL are collected and analyzed by SDS-PAGE and stained with silver or Western staining with the Ni2 + -NTA conjugate up to alkaline phosphatase (Qiagen). The fractions that • f contain the HEX-labeled CHEPO eluted, coalesce and again dialyze against the charge buffer. Alternatively, purification of CHIPO labeled with IgG (or labeled with Fc) can be performed using known chromatography techniques, including for example, column chromatography of Protein A or G protein.

EXAMPLE 7 Preparation of the Antibodies Binding to CHEPO This example illustrates the preparation of the monoclonal antibodies that can bind specifically to a CHEPO. Techniques for the production of monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that can be employed include purified CHEPO, fusion proteins containing CHEPO, and cells expressing recombinant CHEPO on the cell surface. The selection of the immunogen can be prepared by those skilled in the art without undue experimentation. Mice, such as Balb / c, are immunized with the CHEPO immunogen emulsified in a complete adjuvant . Í .Í of Freund and injected subcutaneously or intraperitoneally with an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in an MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the pads of the hind legs of the animals. Immunity was allowed to grow in the mice for 10 to 12 days thereafter with the additional immunogen emulsified in the selected adjuvant. Subsequently, for several weeks, the immunity to the mice was increased with additional immunization injections. Serum samples were obtained periodically from the mice by retro-orbital bleeding for the test in the ELISA assays to detect anti-CHEPO antibodies. After an adequate antibody titer has been detected, animals "positive" for antibodies can be injected with a final intravenous injection of CHEPO. Three to four days later, the mice were sacrificed and the spleen cells harvested. The spleen cells were then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.l, available from the ATCC, No. CRL 1597. The fusions generated hybridoma cells which can then be plated from ^^^ * ¿¿¿¿¿¿¿¿¿¿¿96-well tissue culture containing the HAT medium (hypoxanthine, aminopterin, and thymidine) to inhibit the proliferation of unfused cells, myeloma hybrids, and hybrids of Spleen cells Hybridoma cells can be selected in an ELISA assay for reactivity against CHEPO. The determination of the "positive" hybridoma cells secrete the desired monoclonal antibodies against CHEPO is within the skill in the art. Hybridoma cells, positive, can be injected intraperitoneally into syngeneic Balb / c mice to produce ascites containing the anti-CHEPO monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or in spinner bottles. The purification of the monoclonal antibodies produced in the ascites can be complemented using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the binding of the antibody to protein A or protein G may be employed.

[Tt yf'Ji ^ ff ^ '^' ^ T ^ * '' '' '"' * '' - • - a ^ ** - '^« - A ^ - ^ »A - ^ i * > - ^ - '' "^^^ fe-- > -'- fffTfrfj- ^ i ^^ a .-- ^ ^ Ja- - -.-.- .. ». AA **. Ri ..

EXAMPLE 8 Purification of the CHEPO Polypeptides Using Specific Antibodies The native or recombinant CHEPO polypeptides can be purified by a variety of standard techniques in the art of purification of the protein. For example, the pro-CHEPO polypeptide, the mature CHEPO polypeptide, or the pre-CHEPO polypeptide are purified by immunoaffinity chromatography using antibodies specific for the CHEPO polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-CHEPO polypeptide antibody to an activated chromatographic resin. Polyclonal immunoglobulins are prepared from the immune serum either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Similarly, monoclonal antibodies are prepared from the mouse ascites fluid by precipitation with ammonium sulfate or chromatography on immobilized Protein A. The partially purified immunoglobulin is covalently bound to a chromatographic resin such as SEPHAROSE ™ activated with CnBr U (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derived resin is washed according to the manufacturer's instructions. Such an immunoaffinity column is used in the purification of the CHEPO polypeptide by preparing a fraction from the cells containing the CHEPO polypeptide in a soluble form. This preparation is derived by the solubilization of the whole cell or a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, the soluble CHEPO polypeptide containing a signal sequence can be secreted in a useful amount into the medium in which the cells are grown. A preparation containing the soluble CHEPO polypeptide is passed over the immunoaffinity column, and the column is washed under conditions that allow for preferential absorbance of the CHEPO polypeptide (eg, high ionic strength buffers in the presence of detergents). Then, the column is eluted under conditions that break the binding of the CHEPO polypeptide / antibody (eg, a buffer with low pH such as about pH 2-3, or a high concentration of a chaotrope such as urea or the thiocyanate ion), and the CHEPO polypeptide is collected.

EXAMPLE 9 Drug Selection This invention is particularly useful for screening compounds by using CHEPO polypeptides or binding fragments thereof in any of a variety of drug selection techniques. He The CHEPO polypeptide or fragment used in such a test can be either free in solution, fixed to a solid support, transported on a cell surface, or localized intracellularly. A method of drug selection utilizes eukaryotic or prokaryotic host cells Which are stably transformed with reclosing nucleic acids expressing the CHEPO polypeptide or fragments thereof. The drugs are selected against such transformed cells in competitive binding assays. Such cells, either in a viable or fixed form, can be used to 20 standard link assays. One can measure, for example, the formation of complexes between the CHEPO polypeptide or a fragment thereof and the agent to be tested. Alternatively, one can examine the decrease in formation in the complex between the CHEPO polypeptide and its target cell or target receptors caused by the agent to be tested. Thus, the present invention provides methods for selecting drugs or any other agent that can affect a condition or disorder associated with the CHEPO polypeptide. These methods comprise contacting such an agent with a CHEPO polypeptide or fragment thereof and carrying out assay (I) for the presence of a complex between the agent and the polypeptide or CHEPO fragments, or (ii) for the presence of a complex between the CHEPO polypeptide or fragment and the cell, by methods well known in the art. In such competitive binding assays, the CHEPO polypeptide or fragment is typically labeled. After the appropriate incubation, the free CHEPO polypeptide or fragment is separated from that which is present in linked form, and the amount of the free label or that does not form complexes is a measure of the ability of the particular agent to bind to the CHEPO polypeptide. or to interfere with the CHEPO polypeptide / cell complexes. Another technique for drug selection provides a high efficiency selection for compounds which have an adequate binding affinity to a polypeptide and is described in detail in WO 84/03564, published September 13, 1984. It was briefly established, a large number of different small peptide test compounds are synthesized on a substrate. solid, such as plastic tips or some other surface. As applied to a CHEPO polypeptide, the test compound of a peptide is reacted with the CHEPO ooiipeptide and washed. The bound CHEPO polypeptide is detected by methods well known in the art. The purified CHEPO polypeptide can also be coated directly on plates for use in the aforementioned drug selection techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support. This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding to the CHEPO polypeptide specifically compete with a test compound for binding to a CHEPO pclipeptide or fragments thereof. In this way, the antibodies can be used to detect the presence of any peptide that shares one more antigenic determinants with the CHEPO polypeptide.

EXAMPLE 10 Rational Drug Design The objective of the rational drug design is to produce structural analogs of the biologically active polypeptide of interest (ie, a CHEPO polypeptide) or of small molecules with which it interacts, for example, agonists, antagonists, or inhibitors. Any of these examples can be used as drugs which are more active or stable forms of the CHEPO polypeptide or which allow or interfere with the function of the CHEPO polypeptide in vivo (eg, Hodgson, Bio / Technology, 9: 19 -21 (1991)). In one approach, the three-dimensional structure of the CHEPO polypeptide, or of an inhibitor complex of the CHEPO polypeptide, is determined by X-ray crystallography, by computer modeling or, more typically, by a combination of the two methodologies. Both the shape and charges of the CHEPO polypeptide can be obtained to elucidate the structure and determine the active sites of the molecule. Less often, useful information regarding the structure of the * ** ?? . akí g .-- ....

CHEPO polypeptide can be obtained by modeling based on the structure of homologous proteins. In both cases, the relevant structural information is used to design analogs of molecules similar to the CHEPO polypeptide or to identify efficient inhibitors. Using the examples of rational drug design, molecules having improved activity or stability may be included as shown by Braxton and Wells, Biochemistry, 31: 7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of the native peptides as shown by Athauda et al. , J. Biochem., 113: 742-746 (1993). It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then solve it in its crystal structure. This methodology, in principle, generates a nucleus of drug on the basis of which the design of drugs can be based subsequently. It is possible to deviate the crystallography of the protein together by generating anti-idiotypic antibodies (anti-ids) to a pharmacologically active, functional antibody. Like a mirror image of an image in the mirror, the binding site of the anti-ids would be expected to be an analogue of the original receiver. The anti-ids could then be used to identify and isolate peptides from chemistry or biologically produced libraries or peptides. The isolated peptides would then act as the core of the drug. By virtue of the present invention, sufficient amounts of the CHEPO polypeptide may be available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the amino acid sequence of the CHEPO polypeptide will provide a guide for those employing techniques herein. of computer modeling in place of or in addition to X-ray crystallography. The above written specification is considered to be sufficient to enable someone skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art of the foregoing description and which fall within the scope of the appended claims.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims

CLAIMS Having described the invention as above, the content of the following is claimed as property: 1. An isolated nucleic acid molecule, characterized in that it comprises nucleotides 1 or approximately 82 to approximately 579 of Figure 2 (SEQ. ID NO: 3).
2. An isolated nucleic acid molecule, characterized in that it comprises the nucleotide sequence of Figure 2 (SEQ ID NO: 3).
3. An isolated nucleic acid molecule characterized in that it comprises a nucleotide sequence that encodes the sequence of the amino acid residues from about 1 or approximately 28 to about 193 of Figure 2 (SEQ ID NO: 2) ).
4. A vector characterized in that it comprises the nucleic acid molecule according to any of claims 1, 2, 3, 23 or 26.
5. The vector according to claim 4, characterized in that the nucleic acid molecule is operably linked to the control sequences recognized by a host cell transformed with the vector.
6. - A host cell characterized in that it comprises the vector according to claim 4.
7. The host cell according to claim 6, characterized in that said cell is a CHO cell.
8. The host cell according to claim 6, characterized in that said cell is an E. coli.
9. The host cell according to claim 6, characterized in that said cell is a yeast cell.
10. A process for the production of a polypeptide, characterized in that it comprises culturing the host cell of claim 6 under conditions suitable for the expression of said polypeptide and recovering said polypeptide from the cell culture.
11. An isolated polypeptide, characterized in that it comprises amino acid residues 1 or approximately 28 to approximately 193 of Figure 2 (SEQ ID NO: 2).
12. A chimeric molecule, characterized in that it comprises a polypeptide according to any of claims 11, 24 or 25, fused to a heterologous amino acid sequence.
13. The chimeric molecule according to claim 12, characterized in that the heterologous amino acid sequence is an epitope tag or tag sequence.
14. The chimeric molecule according to claim 12, characterized in that the heterologous amino acid sequence is an Fc region of an immunoglobulin.
15. An antibody characterized in that it binds specifically to the polypeptide according to any of claims 11, 24 or 25.
16. The antibody according to claim 14, characterized in that said antibody is a monoclonal antibody.
17. The antibody according to claim 14, characterized in that said antibody is a humanized antibody.
18. A polypeptide characterized in that it comprises an amino acid sequence selected from the group consisting of: (1) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR NXSXQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT ATLU TLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGE ACRTGDR (SEQ ID NO: 18); (2) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR NXSXQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGE ACRTGDR (SEQ ID NO: 19); (3) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKNFYAWKRN XTXQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTT LLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEA CRTGDR (SEQ ID NO: 20); (4) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR NXTXQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGE ACRTGDR (SEQ ID NO: 21); (5) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR MNXSXQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGE ACRTGDR (SEQ ID NO: 22); (6) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR MNXSXQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGE ACRTGDR (SEQ ID NO: 23); (7) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR MNXTXQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGE ACRTGDR (SEQ ID NO: 25); (8) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR MENXSXAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGE ACRTGDR (SEQ ID NO: 26); (9) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR MENXSXAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAKKEAI S PPDAASAAPLRT I TADT FRKL FRVYSNFLRGKLKL YTGE ACRTGDR (SEQ ID NO: 27); (10) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR MENXTXAVEVWQGIJILLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGE ACRTGDR (SEQ ID NO: 28); (11) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR MENXTXAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGE ACRTGDR (SEQ ID NO: 29); (12) APPRLICDSRVLERYLLEAKE.AENITTGCAEHCSLNENITVPDTKVNFYAWKR MEVNXSXVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAQKEAIS PPDAASAAPLRT I TADT FRKL FRVYSNFLRGKLKL YTGE ACRTGDR (SEQ ID NO: 30); (13) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKR MEVNXSXVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLT TLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYS? FLRGKLKLYTGE ACRTGDR (SEQ ID.? O: 31); and (14) APPRLICDSRVLERYLLEAKEAE? ITTGCAEHCSL? E? ITVPDTKV? FYAWKR MEV? XTXVEVWQGLALLSEAVLRGQALLV? SSQPWEPLQLHVDKAVSGLRSLT TLLRALGAKKEAISPPDAASAAPLRTIT.ADTFRKLFRVYS? FLRGKLKLYTGE ACRTGDR (SEQ ID.? O: 33), where X is any amino acid except proline.
19. The polypeptide according to claim 18, characterized in that it comprises an amino acid sequence selected from the group consisting of: (1) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAE? IT TGCAEHCSL? E? ITVPDTKV? FYAWKR? XSXQQAVEVWQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRT ITADTFRKLFRVYS? FLRGKLKLYTGEACRTGDR (SEQ ID.? O: 34); (2) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAE? IT TGCAEHCSL? E? ITVPDTKV? FYAWKR? XSXQQAVEVWQGLALLSEAVLRGQA LLV? SSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRT ITADTFRKLFRVYS? FLRGKLKLYTGEACRTGDR (SEQ ID.? O: 35); (3) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAE? IT TGCAEHCSL? E? ITVPDTKV? FYAWKR? XTXQQAVEVWQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRT ITADTFRKLFRVYS? FLRGKLKLYTGEACRTGDR (SEQ ID.? O: 36); (4) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAE? IT TGCAEHCSL? E? ITVPDTKV? FYAWKR? XTXQQAVEVWQGLALLSEAVLRGQA LLV? SSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRT ITADTFRKLFRVYS? FLRGKLKLYTGEACRTGDR (SEQ ID.? O: 37); (5) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKE.AE?IT TGCAEHCSL E ITVPDTKV FYAWKRM XSXQAVEVWQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRT ITADTFRKLFRVYS FLRGKLKLYTGEACRTGDR (SEQ ID O:..? 38)?????; (6) IT MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAE TGCAEHCSL E ITVPDTKV FYAWKRM XSXQAVEVWQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRT ITADTFRKLFRVYS FLRGKLKLYTGEACRTGDR (SEQ ID O:..? 39)??????; (7) IT MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAE TGCAEHCSL E ITVPDTKV FYAWKRM XTXQAVEVWQGLALLSEAVLRGQA LLV SSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRT ITADTFRKLFRVYS FLRGKLKLYTGEACRTGDR (SEQ ID O:..? 40)???????; (8) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAE? IT TGCAEHCSL? E? ITVPDTKV? FYAWKRM? XTXQAVEVWQGLALLSEAVLRGQA LLV? SSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRT ITADTFRKLFRVYS? FLRGKLKLYTGEACRTGDR (SEQ ID.? O: 41); ? (9) IT MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAE TGCAEHCSLNENITVfDTKVNFYAWKRMENXSXAVEVWQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRT IT.ADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR (SEQ ID NO:.. 42); (10) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENIT TGCAEHCSLNENITVPDTKVNFYAWKRMENXSXAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRT ITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR (SEQ ID NO: 43); (11) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKE.AENIT TGCAEHCSLNENITVPDTKVNFYAWKRMENXTXAVEVWQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRT ITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR (SEQ ID NO: 44); (12) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENIT TGCAEHCSLNENITVPDTKVNFYAWKRMENXTXAVEVWQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGaAKKEAISPPDAASAAPLRT ITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR (SEQ ID NO:.. 45); (13) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENIT TGCAEHCSLNENITVPDTÍVNFYAWKRMEVNXSXVEVWQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRT ITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR (SEQ ID NO:.. 46); (14) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENIT TGCAEHCSLNENITVPDTKVNFYAWKRMEVNXSXVEVWQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRT ITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR (SEQ ID NO:.. 47); (15) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENIT TGCAEHCSLNENITVPDTKVNFYAWKRMEVNXTXVEWQGLALLSEAVLRGQA LLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRT ITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR (SEQ ID NO:.. 48); 5 (16) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENIT TGCAEHCSLNENITVPDTKVNFYAWKiy iEVNXTXVEVWQGLALLSEAVLRGQA LLV SSQPWEPLQLHVDI AVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRT ITADTFRKLFRVYS FLRGKLKLYTGEACRTGDR (SEQ ID O:..? 49)?, 0 where X is any amino acid except proline.
20. A chimeric molecule characterized in that it comprises a polypeptide according to claim 18 or 19, fused with a heterologous amino acid sequence.
21. The chimeric molecule according to claim 20, characterized in that said heterologous amino acid sequence is a tag or tag sequence of the epitope.
22. The chimeric molecule according to claim 26, characterized in that the heterologous amino acid sequence is an Fc region of an immunoglobulin. g? F ÉÜ = - &., AÍÍ.? A.m? A & amp; - 4 - An isolated nucleic acid molecule, characterized in that it consists of the nucleotide sequence of Figure 2 (SEQ ID NO: 3). 24. An isolated polypeptide, characterized in that it consists of amino acid residues 1 or approximately 28 to approximately 193 of Figure 2 (SEQ ID NO: 2). 25.- An isolated polypeptide encoded by nucleotides 1 or approximately 82 to approximately 579 of Figure 2 (SEQ ID NO: 3). 26.- An isolated nucleic acid molecule, characterized in that it comprises a nucleic acid sequence that encodes the chimeric molecule of any of claims 12, 13, 14, 20, 21 or 22. Íi U jl »A & i- ^^ * X & amp & amp; amp;