KR20020012211A - Novel Chimpanzee Erythropoietin (CHEPO) Polypeptides and Nucleic Acids Encoding the Same - Google Patents

Abstract

The present invention relates to novel chimpanzee erythropoietin polypeptides and nucleic acid molecules encoding these polypeptides. Also provided herein are vectors and host cells comprising the nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the invention fused to heterologous polypeptide sequences, antibodies that bind to the polypeptides of the invention, and methods of producing the polypeptides of the invention do.

Description

[0001] The present invention relates to novel chimpanzee erythropoietin (CHEPO) polypeptides and nucleic acids encoding the same. ≪ Desc / Clms Page number 1 >

Erythropoiesis, or erythropoiesis, continues to occur in humans for a lifetime to compensate for cell destruction. Red blood cell hematopoiesis is a physiological mechanism that is well-suited for adequate tissue oxygenation but is very precisely controlled so that there are enough red blood cells in the blood to not interfere with blood circulation. The formation of red blood cells occurs under the control of the hormone erythropoietin in the bone marrow.

Erythropoietin is an acidic glycoprotein with a molecular weight of about 34,000 daltons and can be produced in three forms, alpha, beta and asialo. The slightly different alpha and beta forms of the carbohydrate component have the same efficacy, biological activity and molecular weight. The asialo form is an alpha or beta form in which the terminal carbohydrate (sialic acid) is removed. Erythropoietin is present in plasma at a very low concentration when the body is in a healthy state (when the tissue is under sufficient oxygen supply from an existing number of red blood cells). This normal low concentration is sufficient to stimulate replacement of erythrocytes that normally disappear through aging.

The amount of erythropoietin present in the circulatory system is increased under hypoxic conditions when oxygen transport by blood cells in the circulatory system is reduced. Hypoxia can occur due to bleeding, destruction of red blood cells due to overexposure to radiation, decreased oxygen uptake due to complications or unconsciousness over time, or a large amount of blood loss through various forms of anemia. Erythropoietin responds to hypoxic stressed tissues and stimulates red cell proliferation by stimulating primary progenitor cells to transform into prolythroblast (proerythroblast, later mature to synthesize hemoglobin and released into the circulatory system as red blood cells) in the bone marrow . When the number of red blood cells in the circulatory system is greater than the number required for normal tissue oxygen demand, erythropoietin in the circulatory system is reduced.

Since erythropoietin is essential for the process of erythropoiesis, the hormone may be useful in the diagnosis and treatment of blood disorders characterized by a low or deficient production of red blood cells (Pennathur-Das, et al. , Blood 63 (5): 1168-71 (1984) and Haddy, Am. Jour. Ped. Hematol. Oncol., 4: 191-196 (1982) (for erythropoietin in therapies that can treat sickle cell disease), and Eschbach et al., J. Clin. Invest. 74 (2): 434-441 (1984) (describing the treatment of erythropoietin on the basis of in vivo response to many plasma circles and the type of anemia associated with chronic kidney failure Suggesting a dose of 10 U EOP / kg per day for 15-40 days as a therapeutic dose). See also Krane, Henry Ford Hosp. Med. J., 31 (3): 177-181 (1983).

Herein we describe identification and characterization of novel erythropoietin polypeptides (referred to herein as CHEPO) derived from chimpanzees.

SUMMARY OF THE INVENTION [

A cDNA clone that encodes a novel chimpanzee erythropoietin polypeptide referred to herein as " CHEPO " and which is homologous to a nucleic acid encoding human erythropoietin was found.

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 (a) a DNA molecule encoding a CHEPO polypeptide having a sequence of about 1 or about 28 to about 193 amino acid residues in Figure 3 (SEQ ID NOS: 2 and 5), or (b) At least about 80%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86% nucleic acid sequence identity to a complement of a DNA molecule. Or about 87% or more, or about 88% or more, or about 89% or more, or about 90% or more, or about 91% or more, or about 92% or more, or about 93% Or about 95% or more, or about 96% or more, or about 97% or more, or about 98% or more, or about 99% or more of the nucleotide sequence.

In another aspect, the isolated nucleic acid molecule comprises (a) a nucleotide sequence encoding a CHEPO polypeptide having a sequence of about 1 or about 28 to about 193 amino acid residues in Figure 3 (SEQ ID NOS: 2 and 5), or (b) ) Complementary to the nucleotide sequence of SEQ ID NO: 2.

In one aspect, the isolated nucleic acid molecule comprises (a) a DNA molecule having a sequence of about 1 or about 82 to about 579 nucleotides of Figure 2 (SEQ ID NO: 3), or (b) At least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86% Or about 88% or greater, or about 89% or greater, or about 90% or greater, or about 91% or greater, or about 92% or greater, or about 93% or greater, or about 94% or greater, or about 95% 96% or more, or about 97% or more, or about 98% or more, or about 99% or more of the nucleotide sequence.

In another aspect, the isolated nucleic acid molecule comprises (a) a nucleotide sequence of about 1 or about 82 to about 579 of Figure 2 (SEQ ID NO: 3), or (b) a complement of the nucleotide sequence of (a).

In yet another aspect, the invention provides a nucleic acid encoding a polypeptide comprising an amino acid 1 of Figure 3 (SEQ ID NOS: 2 and 5), or a nucleotide sequence that hybridizes with a complement of a nucleic acid sequence encoding from about 28 to about 193 and which encodes an active CHEPO polypeptide To an isolated nucleic acid molecule. Preferably hybridization occurs under stringent hybridization and washing conditions.

In another aspect, the invention provides an isolated nucleic acid encoding an active CHEPO polypeptide comprising a nucleotide sequence that hybridizes with a complement of a nucleotide sequence of about 1 or about 82 to about 579 nucleotides in Figure 2 (SEQ ID NO: 3) Preferably, the hybridization occurs under stringent hybridization and washing conditions.

(A) a DNA molecule encoding a CHEPO polypeptide having a sequence of about 1 or about 28 to about 193 amino acid residues in Figure 3 (SEQ ID NOS: 2 and 5), or (b) (a) about 80% or more, or about 81% or more, or about 82% or more, or about 83% or more, or about 84% or more, or about 85% or more nucleic acid sequence identity with a complement of a DNA molecule, or At least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92% (A) or (b) under stringent conditions, provided that the test DNA molecule is hybridized with (a) or (b) under stringent conditions if it is at least about 95%, or at least about 96%, or at least about 97%, or at least about 98% And isolating the test DNA molecule.

(A) about 80% or more, or about 81% or more, or about 82% or more of the amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5) Or greater than or equal to about 83%, or greater than about 84%, or greater than about 85%, or greater than about 86%, or greater than about 87%, or greater than about 88%, or greater than about 89% , Or about 91% or more, or about 92% or more, or about 93% or more, or about 94% or more, or about 95% or more, or about 96% or more, or about 97% or more, or about 98% A nucleotide sequence encoding a polypeptide that is at least about 99% positive, or (b) an isolated nucleic acid molecule comprising (a) a complement of a nucleotide sequence.

In particular aspects, the present invention provides an isolated nucleic acid molecule comprising DNA encoding a N-terminal signal sequence and / or a CHEPO polypeptide free of initiation methionine, or an isolated nucleic acid molecule complementary to such a coding nucleic acid molecule. A signal peptide spanning from about amino acid position 1 to about amino acid position 27 in the sequence of Figure 3 (SEQ ID NOS: 2 and 5) was provisionally found. However, although the C-terminal boundary of the signal peptide may vary, it should be appreciated that the likelihood that no more than about 5 amino acids are present on each side of the signal peptide C-terminal boundary first identified herein is very high, The C-terminal boundary of the peptide can be identified according to the standard commonly used in the art to find this type of amino acid sequence element (see, for example, Nielsen et al., Prot. Eno. 10: 1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14: 4683-4690 (1986)). It should also be noted that, in some cases, the cleavage of the signal sequence of the secreted polypeptide is not entirely uniform, resulting in more than one secretory species. These polypeptides, and polynucleotides encoding them, are also included in the present invention. Thus, for purposes herein, the signal sequence 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) 5). ≪ / RTI > Thus, the mature form of the CHEPO polypeptide included in the present invention comprises a polypeptide comprising the amino acids X to 193 of Figure 3 (SEQ ID NOS: 2 and 5) and variants thereof as described below, wherein X is as shown in Figure 3 And 5). ≪ / RTI > Also included are isolated nucleic acid molecules encoding these polypeptides.

Another embodiment relates to a fragment of a CHEPO polypeptide coding sequence that encodes a fragment of a polypeptide comprising, for example, a CHEPO polypeptide coding sequence that can be used as a hybridization probe, or, optionally, a binding site for an anti-CHEPO antibody. Such nucleic acid fragments typically have a length of at least about 20 nucleotides, or at least about 30 nucleotides, or at least about 40 nucleotides, or at least about 50 nucleotides, or at least about 60 nucleotides, or at least about 70 nucleotides, or at least about 80 nucleotides, Or about 100 nucleotides or more, or about 100 nucleotides or more, or about 110 nucleotides or more, or about 120 nucleotides or more, or about 120 nucleotides or more, or about 130 nucleotides or more, or about 140 nucleotides or more, Or about 180 nucleotides or more, or about 190 nucleotides or more, or about 200 nucleotides or more, or about 250 nucleotides or more, or about 300 nucleotides or more, or about 350 nucleotides or more, or about 400 nucleotides Or about 450 nucleotides or more, or about 500 nucleotides or more, or about 600 nucleotides or more, or about 700 nucleotides or more, or about 800 nucleotides or more, or about 900 nucleotides or more, or about 1000 nucleotides or more, Refers to a plus or minus 10% of the length of the nucleotide sequence referred to. In a preferred embodiment, the nucleotide sequence fragment is derived from any coding region of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1). A novel sequencing of the CHEPO polypeptide-coding nucleotide sequence was performed by sequencing a CHEPO polypeptide-coding nucleotide sequence with another known nucleotide sequence using a sequence alignment program and determining which CHEPO polypeptide-coding nucleotide sequence fragment (s) is novel It should be noted that the manner to be determined.

All such CHEPO polypeptide-coding nucleotide sequences are included herein and can be determined without undue experimentation. Also included herein are CHEPO polypeptide fragments that are encoded by these nucleotide molecule fragments, preferably a CHEPO polypeptide fragment that includes a binding site for an anti-CHEPO antibody.

In another embodiment, the invention provides a vector comprising a nucleotide sequence encoding CHEPO or a variant thereof. This vector may comprise any of the isolated nucleic acid molecules identified above.

Host cells containing such vectors are also provided. For example, the host cell may be a CHO cell, E. coli or yeast. Also provided is a method of producing a CHEPO polypeptide comprising culturing the host cell under conditions suitable for expression of CHEPO and recovering CHEPO from the cell culture.

In another embodiment, the invention provides an isolated CHEPO polypeptide encoded by any of the isolated nucleic acid sequences identified above.

In certain aspects, the invention provides an isolated native sequence CHEPO polypeptide comprising an amino acid sequence comprising about 1 or about 28 to about 193 amino acid residues of Figure 3 (SEQ ID NOS: 2 and 5) in some embodiments.

In another aspect, the invention provides a polypeptide comprising an amino acid sequence identity of at least about 80%, or at least about 81%, or at least about 82% amino acid sequence identity to a sequence of about 1 or about 28 to about 193 amino acid residues in Figure 3 (SEQ ID NOS: 2 and 5) Or about 83%, or about 84% or more, or about 85% or more, or about 86% or more, or about 87% or more, or about 88% or more, or about 89% Or about 91% or greater, or about 92% or greater, or about 93% or greater, or about 94% or greater, or about 95% or greater, or about 96% or greater, or about 97% or greater, or about 98% RTI ID = 0.0 > 99%. ≪ / RTI >

In a further aspect, the invention provides a polypeptide comprising an amino acid sequence having at least about 80%, or at least about 81%, or at least about 82%, as compared to an amino acid sequence of about 1 or about 28 to about 193 amino acid residues in Figure 3 (SEQ ID NOS: 2 and 5) About 83% or more, or about 84% or more, or about 85% or more, or about 86% or more, or about 87% or more, or about 88% or more, or about 89% Or more, or about 92% or more, or about 93% or more, or about 94% or more, or about 95% or more, or about 96% or more, or about 97% or more, or about 98% RTI ID = 0.0 > CHEPO < / RTI >

In particular aspects, the invention provides an isolated CHEPO polypeptide that is free of N-terminal signal sequence and / or initiation methionine and that is encoded by a nucleotide sequence encoding an amino acid sequence such as the amino acid sequence. Methods of producing CHEPO polypeptides are also described herein, including culturing host cells comprising a vector containing a suitable coding nucleic acid molecule under conditions suitable for expression of the CHEPO polypeptide, and recovering the CHEPO polypeptide from the cell culture.

In yet another aspect, the invention provides a nucleic acid molecule comprising a sequence of about 1 or about 28 to about 193 amino acid residues in Figure 3 (SEQ ID NOS: 2 and 5), or a sequence that is biologically active or sufficient to provide a binding site for an anti-CHEPO antibody Wherein a CHEPO polypeptide fragment that is biologically active or that provides a binding site for an anti-CHEPO antibody can be isolated and purified using conventional techniques well known in the art . Preferably, the CHEPO fragment retains the qualitative biological activity of the native CHEPO polypeptide.

(A) a DNA molecule encoding a CHEPO polypeptide having a sequence of about 1 or about 28 to about 193 amino acid residues in Figure 3 (SEQ ID NOS: 2 and 5), or (b) (a) at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 80% nucleic acid sequence identity to a complement of a DNA molecule, Or about 86% or greater, or about 87% or greater, or about 88% or greater, or about 89% or greater, or about 90% or greater, or about 91% or greater, or about 92% or greater, or about 93% (A) or (b) under stringent conditions if the DNA molecule is at least 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98% Ii) culturing the host cell containing the test DNA molecule under conditions suitable for expression of the CHEPO polypeptide, iii) culturing the cell culture with CHEPO By recovering the peptide Lee provides a polypeptide obtained CHEPO.

In another embodiment, the invention provides a chimeric molecule comprising a CHEPO polypeptide fused to a heterologous polypeptide or amino acid sequence, wherein the CHEPO polypeptide may comprise any of the above CHEPO polypeptides, variants or fragments thereof. An example of such a chimeric molecule is a CHEPO polypeptide fused to an epitope tag sequence or the F _C region of an immunoglobulin.

In another embodiment, the invention provides an antibody as defined below which specifically binds to a CHEPO polypeptide as described above. Optionally, the antibody is a monoclonal antibody, antibody fragment or single chain antibody.

In another embodiment, the invention relates to agonists and antagonists of native CHEPO polypeptides as defined below. In a specific embodiment, the agonist or antagonist is an anti-CHEPO antibody or small molecule.

In a further embodiment, the invention relates to a method for identifying an agonist or antagonist for a CHEPO polypeptide, comprising contacting the CHEPO polypeptide with a candidate molecule and observing the biological activity mediated by the CHEPO polypeptide. Preferably, the CHEPO polypeptide is a native CHEPO polypeptide.

In a further embodiment, the present invention relates to a composition comprising a CHEPO polypeptide together with a carrier, an agonist or antagonist of a CHEPO polypeptide as described herein, or an anti-CHEPO antibody. Optionally, the carrier is a pharmaceutically acceptable carrier.

Another embodiment of the present invention is a pharmaceutical composition comprising a CHEPO polypeptide, an agonist or antagonist thereof as described herein, or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament useful for the treatment of a CHEPO polypeptide, an agonist or antagonist thereof, -CHEPO < / RTI > antibody.

Another embodiment of the present invention is directed to a polypeptide comprising (or including) at least one region of a CHEPO polypeptide as compared to a native sequence CHEPO polypeptide, preferably at amino acid position 84 of the CHEPO amino acid sequence shown in Figure 3 (SEQ ID NOs: 2 and 5) Lt; RTI ID = 0.0 > CHEPO < / RTI > In various embodiments, CHEPO variant polypeptides are prepared using well known techniques to generate N- or O-linked glycosylation sites at or near amino acid position 84 of the CHEPO polypeptide sequence. For example, the CHEPO polypeptides included in the present invention include (a) amino acids 81-84 (i.e., Met-Glu-Val-Arg; SEQ ID NO: 6) of the CHEPO amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5) X-Ser-X (SEQ ID NO: 7) or Asn-X-Thr-X (SEQ ID NO: 8), wherein X is any amino acid except Pro; (b) the amino acid 82-85 (i.e., Glu-Val-Arg-Gln; SEQ ID NO: 9) of the CHEPO amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5) has the amino acid sequence Asn-X- Ser- Asn-X-Thr-X (SEQ ID NO: 8), wherein X is any amino acid except Pro; (c) the amino acid sequence 81-84 (i.e., Val-Arg-Gln-Gln; SEQ ID NO: 6) of the CHEPO amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5) has the amino acid sequence Asn-X- Ser- Asn-X-Thr-X (SEQ ID NO: 8), wherein X is any amino acid except Pro; Or (d) the amino acid 81-84 (i.e. Arg-Gln-Gln-Ala; SEQ ID NO: 6) of the CHEPO amino acid sequence shown in Figure 3 (SEQ ID NOs: 2 and 5) has the amino acid sequence Asn-X- Ser- Or Asn-X-Thr-X (SEQ ID NO: 8), wherein X is any amino acid except Pro, to produce N-glycosylation sites at these amino acid positions. Nucleic acids encoding these variant polypeptides, vectors comprising these nucleic acids, and host cells are also encompassed herein.

The present invention relates generally to the identification and isolation of novel chimpanzee erythropoietin polypeptides and nucleic acid molecules encoding these polypeptides and recombinant production of these polypeptides.

1A-C depict nucleotides of isolated genomic DNA molecules comprising the nucleotide sequence encoding the native sequence CHEPO (nucleotides 134-146, 667-812, 1071-1157, 1760-1939 and 2074-2226, except for the remainder) (SEQ ID NO: 1). In addition, the amino acid sequence (SEQ ID NO: 2) encoded by the coding sequence of SEQ ID NO: 1 as well as the position of the initiation codon, exon and intron are also present in the genomic sequence.

Figure 2 shows the cDNA sequence (SEQ ID NO: 3) of the CHEPO molecule and the amino acid sequence (SEQ ID NO: 2) encoded thereby.

Figure 3 is a comparison of the human erythropoietin amino acid sequence (human) (SEQ ID NO: 4) with the erythropoietin (CHEPO) amino acid sequence of the chimpanzee described herein wherein the amino acid position 142 of the CHEPO sequence (SEQ ID NO: 2) or lysine (SEQ ID NO: 5).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [

1. Definitions

The terms " CHEPO polypeptide ", " CHEPO protein " and " CHEPO " as used herein include native sequence CHEPO and CHEPO polypeptide variants (this variant is further defined herein). The CHEPO polypeptide may be isolated from a variety of sources, such as human tissue or other sources, or may be prepared by recombinant and / or synthetic methods.

&Quot; Natural sequence CHEPO " includes polypeptides whose amino acid sequence is identical to native CHEPO. Such native sequence CHEPO may be isolated from chimpanzees or may be produced by recombinant and / or synthetic methods. The term " native sequence CHEPO " specifically encompasses naturally-occurring terminal-truncated or secreted forms (e. G., Extracellular domain sequences), naturally-occurring variant forms (e. G., Alternatively-spliced forms) And natural-occurring allelic variants of CHEPO. In one embodiment of the invention, the native sequence CHEPO is a mature or full-length native sequence CHEPO comprising amino acids 1 to 193 of Figure 3 (SEQ ID NOS: 2 and 5). In addition, the CHEPO polypeptide disclosed in Figure 3 (SEQ ID NOS: 2 and 5), which begins with a methionine residue referred to herein as amino acid position 1, is also referred to as another methionine residue located upstream or downstream from amino acid position 1 in Figure 3 (SEQ ID NOS: 2 and 5) May be used as the starting amino acid residues of the CHEPO polypeptide.

"CHEPO variant polypeptide" refers to a polypeptide comprising (a) residues 1 or about 28 to 193 of the CHEPO polypeptide shown in Figure 3 (SEQ ID NOS: 2 and 5), (b) residues X to 193 of the CHEPO polypeptide shown in Figure 3 Wherein X is any one of amino acid residues 23 to 32 of Figure 3 (SEQ ID NOS: 2 and 5), or (c) an amino acid sequence of another specific fragment derived from the amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5) ≪ RTI ID = 0.0 > 80% < / RTI > Such CHEPO variant polypeptides include, for example, CHEPO polypeptides in which one or more amino acid residues are added to or deleted from one or more internal domains as well as the N- and / or C-termini of the sequence of Figure 3 (SEQ ID NOS: 2 and 5) Typically, the CHEPO variant polypeptide comprises (a) residues 1 or about 28 to 193 of the CHEPO polypeptide shown in Figure 3 (SEQ ID NOS: 2 and 5), (b) residues X of the CHEPO polypeptide shown in Figure 3 193, wherein X is any one of amino acid residues 23 to 32 of Figure 3 (SEQ ID NOS: 2 and 5), or (c) an amino acid sequence of another specific fragment derived from the amino acid sequence shown in Figure 3 At least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86% Or about 95% or more, or about 95% or more, or about 95% or more, or about 95% or more, or about 91% , Or about 96% or more, or about 97% or more, or about 98% or more, or about 99% or more.

CHEPO variant polypeptides do not contain native CHEPO polypeptide sequences. Typically, the length of the CHEPO variant polypeptide is at least about 10 amino acids, at least about 20 amino acids, or at least about 30 amino acids, or at least about 40 amino acids, or at least about 50 amino acids, or at least about 60 amino acids, At least about 70 amino acids, or at least about 80 amino acids, or at least about 90 amino acids, or at least about 100 amino acids, or at least about 150 amino acids, or at least about 200 amino acids, or at least about 300 amino acids, to be.

&Quot; Amino acid sequence identity (%) " for a CHEPO polypeptide sequence identified herein refers to a sequence that aligns the sequence and, if necessary, introduces a gap to obtain maximum sequence identity (% Is defined as the percentage of amino acid residues of the candidate sequence that are identical to the amino acid residues of the CHEPO sequence without being considered as part of the identity.

Alignment to determine amino acid sequence identity (%) may be determined using a variety of methods within the skill of the art, such as publicly available computers such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign Software can be achieved. Those skilled in the art will be able to determine parameters suitable for alignment measurements, including any algorithms necessary to achieve maximum alignment of the full-length sequence to be compared. For purposes herein, however, the amino acid sequence identity (%) is obtained as described below using the sequence comparison computer program ALIGN-2, where the complete source code for the ALIGN-2 program is described in Table 1 below . The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc. The source code shown in Table 1 is registered as a user document in the US Copyright Office (Washington D. C., 20559), and the US copyright registration number is TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, Calif.) Or can be translated from the source code described in Table 1. The ALIGN-2 program can be translated and used on UNIX operating systems, preferably on Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

For purposes of the present application, the amino acid sequence identity (%) of the corresponding amino acid sequence A to the corresponding amino acid sequence B with respect to B and / or B (with respect to the corresponding amino acid sequence B, a constant amino acid sequence identity to B or B) Or a corresponding amino acid sequence A containing or containing the amino acid sequence of SEQ ID NO: 2) is calculated as follows.

X / Y x 100

Where X is the number of amino acid residues recorded as being equally matched by the sequence alignment program ALIGN-2 in the program alignment of A and B, and Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the amino acid sequence identity (%) of A to B is not equal to the amino acid sequence identity (%) of B to A. As examples of amino acid sequence identity calculations, Tables 2 and 3 below show how to calculate the amino acid sequence identity (%) of the amino acid sequence referred to as the comparison protein to the amino acid sequence designated PRO.

Unless otherwise specified, all amino acid sequence identity (%) can be obtained as described above using an ALIGN-2 sequence comparison computer program. However, the amino acid sequence identity (%) can also be calculated using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from the website (http://www.ncbi.nlm.nih.gov.). NCBI-BLAST2 uses several search parameters, such as, for example, unmasked = yes, strand = all, expected occurrence = 10, minimum low complexity length = 15/5, multi- = 0.01, a constant for multi-pass = 25, a drop-off for final gap alignment = 25, and a scoring matrix = BLOSUM62.

When NCBI-BLAST2 is used for amino acid sequence comparison, the amino acid sequence identity of the corresponding amino acid sequence A to B and / or B (in the amino acid sequence B, B and / or B) Which may or may not be represented by the corresponding amino acid sequence A, which has or has a constant amino acid sequence identity (%)) is calculated as follows.

X / Y x 100

Where X is the number of amino acid residues recorded as being equally matched by the sequence alignment program NCBI-BLAST2 in the NCBI-BLAST2 program alignment, and Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the amino acid sequence identity (%) of A to B is not equal to the amino acid sequence identity (%) of B to A.

A "CHEPO variant polynucleotide" or "CHEPO variant nucleic acid sequence" is a nucleic acid molecule that encodes an active CHEPO polypeptide as defined below: (a) a residue 1 or 28 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 the residue 23 of Figure 3 (SEQ ID NOs: 2 and 5) (C) a nucleic acid sequence identity of at least about 80% with a nucleic acid sequence encoding another specific fragment derived from the amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5). Usually, the CHEPO variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 28-193 of the CHEPO polypeptide shown in Figure 3 (SEQ ID NOS: 2 and 5), (b) a CHEPO polypeptide as shown in Figure 3 (SEQ ID NOS: 2 and 5), or (c) an amino acid sequence shown in Figure 3 (SEQ ID NOS: 2 and 5), wherein X is an amino acid residue of any of residues 23 to 32 of Figure 3 Or about 81% or more, or about 82% or more, or about 83% or more, or about 84% or more, or about 80% or more nucleic acid sequence identity to a nucleic acid sequence encoding another specific fragment derived from a nucleic acid sequence Or greater than about 85%, or greater than about 86%, or greater than about 87%, or greater than about 88%, or greater than about 89%, or greater than about 90% , Or about 94% or more, or about 95% or more, or about 96% or more, or about 97% or more, or About 98% or more, or about 99% or more. CHEPO polynucleotide variants do not contain the native CHEPO nucleotide sequence.

Usually, the CHEPO variant polynucleotide is about 30 nucleotides or more in length, or about 60 nucleotides or more, or about 90 nucleotides or more, or about 120 nucleotides or more, or about 150 nucleotides or more, or about 180 nucleotides, Or about 210 or more nucleotides or about 240 or more nucleotides or about 270 or more nucleotides or about 300 or more nucleotides or about 450 or more nucleotides or about 600 or more nucleotides or about 900 or more nucleotides, Or more.

With respect to the CHEPO polypeptide-encoding nucleic acid sequence identified herein, the term " nucleic acid sequence identity (%) " refers to aligning a candidate sequence with a CHEPO polypeptide-encoding nucleic acid sequence, introducing a gap to obtain a maximum of sequence identity if necessary, Is defined as the percentage of nucleotides in the candidate sequence that is the same as the nucleotide of the nucleotide sequence. Alignment to determine nucleic acid sequence identity (%) can be determined using a variety of methods known in the art, such as readily available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or DNASTAR software . ≪ / RTI > Those skilled in the art will be able to determine suitable parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment across the full length sequence to be compared. However, for purposes herein, nucleic acid sequence identity values (%) are obtained using the sequence comparison computer program ALIGN-2 as described below, with the complete source code for the ALIGN-2 program being described in Table 1 below . The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code shown in Table 1 is maintained in the user documentation of the United States Copyright Office (20559 Washington, DC), which is under US Copyright Registration No. TXU510087 It is registered. The ALIGN-2 program is readily available through Genentech, Inc. (South San Francisco, Calif.), Or can be translated from the source code described in Table 1. The ALIGN-2 program can be translated and used in UNIX operating systems, preferably Digital UNIX V4.0D. All sequence comparison parameters are set with the ALIGN-2 program and do not change.

For purposes of the present application, nucleic acid sequence identity (%) of the corresponding nucleic acid sequence C to D and / or D in the corresponding nucleic acid sequence D (in the nucleic acid sequence D, constant nucleic acid sequence identity% Or the corresponding nucleic acid sequence C containing or containing the nucleic acid sequence C) is calculated as follows.

W / Z x 100

Where W is the number of nucleotides that are equally recorded by the sequence alignment program ALIGN-2 in the program alignment of C and D, and Z is the total number of nucleotides in D; It should be noted that if the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, then the nucleic acid sequence identity of C to D is not equal to the nucleic acid sequence identity (%) of D to C. As examples of nucleic acid sequence identity calculations, Tables 4 and 5 show how to calculate the nucleic acid sequence identity (%) of the nucleic acid sequence referred to as the comparative DNA to the nucleic acid sequence referred to as PRO-DNA.

Unless otherwise specified, all nucleic acid sequence identities (%) used herein are obtained as above using an ALIGN-2 sequence comparison computer program. However, the nucleic acid sequence identity (%) can also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from the website (http://www.ncbi.nlm.nih.gov.). NCBI-BLAST2 uses several search parameters, such as, for example, unmasked = yes, strand = all, expected occurrence = 10, minimum low complexity length = 15/5, multi- = 0.01, a constant for multi-pass = 25, a drop-off for final gap alignment = 25, and a scoring matrix = BLOSUM62.

When NCBI-BLAST2 is used for nucleic acid sequence comparison, the nucleotide sequence identity (%) of the corresponding nucleic acid sequence C to D and / or D (to nucleic acid sequence D, D and / or D, Which may or may not be represented by the corresponding nucleic acid sequence C that has or has a constant nucleic acid sequence identity (%)) is calculated as follows.

W / Z x 100

Where W is the number of nucleotides recorded equally matched by the sequence alignment program NCBI-BLAST2 in the program alignment, and Z is the total number of nucleotides in D. It should be noted that if the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, then the nucleic acid sequence identity of C to D is not equal to the nucleic acid sequence identity (%) of D to C.

In another embodiment, the CHEPO variant polynucleotide is a nucleic acid molecule that encodes an active PRO polypeptide capable of hybridizing (preferably in stringent hybridization and washing conditions) to a nucleotide sequence encoding the full-length CHEPO polypeptide disclosed herein. The CHEPO mutant polypeptide CHEPO variant polynucleotides.

The term " positive " in relation to sequence comparisons performed as described above refers to residues of the sequence to be compared which are not only identical but of similar quality. Amino acid residues that record positive values for the amino acid residue of interest are the same residues as the amino acid residue of interest or an amino acid residue that is a preferred substituent of the amino acid residue of interest (defined in Table 6 below).

For purposes of the present application, a positive value (%) of the corresponding amino acid sequence A (corresponding amino acid sequence B, B and / or a certain positive value for B) in the corresponding amino acid sequence B with respect to B and / Or may be represented by the corresponding amino acid sequence A including the amino acid sequence A).

X / Y x 100

Where X is the number of amino acid residues that record the positive value defined above by the sequence alignment program ALIGN-2 in the program alignment of A and B and Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the positive value of A for B is not equal to the positive value of B for A.

&Quot; Isolated " as used to describe the various polypeptides disclosed herein means that the polypeptide has been identified and has been isolated and / or recovered from its natural environmental elements. Preferably, the isolated polypeptide is in a state in which all naturally associated components are not bound. Pollution factors of the polypeptide's natural environment can be substances that generally interfere with the diagnostic or therapeutic use of the polypeptide, including enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the polypeptide is (1) purified to a sufficient degree to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (2) coomassie blue or preferably silver Can be purified to yield only one band on SDS-PAGE under non-reducing or reducing conditions. Isolated polypeptides include polypeptides that are present in the recombinant cell since there will be no more than one natural environmental element of CHEPO. However, usually isolated polypeptides will be obtained by one or more purification steps.

&Quot; Isolated " nucleic acid that encodes a CHEPO polypeptide means a nucleic acid molecule identified and isolated from one or more contaminant nucleic acid molecules that are typically joined in the natural source of the CHEPO polypeptide nucleic acid. Preferably, the isolated nucleic acid is in a state in which all naturally associated components are not bound. The isolated CHEPO-encoding nucleic acid molecule is different from the form or state found in nature. Thus, isolated CHEPO-encoding nucleic acid molecules are distinguished from certain CHEPO-encoding nucleic acid molecules present in natural cells. However, for example, a polypeptide nucleic acid molecule contained in a cell that is present at a chromosomal location different from that of a natural cell and typically expressing CHEPO is included in the isolated CHEPO-encoding nucleic acid molecule.

The term " regulatory sequence " is a DNA sequence necessary for the expression of coding sequences operably linked in a particular host organism. Suitable regulatory sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.

A nucleic acid is " operably linked " when placed in a functional relationship with another nucleic acid sequence. For example, the DNA of a full-length or secretory leader is operably linked to the DNA of the polypeptide when expressed as a whole protein in its pre-secreted form, and the promoter or enhancer is capable of effecting transcription of the polypeptide sequence The ribosome binding site is operably linked to the coding sequence when it is placed to facilitate translation. Generally, " operably linked " means that the DNA sequences to be ligated are located adjacent to each other, and in the case of a secretory leader, not only located adjacent but also within the same reading frame. However, the enhancer does not need to be located contiguously. Linking is accomplished by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

The term " antibody " is used in its broadest sense and specifically includes an anti-CHEPO polypeptide monoclonal antibody (such as an agonist, antagonist and neutralizing antibody), an anti-CHEPO antibody composition with a polyepitope specificity, CHEPO antibodies and fragments of anti-CHEPO antibodies (see below). As used herein, the term " monoclonal antibody " refers to the same individual antibody that comprises a population of substantially allogeneic antibodies, i.e., a population, except for possible spontaneous mutations that may be present in minor amounts.

The " stringency " of the hybridization reaction is readily determinable by those skilled in the art and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, and shorter probes require lower temperatures. Hybridization generally depends on the ability of the denatured DNA to be re-annealed when the complementary strands are in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Thus, the higher the relative temperature, the more stringent the reaction conditions, while the lower the relative temperature, the less stringent the reaction conditions. See Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995) for more information and descriptions regarding the severity of the hybridization reaction.

As used herein, "stringent conditions" or "stringent conditions" means (1) conditions of low ionic strength and high temperature conditions, such as 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate (2) 50 mM sodium phosphate buffer (pH 6.5) / 0.1% bovine serum albumin (PBS) containing formamide, for example, 42 캜, 750 mM sodium chloride and 75 mM sodium citrate at the time of hybridization (3) 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M) in the presence of a modifier such as 50% (vol / vol) formamide prepared from 0.1% Ficoll / 0.1% polyvinylpyrrolidone (50 μg / ml), 0.1% SDS, and 10% dextran sulfate were added to a solution containing 42 mg of sodium citrate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated salmon sperm DNA And 0.2 x SSC (sodium chloride / sodium citrate) at 42 DEG C and 55 < RTI ID = 0.0 > Washed in 50% formamide, to perform a high-stringency wash with 0.1 x SSC containing EDTA at a 55 ℃.

The term " moderate stringency conditions ", as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press 1989), hybridization conditions that are less stringent than those described above Temperature, ionic strength and% SDS). Examples of moderately stringent conditions include 20% formamide, 5 x SSC (150 mM sodium chloride, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate and 20 mg / ml of truncated salmon semen-modified DNA, followed by washing the filter with 1 x SSC at about 37-50 < 0 > C. Skilled artisans will appreciate how to adjust the temperature, ionic strength, and the like necessary to meet such factors as probe length and the like.

As used herein, the term " epitope tagged " refers to a chimeric polypeptide comprising a CHEPO polypeptide fused to a " tag polypeptide ".Quot; tag polypeptide " is sufficiently short that it has sufficient residues to provide an epitope sufficient to make it, but does not interfere with the activity of the fused CHEPO polypeptide. It is desirable that the tag polypeptide is so unique that the antibody to itself does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and typically have about 8 to 50 amino acid residues (preferably about 10 to 20 residues).

The term " immunoadhesin " as used herein refers to an antibody-like molecule that combines the effector function of an immunoglobulin constant domain with the binding specificity of a heterologous protein (adhesin). Structurally, the immunoadhesin comprises a fusion of an immunoglobulin constant domain sequence with an amino acid sequence having the desired binding specificity and not an antigen recognition and binding site of the antibody. The adhered portion of the immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least the binding site of the receptor or ligand. Immunoglobulin constant domain sequences in immunoaffeicin include any of the following: IgG-1, IgG-2, IgG-3, IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, ≪ / RTI >

As used herein, "active" or "activity" refers to the form (s) of a CHEPO polypeptide that retains the biological and / or immunological activity of a natural or naturally occurring CHEPO polypeptide, wherein the "biological" (Inhibitory or promoting ability) by natural or spontaneous CHEPO, not an ability to induce antibody production to an antigenic epitope in the developing CHEPO, and " immunological " activity refers to an antigen in a natural or spontaneous CHEPO Quot; refers to the ability to induce antibody production to a sex epitope. Preferred biological activities include, for example, the ability to modulate erythropoiesis, the ability to bind receptors on the surface of related progenitors of bone marrow and / or other hematopoietic tissues, and / or the proliferation and / .

The term " antagonist " is used in its broadest sense and collectively refers to any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of a native CHEPO polypeptide disclosed herein. Similarly, the term " agonist " is used in its broadest sense and collectively refers to any molecule that mimics the biological activity of the native CHEPO polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include antibodies or antibody fragments of an agonist or antagonist, fragments or amino acid sequence variants of native CHEPO polypeptides, peptides, antisense oligonucleotides, small organic molecules, and the like. A method of identifying an agonist or antagonist of a CHEPO polypeptide can include contacting the CHEPO polypeptide with a candidate agonist molecule or a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the CHEPO polypeptide.

&Quot; Treatment " refers to treatment and prevention or prevention measures performed for the purpose of preventing or slowing (alleviating) a target condition or disease. Subjects requiring treatment include those who are already susceptible to disease or those who are sick, as well as those who are already suffering from the disease.

&Quot; Chronic " administration refers to the administration of the agonist (s) in a continuous manner as opposed to the acute mode, maintaining the initial therapeutic effect (activity) for a prolonged period of time. &Quot; Intermittent " administration refers to a treatment that is performed periodically in nature rather than continuously.

Suitable "mammals" for treatment include, but are not limited to, humans, livestock and rabbits, and mammals including zoo, competition or pets such as dogs, cats, cows, horses, sheep, pigs, goats, Of all animals classified as. A preferred mammal is a human.

Administration "in parallel" with one or more other therapeutic agents encompasses simultaneous (concurrent) administration or sequential administration in any order.

As used herein, " carrier " includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cell or mammal exposed to the dose and concentration employed. A physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptide; Proteins, such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt forming counter ions such as sodium; And / or non-ionic surfactants such as TWEEN, polyethylene glycol (PEG) and PLURONICS.

An " antibody fragment " includes a portion of a whole antibody, preferably an antigen binding region or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments; Diabody; Linear antibodies (Zapata et al., Protein Eng 8 (10): 1057-1062 (1995); single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

Papain degradation of the antibody produces two identical antigen binding fragments, the respective " Fab " fragments with single antigen binding sites, and the remaining named " Fc " fragments reflecting their ability to crystallize readily. Treatment of pepsin produces an F (ab ') ₂ fragment that has two antigen binding sites and is still cross-linked to the antigen.

&Quot; Fv " is a minimal antibody fragment comprising a complete antigen recognition and antigen binding site. This region consists of a dimer consisting of one light chain variable domain and one light chain variable domain that are tightly noncovalently bound. In this structure, three CDRs of each variable domain interact to form an antigen-binding site on the surface of the V _H -V _L dimer. In conclusion, six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv that contains only three CDRs specific for an antigen) has a lower affinity than the entire binding site, but has the ability to recognize and bind to an antigen.

In addition, the Fab fragment contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab fragments differ from Fab fragments in that several residues are added to the carboxy terminus of the heavy chain CH1 domain, which contains at least one cysteine derived from the antibody hinge region. Fab'-SH herein refers to Fab 'in which the cysteine residue (s) of the constant domain retain a free thiol group. The F (ab ') ₂ antibody fragment was originally made up of several pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chain" of an antibody (immunoglobulin) derived from any vertebrate species can be categorized as one of two distinctly different types called kappa (κ) and lambda (λ) based on the amino acid sequence of its constant domain have.

Immunoglobulin can be classified into different classes according to the amino acid sequence of the constant domain of its heavy chain. There are five major classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, some of which subclass (isotype), for example IgG1, IgG2, IgG3, IgG4, IgA and IgA2 .

A "single chain Fv" or "sFv" antibody fragment comprises the V _H and V _L domains of an antibody present in a single polypeptide chain. Preferably, the Fv polypeptide also includes a polypeptide linker between the V _H and V _L domains that allows the sFv to form the necessary structure for antigen binding (see Pluckthan in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

The term "dia body (diabody)" in the same polypeptide chain (V _H -V _L) in the light chain variable domain (V _L) small antibody fragments with two antigen-binding site comprising a heavy chain variable domain (V _H) connected to a . By using a linker that is too short to pair two domains in the same chain, the domain is forcedly paired with the complementary domain of another chain to create two antigen binding sites. Diabodies are described, for example, in EP 404,097, WO 93/11611 and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

An " isolated " antibody is an antibody that has been identified and isolated and / or recovered from elements of its natural environment. Contaminants in his natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones and other monovalent or non-hydrophobic solutes. In a preferred embodiment, the antibody comprises (1) more than 95% by weight, most preferably greater than 99% by weight of the antibody as measured by the Lowry method, or (2) using a spinning cup sequencer To a sufficient degree to obtain at least 15 residues of the N-terminal or internal amino acid, or (3) to a single band by SDS-PAGE under reducing or nonreducing conditions using coomassie blue, or preferably silver salt. It will be refined. An isolated antibody includes an antibody in situ within the recombinant cell, since one or more components of the natural environment of the antibody will not be present. However, the isolated antibody will generally be purified by one or more purification steps.

&Quot; Label " when used herein refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a " labeled " antibody. The label can be detected by itself (radioactive isotope label or fluorescent label) or, if it is an enzyme label, it can catalyze the chemical change of the detectable substrate compound or composition.

&Quot; Solid phase " means a non-aqueous matrix to which an antibody of the invention may be attached. Examples of solid phases include partially or fully formed glasses (e.g., microporous glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrenes, polyvinyl alcohols, and silicones. In certain embodiments, depending upon the context, the solid phase may comprise a well of the assay plate, and in other embodiments, the solid phase is a purification column (e. G., Affinity chromatography column). The term also includes non-continuous solid phases of discrete particles such as those described in U.S. Patent No. 4,275,149.

&Quot; Liposomes " are small vesicles composed of lipids, phospholipids and / or surfactants useful for delivering drugs (e. G., PRO polypeptides or antibodies thereto) to mammals. The components of the liposomes are typically arranged in bilayer form similar to the lipid alignment of biological membranes.

&Quot; Small molecule (small molecule) " is defined herein as having a molecular weight of about 500 daltons or less.

<Table 1>

<Table 2>

<Table 3>

<Table 4>

<Table 5>

II. Compositions and Methods of the Invention

A. Whole length CHEPO polypeptide

The present invention relates to a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as a CHEPO polypeptide. In particular, the cDNA encoding the CHEPO polypeptide was identified and isolated, as described in more detail in the Examples below.

B. CHEPO variant

It is contemplated that, in addition to the full-length native sequence CHEPO polypeptides described herein, it is possible to prepare CHEPO variants. CHEPO variants can be prepared by introducing appropriate nucleotide changes into CHEPO DNA and / or synthesizing the desired CHEPO polypeptide. Those skilled in the art will appreciate that amino acid changes, such as changes in the number or location of glycosylation sites or changes in membrane anchoring properties, can alter post-translational processing of RPO.

Variations in various domains of the full-length native sequence CHEPO or CHEPO described herein can be made, for example, using conservative and non-conservative mutagenesis techniques and instructions as described in U.S. Patent No. 5,364,934. The mutation may be substitution, deletion or insertion of one or more codons encoding CHEPO in which the amino acid sequence of CHEPO is changed compared to the native sequence CHEPO. Optionally, the mutation is generated by substituting one or more amino acids with any other amino acid in one or more CHEPO domains. Amino acid residues that can be inserted, substituted or deleted without adversely affecting the activity of interest can be obtained by comparing the sequence of CHEPO to a sequence of known homologous protein molecules and by minimizing the number of amino acid sequence changes produced in regions of high homology You can decide. Amino acid substitutions can be the substitution of one amino acid with another amino acid having similar structure and / or chemical properties, e. G., Replacement of lysine with serine, i. E. Conservative substitution of amino acids. Insertion or deletion may optionally occur at about 1 to 5 amino acids. Acceptable variations can be determined by systematically inserting, deleting, or substituting amino acids within the sequence and testing the activity of the resulting variant displayed by full-length or mature native sequences.

The subject provides CHEPO polypeptide fragments. For example, when compared to full-length native proteins, these fragments may be truncated at the N-terminus or at the C-terminus and internal residues may be deleted. Some fragments lack amino acid residues that are not essential for the desired biological activity of the CHEPO polypeptides of the invention.

The CHEPO fragment can be prepared by any of many conventional techniques. The target peptide fragments can be chemically synthesized. Another method is an enzymatic degradation method, for example, treating the protein with an enzyme known to cleave the protein at a site defined by a specific amino acid residue, or by cutting the DNA with a suitable restriction enzyme to generate a CHEPO fragment, Lt; / RTI &gt; However, another suitable technique involves isolating and amplifying a DNA fragment encoding a target polypeptide fragment by polymerase chain reaction (PCR). Oligonucleotides that define the target end of a DNA fragment are used as 5 ' and 3 &apos; primers in PCR. Preferably, the CHEPO polypeptide fragment shares one or more biological and / or immunological activities with the native CHEPO polypeptide shown in Figure 3 (SEQ ID NOS: 2 and 5).

In a specific embodiment, conservative substitutions of the subject are shown in Table 6 with the heading of preferred substitution. When the biological activity is changed by such substitution, a more substantial change, which is named as a substitution example in Table 6 below or is described in more detail below for the type of amino acid, is introduced and the product screened.

<Table 6>

Substantial variations in the function or immunological identity of the CHEPO polypeptide include (a) maintaining the structure of the polypeptide backbone in the substitution region, e.g., in sheet or spiral form, or (b) maintaining the charge or hydrophobicity of the molecule at the target site Or (c) selecting a substitution that significantly changes its effect of retaining most of the side chains. Naturally occurring residues can be divided into the following groups according to their common branching characteristics:

(1) hydrophobicity: norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilic: cys, ser, thr;

(3) Acid: asp, glu;

(4) Basicity: asn, gln, his, lys, arg;

(5) Residues that affect chain orientation: gly, pro; And

(6) aromatic; trp, tyr, phe.

Non-conservative substitutions will replace the same kind of component with another kind. In addition, the thus substituted moiety can be introduced into the conservative substitution moiety or, more preferably, can be introduced into the remaining (non-conserved) moiety.

Variations can be made using methods known in the art such as oligonucleotide-mediated (locating) mutagenesis, alanine scanning and PCR mutagenesis. Location-directed mutagenesis [Carter et al., Nucl. Acids Res. , &Lt; / RTI &gt; 13 : 4331 (1986); Zoller et al., Nucl. Acids Res. , 10: 6487 (1987)], cassette mutagenesis [Wells et al, Gene, 34 :.. 315 (1985)], restriction selection mutagenesis [Wells et al, Philos. Trans. R. Soc. London Ser A , 317 : 415 (1986)] or other known techniques into the cloned DNA to prepare the CHEPO polypeptide mutant DNA of the present invention.

In addition, one or more amino acids can be identified along the contiguous sequence using a scanning amino acid assay. Preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Typically, alanine is the preferred scanning amino acid because it is less likely to alter the backbone sequence of the variant, removing side chains outside the beta-carbon [Cunningham and Wells, Science , 244 : 1081-1085 (1989)]. In addition, alanine is preferred because it is usually the most common amino acid. Also, alanine is frequently found in both buried and exposed sites (Creighton, The Proteins , (WH Freeman & Co., NY); Chothia, J. Mol. Biol. , &Lt; / RTI &gt; 150 : 1 (1976)]. An isoteric amino acid can be used if the alanine substitution does not produce a suitable amount of the variant.

C. Variation of CHEPO

Covalent modifications of CHEPO are included within the scope of the present invention. One form of covalent modification includes reacting a target amino acid residue of a CHEPO polypeptide with an organic derivatizing agent capable of reacting with a selected side chain or N terminal or C terminal residue of CHEPO. Derivatization with a bifunctional agent is useful, for example, for cross-linking CHEPO to, or vice versa, a water-insoluble support matrix or surface for use in an anti-CHEPO polypeptide antibody purification method. Commonly used crosslinking agents are, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example esters with 4- Homobifunctional imidoesters including disuccinimidyl esters such as 3,3'-dithiobis (succinimidyl propionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and Methyl-3 - [(p-azidophenyl) dithio] propioimidate.

Other variations include deamidation of the glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of the proline and lysine, phosphorylation of the hydroxyl group of the seryl or threonyl residue, lysine, Methylation of alpha-amino groups of arginine and histidine side chains [TE Creighton, Proteins: Structure and Molecular Properties , WH Freeman & 79-86 (1983)], acetylation of the N-terminal amine and amidation of the C-terminal carboxyl group.

Other types of covalent modifications of CHEPO polypeptides that fall within the scope of the present invention include altering the natural glycosylation pattern of the polypeptide. As used herein, the term " altered native glycosylation pattern " refers to a deletion (deletion of a potential glycosylation site or deletion of a glycosylation by a chemical and / or enzymatic method) of one or more carbohydrate moieties found in the native sequence CHEPO and Or the addition of one or more glycosylation sites that are not present in the native sequence CHEPO. The term also includes qualitative changes in the glycosylation of natural proteins, including changes in the nature and proportions of the various carbohydrate residues present.

Addition of a glycosylation site to a CHEPO polypeptide can be achieved by altering the amino acid sequence. The change can be made, for example, by the addition or substitution of one or more serine or threonine residues in the native sequence CHEPO (for O-linked glycosylation sites). The CHEPO amino acid sequence can be arbitrarily changed through a change at the DNA level by mutating the DNA encoding the CHEPO polypeptide in a preselected base to produce a codon specifically translated into the desired amino acid.

Another means of increasing the number of carbohydrate residues on the CHEPO polypeptide is by chemically or enzymatically coupling the glycoside to the polypeptide. Such methods are described, for example, in WO 87/05330, published September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem. , pp. 259-306 (1981).

Removal of the carbohydrate moieties present in the CHEPO polypeptide can be accomplished chemically or by enzymatic or substitution by mutation of the codons encoding amino acid residues that function as glycosylation targets. Chemical deglycosylation techniques are well known in the art and are described, for example, in Hakimuddin, et al., Arch. Biochem. Biophys. , 259 : 52 (1987) and Edge et al., Anal. Biochem. , 118 : 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved using a variety of endoglycosidases and exoglycosidases [Thotakura et al., Meth. Enzymol. , &Lt; / RTI &gt; 138 : 350 (1987)).

Other classes of covalent modifications of CHEPO can be found in a variety of non-proteinaceous polymers, e. G. In the manner described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337 For example, connecting a CHEPO polypeptide to one of polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.

In addition, the CHEPO of the present invention may be modified in such a manner as to form chimeric molecules comprising CHEPO fused to another heterologous polypeptide or amino acid sequence.

In one embodiment of the invention, such chimeric molecules comprise a fusion of CHEPO with a tag polypeptide that provides an epitope to which the anti-tag antibody can selectively bind. The epitope tag is generally located at the amino or carboxyl terminus of CHEPO. The presence of the epitope tagged form of CHEPO can be detected using an antibody against the tag polypeptide. Further, when an epitope tag is introduced, CHEPO can be easily purified by affinity purification using an affinity matrix that binds to an anti-tag antibody or an epitope tag. A variety of tag polypeptides and their respective antibodies are known in the art. Examples include poly-histidine (poly-his) or poly-his-gly-tag, flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol. , 8: 2159-2165 (1988)] , c-myc tag and hence of 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies [Evan et al, Molecular and Cellular Biology, 5:. 3610-3636 (1985)] , And the herpes simplex virus Glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering , 3 (6): 547-553 (1990)]. Other tag polypeptides include Flag-peptides [Hopp et al., BioTechnology , 6 : 1204-1210 (1988)], KT3 epitope peptides [Martin et al., Science , 255 : 192-194 (1992) Epitope peptides [Skinner et al., J. Biol. Chem. , 266 : 15163-15166 (1991)] and the T7 gene 10 protein tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA , 87 : 6393-6397 (1990).

In another embodiment, the chimeric molecule may comprise a fusion of a specific region of an immunoglobulin or an immunoglobulin with CHEPO. In the case of a bimodal form of chimeric molecule (also referred to as " immunoadhesin "), the fusant may be the Fc region of an IgG molecule. The Ig fusions preferably include substitution of one or more regions of the variable region in the Ig molecule with the soluble (deleted or inactivated transmembrane domain) form of the CHEPO polypeptide. In one particularly preferred embodiment, the immunoglobulin fusions comprise the hinge, CH2 and CH3, or hinge, CH1, CH2 and CH3 regions of the IgG1 molecule. For methods of producing immunoglobulin fusions, see U.S. Patent No. 5,428,130, issued June 27, 1995.

D. Manufacture of CHEPO

The following description relates to a method for producing CHEPO by culturing cells transfected or transfected with a vector containing mainly CHEPO nucleic acid. Of course, CHEPO can be prepared using other methods known in the art. For example, CHEPO sequences or fragments thereof can be prepared by direct peptide synthesis using solid phase techniques (Stewart et al., Solid-Pharmaceutical Peptide Synthesis , WH Freeman Co., San Francisco, CA (1969) Merrifield, J. Am. Chem. Soc. , 85 : 2149-2154 (1963)). In vitro protein synthesis can be performed by manual or automated methods. Automated synthetic methods can be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. The different fragments of CHEPO can be chemically synthesized separately and combined using chemical or enzymatic methods to produce full-length CHEPO.

1. Isolation of DNA encoding CHEPO

DNA encoding CHEPO may be obtained from a cDNA library prepared from tissues that retain CHEPO mRNA and are expected to express it at a detectable level. Thus, human CHEPO DNA can conveniently be obtained from cDNA libraries prepared from human tissue as described in the Examples. The CHEPO coding gene may also be obtained from a genomic library or obtained by known synthetic methods (e. G., Automated nucleic acid synthesis methods).

The library can be screened using probes designed to identify the gene of interest or a protein encoded by the gene (for example, an antibody to the desired CHEPO or an oligonucleotide composed of about 20 to 80 or more bases). Screening of the cDNA or genomic library using the selected probe is performed using standard methods as described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) can do. Other means for isolating the gene encoding the CHEPO is to use a PCR method [Sambrook et al, supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Haror Laboratory Press, 1995).

The following examples illustrate techniques for screening cDNA libraries. The oligonucleotide sequence selected as the probe should be of sufficient length and sufficiently distinct sequence to minimize false results. The oligonucleotides are preferably labeled so that they can be detected upon hybridization to DNA in the library to be screened. The labeling methods are well known in the art and include radioactive labels such as ³² P-labeled ATP, biotinylated or enzymatic labels Use. Hybridization conditions, including moderate stringency and high stringency is provided in the literature (Sambrook et al., Supra).

The sequence identified in the library screening method can be aligned with a public database such as GenBank or other known sequences that may be deposited and available in other proprietary sequence databases. Sequence identity (at the amino acid or nucleotide level) within a defined region of a molecule or across a full-length sequence can be determined using methods known in the art and described herein.

Nucleic acid having protein coding sequence is the first reference to detect the precursor, if used to estimate the amino acid sequence disclosed herein and, if necessary (Sambrook et al., Supra), a normal using the primer extension method, selected cDNA or genomic libraries as described in , And processing an intermediate of the mRNA that has not been reverse transcribed with the cDNA.

2. Selection and Transformation of Host Cells

The host cell is transfected or transformed with the expression or cloning vector described herein for CHEPO production and cultured in a conventional nutrient medium modified to suit the promoter induction, transformant selection or gene amplification encoding the desired sequence. Those skilled in the art can select culture conditions such as medium, temperature, pH, etc., without performing unnecessary experiments. In general, principles, protocols, and techniques for maximizing the productivity of cell cultures are described in Mammalian Cell Biotechnology: a Practical Approach , M. Butler, ed. (IRL Press, 1991) and Sambrook et al., May be found in the above document.

Eukaryotic transfection methods and prokaryotic transformation methods, such as CaCl ₂ , CaPO ₄ , liposome-mediated methods and electroporation, are known to those skilled in the art. Depending on the host cell used, transformation is carried out using standard techniques suitable for the cell. Calcium treatment using the calcium chloride method described in the literature (Sambrook et al., Supra), or electroporation is generally used for prokaryotic cells. Infection with Agrobacterium tumefaciens has been shown to inhibit the growth of certain plant cells as described in Shaw et al., Gene , 23 : 315 (1983), and WO 89/05859, published Jun. 29, &Lt; / RTI &gt; For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology , 52 : 456-457 (1978) can be used. General characteristics of mammalian cell host system transfection are described in U.S. Patent No. 4,399,216. Transformation into yeast is generally described in Van Solingen et al., J. Bact. , 130 : 949 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA) , 76 : 3829 (1979). However, other methods of introducing DNA into cells, such as nuclear microinjection, electroporation, protoplast fusion with bacterial protoplasts, or polycations, such as polybrene and polyornithine, may also be used. See Keown et al., Methods in Enzymology , 185: 527-537 (1990) and Mansour et al., Nature , 336: 348-352 (1988) for a variety of techniques for transforming mammalian cells do.

Suitable host cells for cloning or expressing the DNA in the vector herein include prokaryotic cells, yeast or higher eukaryotic cells. Suitable prokaryotic cells include true bacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, e. Cola, but are not limited thereto. A variety of this. Coli strains, e. E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31, 537); Coli strains W3110 (ATCC 27,325) and K5 772 (ATCC 53,635) are readily available. Other suitable prokaryotic host cells include Escherichia, e. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, Enterobacteriaceae such as martha and maria, and Enterobacteriaceae such as marcescans and Shigella, and Bacillus, Subtilis and non-subtilis. Licheniformis (e.g., B. riciniformis 41P as described in DD 266,710, published April 12, 1989), Pseudomonas such as p. Aeruginosa and Streptomyces. &Lt; / RTI &gt; These examples are merely illustrative and not restrictive. The strain W3110 is a particularly preferred host or parental host since it is a host strain common to recombinant DNA product fermentation. Preferably, the host cell secretes a minimal amount of proteolytic enzyme. For example, strain W3110 can be modified to result in a mutation of the gene encoding the endogenous protein of the host, and an example of such a host is E. coli having the complete genotype tonA. E. coli W3110 strain 1A2, with complete genotype tonA ptr3. E. coli W3110 strain 9E4, complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan ^r . E. coli W3110 strain 27C7 (ATCC 55,244), complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kan ^r . E. coli W3110 strain 37D6, strain 37D6 with a non-kanamycin resistant degP deletion mutant. E. coli W3110 strain 40B4 and the periplasmic protease variants disclosed in U.S. Patent No. 4,946,783, issued August 7, 1990. Coli strains. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotic cells, eukaryotic microbes such as fibrous fungi or yeast are suitable as cloning or expression hosts for CHEPO-coding vectors. Saccharomyces cerevisiae is a lower eukaryotic host microorganism commonly used. Other microorganisms include Schizosaccharomyces pombe (Beach and Nurse, Nature , 290: 140 [1981]; EP 139,383, published May 2, 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al., Bio / Technology , 9: 968-975 (1991)); Lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol. , 737 [1983]), Kay. Fragilis (ATCC 12,424), Kay. Bulgaricus (ATCC 16,045), Kay. Wickeramii (ATCC 24,178), Kay. Waltii (ATCC 56,500), Kay. Drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology , 8: 135 (1990)), Kay. Thermotolerans and K. Marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol. , 28: 265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA , 76: 5259-5263 [1979]); Schwanniomyces such as, for example, Siebenia mume occidentalis (EP 394,538, published October 31, 1990); And filamentous fungi such as Neurospora, Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991) and Aspergillus host, e.g., Listen to this. Biochem. Biophys. Res. Commun. , 112: 284-289 [1983]; Tilburn et al., Gene , 26: 205-221 [1983]; Yelton et al. Proc. Natl. Acad. Sci. USA , 81: 1470-1474 [1984]) and AA. Niger (Kelly and Hynes, EMBO J. , 4: 475-479 [1985]). Methyl auxotrophic yeast is suitable and is a genus consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis and Rhodotorula But not limited to, yeast that can grow on methanol. A list of specific species that are examples of these types of yeast can be found in [C. Anthony, The Biochemistry of Methylotrophs , 269 (1982).

Suitable host cells for the expression of glycosylated CHEPO are derived from multicellular organisms. Examples of invertebrate cells include insect cells and plant cells such as Drosophila S2 and Spodoptera Sf9. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) cells and COS cells. More specific examples include monkey kidney CV1 line (COS-7, ATCC CRL 1651) transformed by SV40, human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen. Virol. , 36: 599 (1997)), Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. 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 mouse mammary tumor (MMT 060562, ATCC CCL51) . One skilled in the art can select suitable host cells.

3. Choosing and Using Replicable Vectors

A nucleic acid (e. G., CDNA or genomic DNA) encoding CHEPO may be inserted into a clonable (amplification of DNA) or a replicable vector for expression. Various vectors can be easily obtained. For example, the vector may be in the form of a plasmid, a cosmid, a viral particle or a phage. Suitable nucleic acid sequences may be inserted into the vector by a variety of methods. Generally, DNA is inserted into the appropriate restriction endonuclease site (s) using techniques known in the art. The vector component generally includes, but is not limited to, a signal sequence, a replication origin, one or more marker genes, an enhancer component, a promoter, and a transcription termination sequence. The preparation of suitable vectors containing one or more of the above ingredients uses standard ligation techniques known to those skilled in the art.

CHEPO can be produced as a fusion polypeptide with a heterologous polypeptide that can be produced by direct recombinant methods as well as other polypeptides or signal sequences that have a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of a vector or it may be part of a CHEPO-coding DNA inserted into a vector. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or thermostable enterotoxin II leaders. For yeast secretion, the signal sequence may be, for example, a yeast invertase leader, an alpha factor leader (including Saccharomyces and Cluveromycetes alpha-factor leader (US Patent 5,010,182)) or an acid phosphatase leader, . It may be a signal sequence as described in C. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990) or WO 90/13646 published Nov. 15, 1990. In mammalian cell expression, signal sequences from secretory polypeptides of mammalian signal sequences, e. G., The same or related species, and viral secretory leader can be used to direct secretion of the protein.

Both the expression and cloning vectors contain nucleic acid sequences that allow the vector to replicate in one or more host cells selected. Such sequences are known for a variety of bacteria, yeast and viruses. The origin of replication from plasmid pBR322 fits most Gram negative bacteria, the 2μ plasmid origin fits yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) clone vectors in mammalian cells useful.

Expression and cloning vectors will typically contain a selectable gene, also referred to as a selectable marker. A representative selection gene, for example, in the case of Bacillus, is a gene encoding D-alanine racemase that (a) is a protein that confers resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, b) proteins that complement nutritional deficiencies, or (c) proteins that supply important nutrients that are not available from complex media.

Examples of suitable selectable markers for mammalian cells are those which enable the identification of cells capable of accepting CHEPO-encoding nucleic acids, e. G. DHFR or thymidine kinase. When wild-type DHFR is used, a suitable host cell is a CHO cell line lacking DHFR activity, see Urlaub et al, Proc. Natl. Acad. Sci. USA , 77: 4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the 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 trp1 gene provides selection markers for mutant strains of yeast lacking the ability to grow into tryptophan (e.g., ATCC 44076 or PEP4-1) [Jones, Genetics , 85: 12 (1977)].

Expression and cloning vectors generally contain a promoter operably linked to a CHEPO coding nucleic acid sequence that directs mRNA synthesis. Promoters recognized by a variety of potential host cells are known. Promoters suitable for use in prokaryotic hosts include the? -Lactamase and lactose promoter systems [Chang et al., Nature , 275: 615 (1978); Goeddel et al., Nature , 281: 544 (1979)], alkaline phosphatase, tryptophan (trp) promoter system [Goeddel, Nucleic acid Res. , &Lt; / RTI &gt; 8: 4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA , 80: 21-25 (1983). In addition, the promoter used in the bacterial system will contain a Shine-Dalgano (SD) sequence operably linked to the DNA encoding CHEPO.

Examples of promoter sequences suitable for use in yeast hosts include 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem. , 255: 2073 (1980)] or other sugar degrading enzymes [Hess et al., J. Adv. Enzyme Reg. , &Lt; / RTI &gt; 7: 149 (1968); Holland, Biochemistry , 17: 4900 (1978)) such as enolase, glyceraldehyde-3-phosphate dihydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6 -Phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosulfate isomerase, phosphoglucose isomerase and glucokinase.

Other yeast promoters that are inducible promoters with the additional advantage of transcription controlled by growth conditions include alcohol dehydrogenase 2, isocytocrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism, metallothionein, glyceraldehyde -3-phosphate dehydrogenase, and an enzyme that acts on maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

CHEPO transcription from vectors in mammalian host cells can be carried out using viruses, e. G., Poliomaviruses, papillomaviruses (UK 2,211,504 published 5 July 1989), adenoviruses (e. G. Adenovirus 2) A promoter obtained from the genome of virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and monkey virus 40 (SV40), heterologous mammalian promoters such as actin promoter or immunoglobulin promoter and heat- &Lt; / RTI &gt; which is suitable for the host cell system.

Transcription of DNA encoding CHEPO by higher eukaryotic cells can be increased by inserting an enhancer sequence into the vector. Enhancers are generally cis-acting components of DNA of about 10 to 300 bp that act on the promoter to increase transcription. Many enhancer sequences are known to result from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, an enhancer will usually be used which is derived from eukaryotic viruses. Examples include the SV40 enhancer (bp 100-270) on the back of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the back of the origin of replication, and the adenovirus enhancer. The enhancer may be spliced to the vector at the 5 'or 3' position in the CHEPO coding sequence, but is preferably located at the 5 'site from the promoter.

In addition, expression vectors used in eukaryotic host cells (polynuclear cells derived from yeast, fungi, insects, plants, animals, humans or other multicellular organisms) may contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are typically obtained from the 5 'and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions include nucleotide fragments that are transcribed as polyadenylation fragments in the untranslated portion of the mRNA encoding CHEPO.

Other methods, vectors, and host cells suitable for application in the synthesis of CHEPO in recombinant vertebrate cell culture 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.

4. Detection of gene amplification / expression

Gene amplification and / or expression can be performed, for example, by conventional Southern blotting, northern blotting to quantitate transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA , 77: 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization using appropriately labeled probes based on the sequences provided herein. Alternatively, an antibody capable of recognizing a particular double-strand, including DNA duplexes, RNA duplexes and DNA-RNA hybrid duplexes or DNA-protein duplexes, can be used. In other words, an antibody can be labeled and the double strand bound to the surface to perform an assay that can detect the presence of an antibody bound to the double strand when a double strand is formed on the surface.

Alternatively, gene expression can be measured by analysis of cell culture fluids or body fluids to directly quantitate the expression of the gene product and immunological methods such as immunohistochemical staining of cells or tissue sections. Antibodies useful for immunohistochemical staining and / or sample fluid analysis may be monoclonal or polyclonal antibodies and may be prepared in any mammal. Conveniently, an antibody against a native CHEPO polypeptide or a synthetic peptide based on the DNA sequence provided herein or an exogenous sequence fused to CHEPO DNA and encoding a particular antibody epitope can be produced.

5. Purification of the polypeptide

The form of CHEPO can be recovered from the culture medium or the bacterial solution for host cells. When bound to the membrane, it can be released from the membrane by using a suitable detergent solution (e.g. Triton-X 100) or by cleavage of the enzyme. Cells used for the expression of CHEPO may be pulverized by various physical or chemical means such as freeze-thaw cycles, sonication, mechanical grinding or cell homogenization.

It may be desirable to purify CHEPO from recombinant cell proteins or polypeptides. Examples of suitable purification methods include fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resins such as chromatography on DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, Gel filtration using G-75, a protein A Sepharose column for removing contaminants such as IgG, and a metal chelating column for binding the epitope tag form of CHEPO. A variety of protein purification methods can be used, such methods are well known in the art and are described, for example, in Deutscher, Methods in Enzymology , 182 (1990); Scopes, Protein Purification: Principles and Practice , Springer-Verlag, New York (1982). The purification step (s) selected will depend, for example, on the production method used and on the characteristics of the particular CHEPO produced.

Uses of E. CHEPO

The nucleotide sequence (or its complement) encoding CHEPO has a variety of uses in the field of molecular biology, including chromosome and gene mapping, and in the production of antisense RNA and DNA, including as a hybridization probe. In addition, CHEPO-encoding nucleic acids will also be useful for the production of CHEPO polypeptides by recombinant techniques as described herein.

The full length native sequence CHEPO gene or fragment thereof may be isolated from full length CHEPO cDNA or other cDNA (e. G., A gene encoding CHEPO from a naturally occurring variant or other species of CHEPO) having the desired sequence identity with the native CHEPO sequence disclosed herein Can be used as a hybridization probe for the cDNA library to be used. Optionally, the length of the probe will be from about 20 to about 50 bases. The hybridization probe may be derived from a genomic sequence comprising at least a portion of a full-length natural nucleotide sequence, wherein the region may be determined without undue experimentation, or a promoter, enhancer component and intron of the native sequence CHEPO. For example, the screening method will include isolating the coding region of the CHEPO gene using a known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes can be labeled with a variety of labels, including enzymatic labels such as alkaline phosphatase coupled to probes via radioactive nucleotides such as ³² P or ³⁵ S or avidin / biotin coupling systems. A library of human, cDNA, or mRNA can be screened using a labeled probe having a sequence complementary to the CHEPO gene of the present invention to determine the member of the library to which the probe hybridizes. The hybridization technique is described in more detail in the following examples.

Any EST sequence disclosed herein may be used analogously as a probe using the methods described herein.

Other useful fragments of the CHEPO nucleic acid include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (RNA or DNA) capable of binding to a target CHEPO mRNA (sense) or CHEPO DNA (antisense) sequence. An antisense or sense oligonucleotide according to the present invention comprises a fragment of the coding region of CHEPO DNA. This fragment generally comprises at least about 14 nucleotides, preferably about 14 to 30 nucleotides. The ability to derive antisense or sense oligonucleotides based on cDNA sequences encoding the protein is described, for example, in Stein and Cohen ( Cancer Res. 48: 2659, 1988) and van der Krol et al. ( BioTechniques 6: 958, 1988).

The binding of the antisense or sense oligonucleotide to the target nucleic acid sequence forms a double strand that blocks transcription or translation of the target sequence by one of several methods or other methods including enhanced resolution of the double strand, early termination of transcription or translation . Thus, antisense oligonucleotides can be used to block the expression of CHEPO proteins. The antisense or sense oligonucleotides also include oligonucleotides having a modified sugar-phosphodiester backbone (or other sugar chains, for example, the sugar chains disclosed in WO 91/06629), wherein such sugar chains are internal nucleases There is resistance to the subject. Such oligonucleotides with a resistant sugar chain retain sequence homology that is stable in vivo (i.e., resistant to enzymatic degradation) but capable of binding to a target nucleotide sequence.

Other examples of sense or antisense oligonucleotides include, but are not limited to, residues disclosed in WO 90/10048 and other residues that increase the affinity of the oligonucleotide for the target nucleic acid sequence, such as poly- (L-lysine) Covalently linked oligonucleotides are included. In addition, an intercalator, such as elliticin, and an alkylating agent or metal complex can be linked to a sense or antisense oligonucleotide to alter the binding specificity of the antisense or sense oligonucleotide to the target nucleotide sequence.

For example, CaPO _4-on cells containing the target nucleic acid sequence by using a gene transfer vector, such as a bar (Epstein-Barr) virus-mediated DNA transfection method, electret troponin of any gene transfer method, including migration or Epstein Antisense or sense oligonucleotides can be introduced. In one preferred method, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. Cells containing the target nucleic acid sequence are contacted with the recombinant retroviral vector in vivo or ex vivo. Suitable retroviral vectors include vectors derived from murine and retroviral M-MuLV, N2 (a retrovirus derived from M-MuLV), or a double copy vector referred to as DCT5A, DCT5B and DCT5C (see WO 90/13641) But are not limited to.

In addition, as disclosed in WO 91/04753, a sense or antisense oligonucleotide can be introduced into a cell comprising a target nucleotide sequence by complexing with a ligand binding molecule. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, proliferation factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the linkage of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind its corresponding molecule or receptor, nor does it block the intracellular entry of the sense or antisense oligonucleotide or its complex form.

Alternatively, a sense or antisense oligonucleotide can be introduced into a cell containing a target nucleic acid sequence by forming an oligonucleotide-lipid complex, as disclosed in WO 90/10448. Such sense or antisense oligonucleotide-lipid complexes are preferably separated by an internal lipase in the cells.

The probe can also be used in PCR technology to generate a set of sequences for the identification of closely related CHEPO coding sequences.

In addition, a nucleotide sequence encoding CHEPO can be used to prepare a hybridization probe for mapping genes coding for CHEPO and for genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein can be mapped to specific regions of chromosomes and chromosomes using known techniques such as in situ hybridization, linkage analysis to known chromosome markers, and hybridization screening using libraries.

If the coding sequence for CHEPO encodes a protein that binds to another protein (e.g., where CHEPO is a receptor), then CHEPO can be used in assays to identify other proteins or molecules involved in such binding interactions . By this method, inhibitors of receptor / ligand binding interactions can also be identified. In addition, proteins involved in the binding interactions can be used to screen for small molecule inhibitors or agonists of peptides or binding interactions. In addition, similar ligands can be isolated using the receptor CHEPO. Screening assays can be designed to find lead compounds that mimic the biological activity of the receptor on native CHEPO or CHEPO. Such screening assays will include high throughput screening assays for chemical libraries and will be particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell-based assays, which are characterized in the art.

In addition, nucleic acids encoding CHEPO or its variants can be used to generate transgenic animals or " knock out " animals useful in the development and screening of useful therapeutic agents. Transgenic animals (e. G., Mice or rats) are animals with cells that contain a transgene, the transgene being introduced into the animal or an ancestor of the animal in the fetal phase, e. G. do. The transgene is DNA that is integrated into the genome of the cell, from which transgenic animals develop. In one embodiment, the genomic DNA encoding CHEPO can be cloned according to established techniques using cDNA encoding CHEPO, and transgenic cells containing cells expressing the DNA encoding CHEPO using this genomic sequence Animals can be created. In particular, methods for producing transgenic animals such as mice or rats are conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, certain cells are targeted for the introduction of a CHEPO transgene with a tissue-specific enhancer. Transgenic animals containing transfection genes encoding one copy of CHEPO introduced into the animal's reproductive line at the embryonic stage can be used to investigate the increased expression of CHEPO-encoding DNA. Such animals can be used, for example, as test animals for reagents that are believed to protect against pathological symptoms associated with overexpression of CHEPO polypeptides. According to this aspect of the invention, treating the animal with the reagent results in a reduction in the incidence of pathological symptoms compared to untreated animals carrying the transgene, which indicates potential therapeutic intervention for the pathological symptoms.

Alternatively, a non-human analogue of CHEPO may be used to introduce a defective or modified gene encoding CHEPO as a result of homologous recombination between the endogenous gene encoding CHEPO and the modified genomic DNA encoding CHEPO introduced into the embryonic stem cell of this animal CHEPO "knock-out" can be used to make animals. For example, genomic DNA encoding CHEPO can be cloned according to established techniques using cDNA encoding CHEPO. Some of the genomic DNA encoding CHEPO may be deleted or substituted with other genes, such as genes encoding an optional marker that can be used to monitor integration. Generally, unmodified flanking DNA (at both the 5 'and 3' ends) of thousands of bases is included in the vector (see, for example, Thomas and Capecchi, Cell , 51: 503 (1987)). This vector is introduced into the embryonic cell line (for example, by electroporation), and cells in which the introduced DNA and the endogenous DNA are homologously recombined are selected (see, for example, Li et al., Cell , 69 : 915 (1992)). Selected cells are then injected into blastocysts of animals (e. G., Mice or rats) to form aggregation chimeras (see, for example, Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ Robertson, , Oxford, 1987), pp. 113-152). The chimeric embryos were then transplanted into suitable primate surrogate animal to produce " knock-out " animals. Progeny possessing homologous recombinant DNA in germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain homologous recombinant DNA. Rust-out animals are characterized, for example, by their ability to defend against certain pathological conditions and the occurrence of pathological symptoms due to the absence of CHEPO polypeptides.

The nucleic acid encoding the polypeptide may also be used in gene therapy. When used in gene therapy, a gene is introduced into a cell to achieve, for example, a defective gene in order to achieve in vivo synthesis of a therapeutically effective gene product. &Quot; Gene therapy " encompasses both traditional gene therapy where a sustained effect is achieved by a single treatment, and administration of a gene therapy agent, including once or repeated administration of therapeutically effective DNA or mRNA. Antisense RNA and DNA can be used as therapeutic agents to block the expression of a specific gene in vivo. It has already been shown that the absorption of short antisense oligonucleotides through cell membranes is limited, so that despite their low intracellular concentrations of these oligonucleotides, they can act as inhibitors when introduced into cells (Zamecnik et al., Proc. Natl. Acad Sci. USA 83, 4143-4146 (1986)). Such oligonucleotides can increase the uptake of oligonucleotides by modification, for example, by replacing their negatively charged phosphodiester groups with non-charged groups.

There are a variety of techniques that can be used to introduce nucleic acids into living cells. This technique depends on whether the nucleic acid is delivered to the cultured cells in vitro or to the cells of the desired host in vivo. Suitable techniques for delivering nucleic acids into mammalian cells in vitro include liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. Currently preferred as in vivo gene transfer techniques are transfection using virus (typically retroviral) vectors and viral coat protein-liposome-mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 1993). In some cases, it is desirable to provide the nucleic acid source with an agent that targets the target cell, such as a cell surface membrane protein or an antibody specific for the target cell, a ligand for a receptor on the target cell, and the like. When liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis can be used for targeting and / or promoted uptake, examples of which include, but are not limited to, An antibody to a protein that undergoes internalization during circulation, a protein that targets the intracellular location and increases intracellular half-life, and the like. Receptor-mediated endocytosis techniques are described for example in Wu et al., J. Biol. Chem. 262, 4429-4432 (1987), and Wagner et al., Proc. Natl. Acad. -3414 (1990). For a review of gene marking and gene therapy protocols, see Anderson et al., Science 256, 808-813 (1992).

The CHEPO polypeptides disclosed herein may also be used as molecular weight markers for protein electrophoresis.

Nucleic acid molecules or fragments thereof encoding the CHEPO polypeptides disclosed herein are useful for identification of chromosomes. In this regard, there is a continuing need to identify new chromosomal markers because the available chromosomal marking reagents based on actual sequence data are currently relatively small. Each of the CHEPO nucleic acid molecules of the present invention can be used as a chromosome marker.

The CHEPO polypeptides and nucleic acid molecules of the present invention may also be used for tissue typing, wherein the CHEPO polypeptides of the present invention may be differentially expressed in one tissue compared to other tissues. The CHEPO nucleic acid molecule will be used to generate probes for PCR, Northern analysis, Southern analysis and Western analysis.

The CHEPO polypeptides disclosed herein may also be used as therapeutic agents. The CHEPO polypeptides of the present invention may be formulated according to known methods to produce pharmaceutically useful compositions wherein the CHEPO product is combined with a pharmaceutically acceptable carrier vehicle. Therapeutic formulations are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the active ingredient having the desired degree of purity with any physiologically acceptable carrier, excipient or stabilizer ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. 1980). Acceptable carriers, excipients or stabilizers are non-toxic to the patient at the dosages and concentrations employed and include antioxidants such as phosphates, citrates and other organic acids, ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, serum albumin , Proteins such as gelatin or immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and carbohydrates including glucose, mannose, or dextrin, A chelating agent such as EDTA, a sugar alcohol such as mannitol or sorbitol, a salt-forming counter ion such as sodium, and / or a nonionic interface such as Tween (TM), Pluronics (registered trademark) Active agents and the like.

The formulation used for in vivo administration should be sterilized. This is easily accomplished by filtration through sterile filtration membranes before or after lyophilization and reconstitution.

The therapeutic compositions herein may be placed in a container with a sterile access port, for example a vial containing an intravenous solution bag or a stopper pierceable by a hypodermic needle.

The route of administration may be a known method, for example, by injection or injection by intravenous, intraperitoneal, intracerebral, intramuscular, intravenous, intraarterial, or intralesional routes, topical administration, or sustained release.

The dosage of the pharmaceutical composition of the present invention and the desired drug concentration may vary depending on the particular application for which it is intended. Determination of the appropriate dosage or route of administration is well known to those skilled in the art. The results of animal experiments provide reliable guidance in determining effective doses for human treatment. An effective dose scaling of the species is described in Mordenti, J. and Chappell, W. " The use of interspecies scaling in toxicokinetics " in Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, , pp. 42-96).

When a CHEPO polypeptide or agonist or antagonist thereof is used in vivo, the typical dosage will depend on the route of administration and will be in the range of about 10 ng to 100 mg per kg of body weight of the mammal, or preferably about 1 Mu] g / kg to 10 mg / kg. Instructions for specific dosages and delivery methods are provided in the literature, for example, in U.S. Pat. Nos. 4,657,760, 5,206,344 or 5,225,212. It is contemplated that other agents may be effective for other therapeutic compounds and other conditions, and administration targeting one organ or tissue may require delivery in a different manner than administration to, for example, another organ or tissue do.

Microencapsulation of CHEPO polypeptides is contemplated where sustained release administration of a CHEPO polypeptide is required for an agent having release characteristics suitable for the treatment of a particular disease or disorder requiring administration of the CHEPO polypeptide. Microencapsulation of recombinant proteins for delayed release has been successfully performed using human growth hormone (rhGH), interferon (rhIFN), interleukin-2 and MN rgp120 (Johnson et al., Nat. Med. , 2: 795- Cleland, " Design and Production of Single Immunization Vaccines Using &quot;(1996); Yasuda, Biomed. Ther. , 27: 1221-1223 (1993); Hora et al., Bio / Technology , 8: 755-758 Polyactide Polyglycolide Microsphere Systems, "in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp 439-462:. WO 97/03692, WO 96/40072, WO 96 / 07399 and U.S. Patent No. 5,654,010).

The sustained-release formulation of the protein has been developed using poly-lactic acid-co-glycolic acid (PLGA) polymers due to its biocompatibility and wide biodegradability properties. Lactic acid and glycolic acid, degradation products of PLGA, can be rapidly lost in the human body. The resolution of the polymer can also be adjusted to months to years depending on its molecular weight and 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).

The present invention includes a method for screening compounds (antagonists) for identifying compounds (agonists) mimicking a CHEPO polypeptide and for preventing the effects of CHEPO polypeptides. Screening assays for antagonist drug candidates were designed to identify compounds that bind or complex with CHEPO polypeptides encoded by the genes identified herein or that interfere with the interaction of coded polypeptides with other intracellular proteins. Such screening assays include assays capable of high throughput screening for chemical libraries, which would be particularly suitable for identifying small molecule drug candidates.

Such assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell-based assays, which are characterized in the art. The commonality of all assays for antagonists is that they require contact of the CHEPO polypeptide encoded by the nucleic acid identified herein with the drug candidate for a sufficient time and under conditions sufficient to interact.

In the binding assay, the interaction is binding, and the complex formed can be isolated or detected in the reaction mixture. In certain embodiments, CHEPO polypeptides or drug candidates encoded by the genes identified herein are fixed to a solid phase, e. G., A microtiter plate, by covalent attachment or noncovalent attachment. Non-covalent attachment is generally achieved by coating the solid surface with a solution of CHEPO polypeptide and drying. Alternatively, a monoclonal antibody specific for an immobilized antibody, e. G., A fixed CHEPO polypeptide, can be used to anchor it to a solid surface. This assay is carried out by adding a non-immobilizable component which can be labeled with a detectable label, to a coding surface comprising a fixed component, for example an anchored component. When the reaction is terminated, unreacted components are removed, for example by washing, and complexes anchored to the solid surface are detected. Detection of a label immobilized on a surface indicates that a complexing reaction has taken place if the original, non-immobilized component carries a detectable label. If the original non-immobilized component does not carry the label, for example, a labeled antibody that specifically binds to the immobilized complex can be used to detect the complexation reaction.

If the candidate compound interacts with but does not bind to a particular CHEPO polypeptide encoded by the gene identified herein, the interaction of the candidate compound with the polypeptide may be analyzed by well known methods for detecting protein-protein interactions . Such assays include conventional methods, such as cross-linking, co-immunoprecipitation, and co-purification through gradient or chromatographic columns. Protein-protein interactions have also been reported by Fields and co-workers (Fields and Song, Nature (London) , 340: 245-246 (1989); Chien et al., Proc. Natl. Acad. : 9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA , 89: 5789-5793 (1991)). Many transcription activators, such as yeast GAL4, consist of two physically distinct modular domains, one acting as a DNA-binding domain and the other acting as a transcription-active domain. The yeast expression system described in the above publications (generally referred to as "2-hybrid system") utilizes this property and uses a 2-hybrid protein, one of which is the target protein having the DNA- And the other is the fusion of the candidate active protein with the active domain. The expression of the GAL1-lacZ reporter gene under the control of the GAL4-active promoter depends on the reconstitution of the GAL4 activity through protein-protein interactions. Colonies containing interacting polypeptides are detected using a chromogenic substrate for beta -galactosidase. A MATCHMAKER (registered trademark) that confirms protein-protein interactions between two specific proteins using the 2-hybrid technique is available from Clontech. In addition, this system can be extended to map protein domains involved in specific protein interactions, as well as to pinpoint the position of amino acid residues critical to such interactions.

In the following way, compounds which interfere with the interaction of other genes, which encode CHEPO polypeptides identified herein, with other intracellular or extracellular components can be tested. A reaction mixture comprising the product of the gene encoding the CHEPO polypeptide and the intracellular or extracellular component was prepared under conditions and for a time allowing interaction and binding of the two products. To test the ability of the candidate compound to inhibit binding, the reaction was carried out in the presence and absence of the test compound. Placebo can also be added to a third reaction mixture that serves as a positive control. Binding (complex formation) between intracellular or extracellular components and test compounds present in the mixture was monitored as described above. A complex formed in the control reaction but no complex formed in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound with its reaction counterpart.

To analyze the antagonist, the CHEPO polypeptide could be added to the cells along with the compound to screen for the specific activity, and the ability of the compound to inhibit the desired activity in the presence of the CHEPO polypeptide showed that this compound is an antagonist to the CHEPO polypeptide. Alternatively, antagonists could be detected by binding CHEPO polypeptides and potential antagonists with membrane-bound CHEPO polypeptide receptors or recombinant receptors under appropriate conditions for competitive inhibition analysis. CHEPO polypeptides can be labeled, for example, with radioactive isotopes, to determine the effect of potential antagonists using the number of CHEPO polypeptide molecules that bind to the receptor. Genes encoding the receptor could be identified by several methods known to those skilled in the art, for example, the ligand panning method and the FACS taxonomy (Coligan et al., Current Protocols in Immun. , 1 (2): Chapter 5 (1991) . Preferably, expression cloning is used wherein the polyadenylated RNA is prepared from cells that are reactive to the CHEPO polypeptide, and the cDNA library generated from the RNA is classified as a PUO and is a COS cell that is unreactive to the CHEPO polypeptide or It is used to transfect other cells. Transfected cells proliferating on a glass slide are exposed to the labeled CHEPO polypeptide. The CHEPO polypeptide could be labeled by several methods including iodination or encapsulation of the recognition site on the site-specific protein kinase. After immobilization and incubation, the slides were analyzed by autoradiography. Positive pools were identified to produce sub-pools and re-transfected using interactive sub-fooing and re-screening methods, resulting in a single clone encoding the putative receptor.

In another way to identify the receptor, the labeled CHEPO polypeptide could be photo-chemically-coupled with an extractant sample expressing the cell membrane or receptor molecule. The crosslinked material was analyzed by PAGE and exposed to x-ray film. The labeled complexes containing the receptor could be cut and digested into peptide fragments for protein micro-sequencing. By designing a set of degenerate oligonucleotide probes using the amino acid sequence obtained from micro-sequencing, a cDNA library could be screened and genes encoding the putative receptor could be identified.

In a different antagonist assay, mammalian cells or membrane samples expressing the receptor in the presence of the candidate compound were incubated with the labeled CHEPO polypeptide. Thereafter, the ability of the compound to enhance or block the interaction could be measured.

More specific examples of potential antagonists include oligonucleotides that bind to fusions of immunoglobulins and CHEPO polypeptides, and in particular poly- and monoclonal antibodies, and antibody fragments, side chain antibodies, anti-genotype antibodies, and antibodies or fragments thereof Chimeric or humanized forms of human antibodies, and antibodies, including human antibodies and antibody fragments. Alternatively, a potential antagonist may be a mutated form of a CHEPO polypeptide that competitively inhibits the action of a CHEPO polypeptide, though it does not pertain to a closely related protein, e. G., A receptor.

Another potential CHEPO polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology wherein an antisense RNA or DNA molecule acts to directly block translation of the mRNA by hybridizing with the target mRNA to prevent protein translation . Antisense technology can be used to regulate gene expression through triple helix formation or antisense DNA or RNA, all based on the binding of polynucleotides to DNA or RNA. For example, antisense RNA oligonucleotides of about 10 to 40 bases in length are designed using a 5 'coding portion of a polynucleotide sequence encoding a mature CHEPO polypeptide herein. DNA oligonucleotides are designed to be complementary to the gene regions involved in transcription (triple helix-Lee et al., Nucl. Acids Res. , 6: 3073 (1979); Cooney et al., Science , 241: 456 (1988); Dervan et al., Science , 251: 1360 (1991)) to prevent the transcription and production of CHEPO polypeptides. Antisense RNA oligonucleotides hybridize with their mRNAs in vivo to block translation of mRNA molecules into CHEPO polypeptides (antisense - Okano, Neurochem. , 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression Boca Raton, FL, 1988). In addition, by expressing the antisense RNA or DNA in vivo by transferring the oligonucleotide described above to cells, the production of CHEPO polypeptide could be inhibited. Where antisense DNA is used, oligodeoxyribonucleotides derived from transcription-initiation sites, for example between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that block the normal biological activity of CHEPO polypeptides by binding to active sites, receptor binding sites, or growth factors or other related binding sites of CHEPO polypeptides. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.

Ribozymes are enzyme RNA molecules capable of catalyzing specific cleavage of RNA. Ribozymes function by sequencing-specific hybridization with complementary target RNA followed by cleavage by endonuclease. A known technique could identify specific ribozyme cleavage sites within a potential RNA target. For further details, see, for example, Rossi, Current Biology , 4: 469-471 (1994) and PCT publication No. WO 97/33551 (published September 18, 1997).

The triple helical nucleic acid molecule used to inhibit transcription must be single-stranded and must be composed of deoxynucleotides. The base composition of these oligonucleotides is designed to promote triple helix formation through the Hoogsteen base pairing rule. Triple helix formation generally requires a purine or pyrimidine stretch of considerable size on one of the double strands. For further details, see, for example, PCT publication No. WO 97/33551, supra.

The small molecules may be identified by any one or more of the screening assays described above and / or by any other screening technique well known to those skilled in the art.

F. Anti-CHEPO antibody

The present invention further provides an anti-CHEPO antibody. Examples of antibodies include polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies.

1. polyclonal antibody

The anti-CHEPO antibody may comprise a polyclonal antibody. Methods for producing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be produced in mammals, for example, by injection of one or more immunizing agents and, if desired, adjuvants. In general, the immunizing agent and / or adjuvant will be injected into the mammal by subcutaneous or intraperitoneal injection. The immunizing agent may comprise a CHEPO polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the immunized mammal. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, sorbitol globulin, and soybean trypsin inhibitor. Examples of adjuvants that may be used include Freud's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose decolinomicolate). Immunization methods may be selected by those skilled in the art without undue experimentation.

2. Monoclonal antibody

Anti-CHEPO antibodies may alternatively be monoclonal antibodies. Monoclonal antibodies can be prepared using the hybridoma method as described in Kohler and Milstein, Nature , 256 : 495 (1975). In the hybridoma method, mice, hamsters or other suitable host animals are usually immunized with an immunizing agent to induce lymphocytes capable of producing or producing an antibody that specifically binds to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

The immunizing agent will generally comprise a CHEPO polypeptide or a fusion protein thereof. In general, peripheral blood lymphocytes (" PBL &quot;) are used when cells of human origin are desired, but spleen cells or lymph nodes are used when a non-human mammalian source is desired. Subsequently, a suitable fusion agent, such as polyethylene glycol, is used to fuse lymphocytes and immortalized cell lines to form hybridoma cells (Goding, Monoclonal Antibodies: Principle and Practice , Academic Press, (1986) pp. 59-103]. Immortalized cell lines are generally transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Generally, mouse or mouse myeloma cell lines are used. The hybridoma cells may preferably be cultured in a suitable culture medium containing one or more substances that inhibit the proliferation or survival of unfused, immortalized cells. For example, if the parental cell lacks hypoxanthine guanine phospholibosyl transferase (HGPRT or HPRT) enzyme, the hybridoma culture medium (" HAT medium &quot;) typically contains hypoxanthine, aminopterin, and thymidine These substances inhibit the proliferation of HGPRT-deficient cells.

Preferred immortalized cell lines are those that efficiently fuse, remain stable to high levels of antibody production by the selected antibody-producing cells, and susceptible to media such as HAT medium. A more preferred immortalized cell line is, for example, a murine myeloma cell line available from the Salk Institute Cell Distribution Center (San Diego, CA) and the American Type Culture Collection (Manassas, Va., USA) . In addition, human myeloma cell lines and mouse-human heteromyeloma cell lines have been described 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) pp. 51-63].

The culture medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against CHEPO. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) . Such techniques and analyzes are well known in the art. The binding affinity of the monoclonal antibody can be measured, for example, by Scatchard analysis (Munson and Pollard, Anal. Biochem. , 107 : 220 (1980)).

After identifying the desired hybridoma cells, the clones were subcloned by limiting dilution procedures and cultured by standard methods [Goding, supra ]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Alternatively, the hybridoma cells may be cultivated in vivo as multiple tumors in a mammal.

The monoclonal antibody secreted by the subclone may be purified by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography Can be isolated and purified from the culture medium or from the plural liquids.

In addition, monoclonal antibodies can be prepared by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be readily obtained by using a conventional method (for example, by using an oligonucleotide probe capable of specifically binding to a gene encoding a heavy chain and a light chain of a mouse antibody) Can be isolated and sequenced. The hybridoma cells of the present invention serve as a desirable source of such DNA. Once isolated, the DNA can be introduced into expression vectors and then transfected into expression vectors into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulin proteins, Monoclonal antibodies can be synthesized from host cells. In addition, DNA, for example, replacing the homologous murine sequences portion Province in the coding sequence for human heavy and light chain constant domain, or (U.S. Patent Nos. 4,816,567;. Morrison et al, supra), or a non-immunoglobulin polypeptide Can be modified by covalently linking all or part of the coding sequence to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can substitute for the constant domain of an antibody of the invention or substitute for a variable domain of an antibody-binding site of an antibody of the invention to generate a chimeric dipeptide antibody.

The antibody may be a monovalent antibody. Methods for making monovalent antibodies are known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. To prevent heavy chain cross-linking, the heavy chain is typically truncated at any point in the Fc region. Alternatively, the related cysteine residue is substituted or deleted with another amino acid residue to prevent cross-linking.

In addition, in vitro methods are suitable for the production of antibodies. The degradation of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using techniques commonly known in the art.

3. Human and humanized antibodies

In addition, the anti-CHEPO antibodies of the present invention may comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies include, but are not limited to, chimeric immunoglobulins, immunoglobulin chains comprising minimal sequence derived from non-human immunoglobulin or fragments thereof (e.g., Fv, Fab , Fab ', F (ab') ₂ or other antigen-binding small sequences of antibodies). Humanized antibodies are human immunogens that have residues of the complementarity determining region (CDR) of the receptor replaced with residues from the CDRs of non-human species (donor antibody) such as mice, rats or rabbits having the desired specificity, affinity and ability Globulin (accepting antibody). In some cases, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may comprise residues that are not found in the recipient antibody or in the CDR or framework sequences introduced. In general, a humanized antibody will comprise substantially all of one or more, typically two or more, variable domains, wherein all or substantially all of the CDR regions correspond to regions of non-human immunoglobulin and all or substantially all FRs The region corresponds to the region of the human immunoglobulin consensus sequence. Also, a humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), generally at least a portion of a human immunoglobulin region (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 humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced thereto from a non-human source. These non-human amino acid residues are often referred to as " import " residues and are typically obtained from the " import " Humanization can be performed essentially as described by Winter et al., Nature , 321 : 522-525 (1986); by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Riechmann et al., Nature , 332 : 323-327 (1988); Verhoeyen et al., Science , 239 : 1534-1536 (1988). Thus, such " humanized " antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially less intact human variable domains are replaced by corresponding sequences from non-human species. In fact, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues have been replaced with residues from similar sites in rodent antibodies.

Human antibodies can also be prepared using a variety of techniques known in the art including phage display libraries [Hoogenboom and Winter, J. Mol. Biol. , 227 : 381 (1991); Marks et al., J. Mol. Bio. , 222 : 581 (1991)). Also, techniques such as Coulter et al. And Boerner et al. Can also be used to prepare human monoclonal antibodies (Cole 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 produced by introducing a human immunoglobulin locus in a transgenic animal, such as a mouse, After antigen administration, human antibody production was observed, which was very similar to that observed in humans in all respects, including gene rearrangement, assembly, and antibody listing. See, for example, U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and scientific publications, Marks et al., Bio / Technology 10 , 779 -783 (1992); Lonberg et &lt; RTI ID = 0.0 &gt; Nature Biotechnology 14 , 845-51 (1996); Neuberger, Nature Biotechnology 14 , 826 (1996); Nature 368 , 856-859 (1994), Morrison, Nature 368 , 812-13 Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995)).

4. Bispecific antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies having binding specificity for two or more different antigens. In this case, one of the binding specificities is for CHEPO and the other is for any other antigen, preferably a cell surface protein or receptor or receptor subunit.

Methods for producing bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain / light-chain pairs, wherein the two heavy chains have different specificities [Millstein and Cuello, Nature , 305 : 537-539 (1983)]. Due to the random classification of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has an accurate bispecific structure. Purification of the correct molecule is generally carried out by an affinity chromatography step. A similar method is described in WO 93/08829 (published May 13, 1993) and Traunecker et al., EMBO J. , 10: 3655-3659 (1991).

The variable domain (antibody-antigen binding site) of the antibody with the desired binding specificity can be fused to the immunoglobulin constant domain sequence. This fusion is preferably fused with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2 and CH3 regions. It is preferred that the first heavy chain constant region (CHl) comprising the site necessary for light chain binding is present in at least one of the fusions. The immunoglobulin heavy chain fusions and, if desired, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. For more details on producing bispecific antibodies see, for example, Suresh et al., Methods in Enzymology , 121 : 210 (1986).

According to another method disclosed in WO 96/27011, the interface between a pair of antibody molecules can be manipulated to maximize the percentage of heterodimers recovered from the recombinant cell culture. A preferred interface comprises at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine), a complementary " cavity " of the same or similar size to the larger side chain was generated at the interface of the second antibody molecule. This provides a mechanism to increase the yield of the heterodimer above the yield of other undesired end-products such as homodimers.

Bispecific antibodies can be prepared from full-length antibodies or antibody fragments (e. G., F (ab ') ₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be prepared using chemical linkages. In the literature (Brennan et al., Science 229: 81 (1985)) describes a method of generating F (ab ') ₂ fragments by truncation of intact antibody by protein hydrolysis. These fragments are reduced in the presence of sodium arsenite, a dithiol complex, to stabilize nearby dithiols and prevent disulfide formation in the bonsai. The resulting Fab 'fragment was then converted to a thionitrobenzoate (TNB) derivative. Thereafter, one of the Fab'-TNB derivatives was re-converted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as a material for selective immobilization of the enzyme.

this. Fab 'fragments were directly recovered from the E. coli and chemically coupled to form bispecific antibodies. A complete humanized bispecific antibody F (ab ') ₂ molecule is disclosed in Shalaby et al., J. Exp. Med. 175: 217-225 (1992). Each Fab 'fragment was secreted separately from E. coli and ligated by the in vitro chemical method indicated to form a bispecific antibody. The bispecific antibodies thus formed were capable of binding to ErbB2 receptor overexpressing cells and normal human T cells, as well as initiating the lytic activity of human cytotoxic lymphocytes against human breast cancer targets.

In addition, several techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have been disclosed. For example, bispecific antibodies were prepared using a leucine zipper (Kostelny et al., J. Immunol . 148 (5): 1547-1553 (1992)). By gene fusion, leucine zipper peptides from the Fos and Jun proteins were ligated to the Fab 'portion of two different antibodies. The hinge region of the antibody homodimer was reduced to form monomers and then reoxidized to form antibody heterodimers. This method can also be used as a method for producing antibody homodimers. The "diabody" technique described in Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides another mechanism for making bispecific antibody fragments. This fragment contains a heavy chain variable domain (V _H ) linked to the light chain variable domain (V _L ) by the linker, which is too short to pair the two domains to the same chain. Therefore, V _H and V _L domains of one fragment are formed complementary to the V _H and V _L domain pairs and of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments using short chain Fv (sFv) dimers has been reported (Gruber et al., J. Immunol. 152: 5368 (1994)). Antibodies with a bivalent or higher valency are also contemplated. For example, triple specific antibodies can be made (Tutt et al., J. Immunol. 147: 60 (1991)).

Exemplary bispecific antibodies may bind to two different epitopes of the CHEPO polypeptide provided herein. Alternatively, in order to focus on the intracellular defense mechanism for cells expressing a particular CHEPO polypeptide, the arm of the anti-CHEPO polypeptide may be conjugated to a leukocyte-initiating molecule such as a T-cell receptor molecule (e.g., CD2, CD3, CD28 Or B7) or an arm that binds an Fc receptor (Fc [gamma] R) of IgG, such as Fc [gamma] RI (CD64), Fc [gamma] RII (CD32) and Fc [gamma] RIII (CD16). Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing a particular CHEPO polypeptide. Such antibodies have arms that combine with CHEPO-linked arms and cytotoxic agents or radionuclide chelators such as EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest binds the CHEPO polypeptide and further binds to the tissue factor (TF).

5. Heterozygous antibodies

Heterozygous antibodies are also included within the scope of the present invention. A heterozygous antibody consists of two covalently linked antibodies. Such antibodies are useful, for example, for targeting immune system cells to undesired cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089 Respectively. It is believed that the heterologous antibody can be prepared in vitro using methods known in the art of synthetic protein chemistry, including methods using cross-linking agents. For example, immunotoxins can be prepared by forming disulfide exchange reactions or thioether linkages. Reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and reagents disclosed in U.S. Patent No. 4,676,980.

6. Operation of effector function

It may be desirable to modify the antibody of the invention to function as an effector, for example, to increase the effect of the antibody in the treatment of cancer. For example, a cysteine residue can be introduced into the Fc region to form interchain disulfide bonds within this region. The homodimeric antibody thus generated enhances the internalization capacity and increases complement-mediated cell death and antibody-dependent cellular cytotoxicity (Carcon et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, J. Immunol. , 148: 2918-2922 (1992)). In addition, heterodimeric crosslinking as described in Wolff et al. Cancer Research , 53 : 2560-2565 (1993) can be used to prepare homodimeric antibodies with enhanced anti-tumor activity. Alternatively, antibodies with dual Fc regions can be engineered to increase complement fungi and ADCC capabilities (see Stevenson et al., Anti-Cancer Drug Design , 3: 219-230 (1989) .

7. Immunoconjugate

The present invention also relates to a method of treating or preventing a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active bacterial, fungal, plant or animal toxin, or a fragment thereof) or a radioactive isotope (i.e., Lt; RTI ID = 0.0 &gt; immunoglobulin &lt; / RTI &gt;

Chemotherapeutic agents useful for the production of the immunogenic composition are described above. Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), lysine A chain, Aleurites fordii protein, Dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), bitter melon (momordica charantia) inhibitor, Cicin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restricotisin, penomycin, enomycin, and tricothecenes. Several radionuclides can be used in the preparation of radiolabeled antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

Various bifunctional protein-coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (For example, dimethyl adipimidate HCL), active esters (e.g., disuccinimidylsuberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., (P-diazonium benzoyl) -ethylenediamine), a diisocyanate (e.g., toluene 2,6-diisocyanate), and a bis-diazonium derivative (e.g., A complex of an antibody and a cytotoxic agent can be made using a bis-active fluorine compound (for example, 1,5-difluoro-2,4-dinitrobenzene). See, for example, Vitetta et al , Science , 238 : 1098 (1987)). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is a typical chelating agent linking a radioactive nucleotide and an antibody (see WO 94/11026 do).

In another embodiment, an antibody and a " receptor " (e.g., streptavidin) used for tumor preliminary targeting can be linked, wherein the antibody- receptor complex is administered to the subject, Unbound complexes are removed and a " ligand " (e.g., avidin) linked to a cytotoxic agent (e. G., A radionucleotide) is administered.

8. Immunoliposome

The antibodies disclosed herein may also be formulated into an immunoliposome. Liposomes containing this antibody can be prepared by methods known in the art, for example, by Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad USA, 77: 4030 (1980), and U.S. Patent Nos. 4,485,045 and 4,544,545). Liposomes with increased circulation time are disclosed in U.S. Patent No. 5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation using lipid compositions containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposome was extruded through a filter having a predetermined pore size to obtain a liposome having a predetermined diameter. Fab 'fragments of the antibodies of the invention can be linked to liposomes via a disulfide-interchange reaction as described in Martin et al., J. Biol. Chem. , 257 : 286-288 (1982). Chemotherapeutic agents (e. G., Doxorubicin) are optionally included in liposomes (see Gabizon et al., J. National Cancer Inst. , 81 (19): 1484 (1989)).

9. Antibody pharmaceutical composition

To treat various diseases, antibodies specifically binding to CHEPO polypeptides identified herein and other molecules identified by the screening assays described above may be administered in the form of pharmaceutical compositions.

When the CHEPO polypeptide is in the cell and all of the antibody is used as an inhibitor, the internalizing antibody is preferred. However, lipofection or liposomes can also be used to deliver an antibody or antibody fragment into a cell. When an antibody fragment is used, a minimal inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable-region sequence of the antibody, a peptide molecule with the ability to bind to a target protein sequence can be designed. Such peptides can be chemically synthesized and / or produced by recombinant DNA techniques (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA , 90: 7889-7893 (1993) ). In addition, the formulations herein may comprise one or more active compounds, preferably compounds with complementary activity that do not adversely affect one another, as necessary for the particular condition being treated. Alternatively, or additionally, the composition may comprise a substance that enhances its function, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a proliferation inhibitor. Such molecules are suitably combined in an amount effective for the intended purpose.

The active ingredient may be formulated, for example, in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively, for example by coacervation techniques or interfacial polymerization, May be encapsulated in microcapsules prepared by hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules. Such techniques are described in Remington ' s Pharmaceutical Sciences , supra.

Preparations used for intravenous administration should be sterilized. This is easily accomplished by filtration through a sterile filter membrane.

Sustained release formulations may also be prepared. Suitable examples of sustained-release preparations include molded articles of solid hydrophobic polymers containing the antibody, such as semipermeable matrices in the form of films or microcapsules. Examples of sustained release matrices are polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), L- And lactic acid-glycolic acid copolymers such as LUPRON DEPOT (registered trademark) (lactic acid-glycolic acid copolymer and looprolide acetate), non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer, Possible microspheres), and poly-D - (-) - 3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid release molecules for more than 100 days, but some hydrogels release proteins for shorter periods of time. If the encapsulated antibody remains in the body for a long period of time, it can be denatured or aggregated as a result of exposure to moisture at 37 &lt; 0 &gt; C to reduce biological activity and alter immunogenicity. Rational strategies can be designed according to the relevant mechanisms for stabilization. For example, if it is found that the cohesion mechanism is SS bond formation in the bonsai through thio-disulfide interchange, it is contemplated that modifications of the sulfhydryl moiety, lyophilization from an acidic solution, control of moisture content, Stabilization can be achieved by the development of a matrix composition.

G. Uses of anti-CHEPO antibodies

The anti-CHEPO antibodies of the present invention have a variety of utility. For example, an anti-CHEPO antibody can be used for diagnostic assays for CHEPO, e.g., detection of its expression in certain cells, tissues or serum. A variety of diagnostic analytical techniques known in the art can be used, such as competition binding assays, direct or indirect sandwich assays, and immunoprecipitation assays performed on heterologous or homologous forms [Zola, Monoclonal Antibodies: A Manual of Techniques , CRC Press, (1987) pp. 147-158]. Antibodies used in diagnostic assays can be labeled with detectable moieties. The detectable moiety should be capable of generating a detectable signal, either directly or indirectly. For example, the detectable moiety may be a radioactive isotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I, a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, Luciferin, or enzymes such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Hunter et al., Nature , 144 : 945 (1962); David et al., Biochemistry , 13 : 1014 (1974); Pain et al., J. Immunol. Meth. , &Lt; / RTI &gt; 40 : 219 (1981); And Nygren, J. Histochem. and Cytochem. , 30 : 407 (1982)), as well as methods known in the art for binding antibodies to detectable moieties.

In addition, anti-CHEPO antibodies are also useful for affinity purification of CHEPO from recombinant cell culture fluids or natural sources. In this process, the antibody against CHEPO is immobilized on a suitable support for the Sephadex resin or filter paper using methods known in the art. The immobilized antibody is then contacted with a sample containing CHEPO to be purified and then washed with a suitable solvent to substantially remove all substances in the sample except the CHEPO polypeptide bound to the immobilized antibody. Finally, the support is washed with another suitable solvent to release the CHEPO polypeptide from the antibody.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

The contents of all patents and documents cited in this specification are incorporated herein by reference.

Example 1: Isolation of nucleic acid encoding CHEPO

Genomic DNA was isolated from two species of chimpanzee cell lines (ATCC CRL 1609 and CRL 1857) using a Qiagen kit (cat &num; 10262) according to the manufacturer's instructions. Then, PCR was performed using 1 g of genomic DNA as a template and the following primer pairs to obtain the chimp EPO gene as three isolated fragments.

EPO.F: 5'-ACCGCGCCCCCTGGACAG-3 '(SEQ ID NO: 12)

EPO.INT1.R: 5'-CATCCACTTCTCCGGCCAAACTTCA-3 '(SEQ ID NO: 13)

EPO.INTlF: 5'-TTTGGCCGGAGAAGTGGATGC-3 '(SEQ ID NO: 14)

EPO.INT4R: 5'-TCACTCACTCACTCATTCATTCATTCATTCA-3 '(SEQ ID NO: 15)

EPO.INT4F: 5'-GTTGAATGAATGATTGAATGAATGAGTGA-3 '(SEQ ID NO: 16)

EPO.R: 5'-GCACTGGAGTGTCCATGGGACAG-3 '(SEQ ID NO: 17)

Each PCR reaction contained 5 μl of 10 × PCR buffer (Perkin Elmer), 1 μl of dNTP (20 mM), 1 μl of genomic DNA, 1 μl of each primer, 1 μl of the polymerase (Clontech) and 1 μl of H ₂ O &lt; / RTI &gt; First, the reaction product was denatured at 94 ° C for 4 minutes, and then amplified by repeating a cycle consisting of 94 ° C for 1 minute, 65 ° C or 66 ° C for 1 minute, and 72 ° C for 1 minute for 40 times. The last extension step was performed at 72 ° C for 5 minutes. The reaction was then analyzed on an agarose gel. PCR products of 500 bp, 1200 bp and 750 bp were observed for each PCR product, respectively. The PCR reaction was then purified using a Wizard kit (Promega cat #: A7170) and sequenced directly. Sequencing of the PCR products was performed using an Applied Biosystems 377 DNA sequencer (PEI Applied Biosystems, Foster City, Calif.). The chemical principles used were die terminator cycle sequencing using dRodamine and BIG DYE terminators (PEI Applied Biosystems, Foster City, Calif.). Sequence assemblies and translations were performed using Sequencher (Gene Codes, Ann Arbor, Mich.).

Five coding exons were identified by homology with the human erythropoietin sequence and this exon was assembled into the predicted full - length cDNA. The length of the coding region of the CHEPO cDNA is 579 nucleotides (Figure 1) and encodes a predicted protein of 193 amino acids (Figure 3). There are three potential signal cleavage sites located after amino acid residues 22, 24, and 27. When compared to the N-terminus of human EPO, there is a terminus likely to correspond to the cleavage site of the chimpanzee EPO. There is a mature protein of 166 amino acids that contains three potential N-glycosylation sites followed by a hydrophobic signal peptide of 27 amino acids. A single nucleotide polymorphism is present in the predicted sequence from CRL 1609 and changes the protein sequence at amino acid position 142 from Q to K. The alignment of the human sequence with the chimpanzee EPO protein shows a single change in amino acid position 84 (Figure 3).

Example 2: Use of CHEPO as a hybridization probe

The following method describes the use of a nucleotide sequence encoding CHEPO as a hybridization probe.

DNA containing the coding sequence of full-length or mature CHEPO (as shown in Figure 2, SEQ ID NO: 3), or using homologous DNA (e. G., A native variant of CHEPO in a human tissue cDNA library or human tissue genomic library Coding) were used as probes in screening.

Hybridization and washing of filters containing these library DNAs were performed under the following stringent conditions. The radiolabeled CHEPO induction probe was dialyzed against a solution of 50% formamide, 5 x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2 x Denhardt's solution and 10% dextran sulfate Lt; RTI ID = 0.0 &gt; 42 C &lt; / RTI &gt; for 20 hours. The filters were washed at 42 [deg.] C in an aqueous solution of 0.1 x SSC and 0.1% SDS.

DNA having the desired sequence identity with the DNA encoding the full length native sequence CHEPO was then identified by standard methods known in the art.

Example 3: Expression of CHEPO in E. coli

In this embodiment, Lt; RTI ID = 0.0 &gt; CHEPO &lt; / RTI &gt; polypeptide by recombinant expression in E. coli.

First, the DNA sequence (SEQ ID NO: 3) encoding the desired CHEPO was amplified using the selected PCR primers. The primer should contain a restriction enzyme site corresponding to the restriction enzyme site on the selected expression vector. Multiple expression vectors may be used. Examples of suitable vectors include pBR322 [which contains the ampicillin and tetracycline resistance gene. Derived from E. coli; Bolivar et al., Gene 2:95 (1977)]. The vector was digested with restriction enzymes and dephosphorylated. The PCR amplified sequence was then ligated to the vector. The vector preferably comprises a sequence encoding an antibiotic resistance gene, a trp promoter, a polythreader (including the first six STII codons, a polyh sequence and an enterokinase cleavage site), a CHEPO coding region, a lambda transcription terminator and an argU gene something to do.

The ligation mixture was then purified using the method described by Sambrook et al. (Supra). And used to transform E. coli strains. Transformants capable of growing in LB medium were identified and colonies with antibiotic resistance were selected. Plasmid DNA was isolated and identified using restriction analysis and DNA sequencing.

Selected clones were cultured overnight in a liquid culture medium such as LB broth supplemented with antibiotics. This culture was used for larger seed culture inoculation. The cells were then incubated to the desired optical density while the expression promoter was running.

After culturing the cells for several hours or more, cells can be recovered by centrifugation. The cell pellet obtained by centrifugation was dissolved using various reagents known in the art, and the dissolved CHEPO protein was purified using a metal chelating column under conditions in which the protein binds strongly.

CHEPO uses this process to Can be expressed in a form in which a poly-His tag is added in a colage. The DNA encoding CHEPO is initially amplified using the selected PCR primers. Primers included restriction sites corresponding to restriction sites on selected expression vectors and other useful sequences that provided efficient and reliable translation initiation, rapid purification in metal chelate columns, proteolytic cleavage using enterokinase. The PCR-amplified, poly-His tagged sequence was then ligated to the expression vector, The transformant was transformed into LB medium (carbenicillin) containing 50 mg / ml of carbenicillin, and the transformant was transformed into LB medium containing 50 mg / ml carbenicillin. OD600 of 3 to until it reaches the 5 shake cultured at 30 ℃. then, 3.57 in the culture CRAP media (500 ㎖ water in _{g (NH 4) 2 SO 4} , 0.71 g sodium citrate · 2H ₂ O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF, further 110 mM MPOS, pH 7.3, 0.55% (w / v) glucose and 7 mM MgSO ₄ ) The cells were shake-cultured for 30 hours at 30 ° C. The samples were taken and confirmed by SDS-PAGE analysis, and the cells were pelleted by centrifugation of the bulk culture.

0.5 to 1 L fermentation broth. The coli paste (6 to 10 g pellet) was resuspended in 10 volumes (w / v) of 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium sodium tartrate were added to final concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred at 4 째 C overnight. At this stage, a denatured protein with all cysteine residues blocked by sulfation was produced. The solution was centrifuged at 40,000 rpm for 30 minutes in a Beckman ultracentrifuge. The supernatant was diluted with a 3-5 fold volume of metal chelating column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and clarified by filtration through a 0.22 micron filter. The clarified extract was loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated with metal chelate column buffer. The column was washed with additional buffer (pH 7.4) containing 50 mM imidazole (Calbiochem, Utrol grade). The protein was eluted with a buffer containing 250 mM imidazole and the fractions containing the desired protein were pooled and stored at 4 &lt; 0 &gt; C. The protein concentration was measured at 280 nm absorbance using the extinction coefficient calculated according to the amino acid sequence.

Samples were slowly diluted with freshly prepared refolding buffer consisting of 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA to refold the proteins. The refolding volume was determined such that the final protein concentration was 50-100 占 퐂 / ml. The refolding solution was slowly stirred at 4 캜 for about 12 to 36 hours. The refolding reaction was stopped by adding TFA to a final concentration of 0.4% (about pH 3). Before further protein purification, the solution was filtered through a 0.22 micron filter and acetonitrile was added to a final concentration of 2-10%. The refolded protein was chromatographed on a Poros R1 / H reverse phase column using a mobile buffer of 0.1% TFA, eluting with a gradient of acetonitrile concentration from 10 to 80%. A fraction of the fraction showing A280 absorbance was analyzed on SDS polyacrylamide gel to recover fractions containing homogeneously refolded proteins. Generally, the protein form that is properly refolded in most proteins elutes at the lowest concentration of acetonitrile, since the refolded protein most closely fits with the hydrophobic interior portion that is protected from interaction with the reverse phase resin. The aggregated proteins are usually eluted at high acetonitrile concentrations. The reverse phase step not only separates the protein of the wrongly folded form from the protein of the desired type, but also removes the endotoxin from the sample.

The fraction containing the folded CHEPO polypeptide was recovered and the solution was gently applied with a nitrogen stream to remove the acetonitrile. Proteins were formulated with 20 mM Hepes, pH 6.8, containing 0.14 M sodium chloride and 4% mannitol, by gel filtration using G25 Superfine (Pharmacia) resin equilibrated with dialysis or formulation buffer and sterile filtered.

Example 4 Expression of CHEPO in Mammalian Cells

This example illustrates a method for producing a possible glycosylated form of a CHEPO polypeptide by recombinant expression in mammalian cells.

The vector pRK5 (1989, 3.15, published EP 307,247) was used as an expression vector. Optionally, CHEPO DNA was ligated to pRK5 using the selected restriction enzymes using the ligation method described by Sambrook et al. (Supra), and CHEPO DNA was inserted. The resulting vector was referred to as pRK5-CHEPO polypeptide.

In one embodiment, 293 cells can be selected as host cells. Human 293 cells (ATCC CCL 1573) were cultured in confluent tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally with nutrients and / or antibiotics. Approximately 10 μg of pRK5-CHEPO DNA was mixed with approximately 1 μg of DNA encoding the VA RNA gene [Thimmappaya et al., Cell , 31 : 543 (1982)] and incubated with 500 μl of 1 mM Tris-HCl, 0.227 M CaCl ₂ . 500 쨉 l of 50 mM HEPES (pH 7.35), 280 mM NaCl, and 1.5 mM NaPO ₄ were added dropwise to the mixture to form a precipitate at 25 째 C for 10 minutes. The precipitate was suspended and added to 293 cells and allowed to stand at 37 DEG C for about 4 hours. The medium was aspirated off and 2 ml of 20% glycerol in PBS was added for 30 seconds. Subsequently, 293 cells were washed with serum-free medium, fresh medium was added, and cells were cultured for 5 days.

After about 24 hours, transfection was replaced with medium to remove the culture medium contains (only) medium or 200 μCi / ㎖ ³⁵ S- cysteine and 200 μCi / ㎖ ³⁵ S- methionine. After 12 hours incubation, the conditioned medium was collected, concentrated on a rotary filter and loaded onto 15% SDS gel. The treated gel was dried and exposed to film for a period of time to confirm the presence of the CHEPO polypeptide. The culture containing the transfected cells was further cultured (in serum-free medium) and the medium was tested with the selected biochemical assay.

Alternatively, Somparyrac et al., Proc. Natl. Acad. Sci. , 12 : 7575 (1981)], the CHEPO could be transiently introduced into 293 cells using the dextran sulfate method described above. 293 cells were cultured to the highest density in a spinning flask and 700 μg pRK5-CHEPO DNA was added. Cells were first centrifuged, concentrated from spin-flask and washed with PBS. The DNA-dextran precipitate was incubated on the cell pellet for 4 hours. Cells were treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and then placed in a spinning flask containing tissue culture medium, 5 ug / ml bovine insulin, and 0.1 ug / ml bovine transferrin. After about 4 days, the conditioned medium was centrifuged and filtered to remove cells and lysates. Samples containing the expressed CHEPO were concentrated and purified by a selected method, e. G., Dialysis and / or column chromatography.

In another embodiment, CHEPO can be expressed in CHO cells. The pRK5-CHEPO known reagents such as can be transfected into CHO cells using the CaPO ₄ or DEAE-dextran. As mentioned above, cells can be cultured and the culture medium replaced with medium alone or in a medium containing a radioactive label such as ³⁵ S-methionine. After confirming the presence of the expressed polypeptide, the culture medium is replaced with serum-free medium. Preferably, the culture is incubated for about 6 days and the conditioned medium is recovered. The medium containing the expressed CHEPO may be concentrated and purified by a selected method.

In addition, epitope tagged CHEPO can be expressed in CHO host cells. CHEPO can be subcloned from the pRK5 vector. Insertion of a subclone may be fused to a baculovirus expression vector in a form having a selected epitope tag, such as a poly-his tag, via PCR. The CHEPO insert with the poly-his tag may be subcloned into an SV40 inducible vector containing a selection marker such as DHFR to select a stable clone. Finally, CHO cells can be transfected with an SV40 inducible vector (as above). May be labeled in the same manner as described above to confirm expression. The culture medium containing the expressed poly-His-tagged CHEPO can then be concentrated and purified by a selected method such as Ni ²⁺ -chelate affinity chromatography.

CHEPO can also be expressed in CHO and / or COS cells by transient expression processes or in CHO cells by other stable expression methods.

It was stably expressed in CHO cells using the following experimental method. The protein is expressed and / or expressed as an IgG construct (immunohedressin) fused to a IgGl constant region sequence comprising the hinge, CH2 and CH2 domains, and / His tag was expressed in an appended form.

Following PCR amplification, each DNA was subcloned into a CHO expression vector using the standard method described by Ausubel et al., Current Protocols of Molecular Biology , Unit 3.16, Johnn Wiley and Sons (1997). The CHO expression vector is prepared to have restriction sites suitable for 5 'and 3' of the desired DNA so that restriction sites of the cDNA can be conveniently shuttled. The vector used for expression in CHO cells is described in Lucas et al., Nucl. Acids Res. 24: 9 (1774-1779 (1996)), and the SV40 early promoter / enhancer was used to express the desired cDNA and dihydrofolate reductase (DHFR). A plasmid transfected with DHFR expression Can be selected.

Twelve micrograms of the desired plasmid DNA was introduced into approximately 10 million CHO cells using the commercially available transfection reagent Superfect TM (Qiagen), Dosper TM or Fugene TM (Boehringer Mannheim). Cells were cultured according to the method described by Lucas et al., Supra. About 3 x 10 &lt;&quot; ⁷ &gt; cells were then frozen in ampoules according to the method described below for culturing and producing the cells.

Plasmid DNA-containing ampoules were dissolved in a water bath and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 ml of the medium and centrifuged at 1000 rpm for 5 minutes. The supernatant was inhaled and the cells resuspended in 10 ml of selective medium (0.2 [mu] m filtered PS20 containing 0.2 [mu] m dialyzed 5% fetal bovine serum). Cells were dispensed into 100 ml spinner containing 90 ml selection medium. After 1-2 days, the cells were transferred to a 250 ml spinner filled with 150 ml of selective medium and incubated at 37 [deg.] C. The 250 ㎖, 3 x 10 ⁵ cells / ㎖ to 500 ㎖ and 2000 ㎖ spinner after 2 to 3 days were inoculated. The cell culture was centrifuged, replaced with fresh medium, and resuspended in the production medium. Any suitable CHO medium can be used, but the production medium described in published U. S. Patent No. 5,122, 469 (June 16, 1992) was actually used. And inoculated to a 3 L production spinner at a density of 1.2 x 10 &lt; ⁶ &gt; cells / mL. Cell number and pH were measured at day 0. On the first day, a sample was taken from the spinner and the application of the filtered air was started. On the second day, samples were taken from the spinner and the temperature was changed to 33 ° C, and 30 ml of 500 g / L-glucose and 0.6 ml of 10% defoamer (eg 35% polydimethylsiloxane emulsion, Dow Corning 365 medical grade emulsion) were added. During the entire production period, the pH should be maintained at about 7.2. After 10 days or when the survival rate dropped to less than 70%, the cell culture was recovered by centrifugation and filtered through a 0.22 mu m filter. The filtrate was stored at 4 [deg.] C until loading on the purification column.

For poly-his tagged constructs, proteins were purified using Ni-NTA column (Qiagen). Imidazole was added to the conditioned medium at a concentration of 5 mM before purification. The conditioned media was injected into a 6 ml Ni-NTA column equilibrated with 20 mM Hepes, pH 7.4 buffer containing 0.3 M NaCl and 5 mM imidazole at 4 ° C at a flow rate of 4-5 ml / min. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The purified protein was then removed in 25 ml of a G25 supernatant (Pharmacia) column in a storage buffer (pH 6.8) containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol and stored at -80 ° C.

The immunoadhesin (including Fc) construct was purified from the conditioned medium as follows. The conditioned medium was injected into a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer (pH 6.8). After loading, the column was washed with equilibration buffer and eluted with 100 mM citric acid (pH 3.5). The eluted protein was recovered in 1 ml fractions in a tube containing 275 l of 1 M Tris buffer (pH 9) and immediately neutralized. The highly purified protein was then removed with the storage buffer in the manner described above for the poly-His tagged protein. Homology was confirmed by N-terminal amino acid sequence analysis performed with SDS-polyacrylamide gel and Edman degradation.

Example 5 Expression of CHEPO Polypeptides in Yeast

The following method describes the recombinant expression of the desired CHEPO in yeast.

First, a yeast expression vector for the intracellular production or secretion of CHEPO was prepared from the ADH2 / GAPDH promoter. The DNA encoding CHEPO and the promoter was inserted into the appropriate restriction enzyme site of the plasmid selected for intracellular expression of the CHEPO polypeptide. In the case of secretion, the DNA encoding CHEPO for CHEPO expression is cloned into the ADH2 / GAPDH promoter, native CHEPO signal peptide or other mammalian signal peptide such as yeast alpha-factor or invertase secretion signal / leader sequence and linker sequence If desired) with the DNA encoding the selected plasmid.

Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatant can be precipitated with 10% trichloroacetic acid, separated by SDS-PAGE and then stained with Coomassie blue to analyze the gel.

Subsequently, the yeast cells are removed from the fermentation medium by centrifugation and the medium is concentrated using the selected cartridge filter to isolate and purify the recombinant CHEPO. The concentrate containing CHEPO may be further purified using a selected column chromatography resin.

Example 6 Expression of CHEPO in Insect Cells Infected with Baculovirus

The following method describes the recombinant expression of CHEPO in baculovirus-infected insect cells.

The sequence encoding CHEPO was fused upstream of the epitope tag included in the baculovirus expression vector. The epitope tag includes a poly-his tag and an immunoglobulin tag (such as the Fc region of an IgG). Several plasmids can be used, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, a desired portion of the coding sequence of CHEPO or CHEPO, such as the sequence encoding the extracellular domain of the transmembrane protein or the sequence coding for the mature protein, is amplified by PCR using primers complementary to the 5 'and 3' regions Respectively. The 5 ' primer may comprise an adjacent (selected) restriction enzyme site. The product was then digested with the selected restriction enzymes and subcloned into an expression vector.

Recombinant baculovirus was transformed with Spodoptera frugiperda (" Sf9 &quot;) cells (ATCC CRL 1711) using the above plasmid and BaculoGold (R) virus DNA (Pharmingen) using lipofectin (purchased from GIBCO- Lt; / RTI &gt; After incubation at 28 ° C for 4 to 5 days, the released virus is recovered and used for subsequent amplification. Viral infections and protein expression were performed according to O'Reilley et al., Baculovirus Expression vectors : A laboratory Manual , Oxford: Oxford University Press (1994).

The expressed poly-hero-tagged CHEPO can then be purified, for example, by Ni ²⁺ -chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells according to Rupert et al., Nature , 362 : 175-179 (1993). Briefly, washed Sf9 cells sonication buffer (25 ㎖ Hepes, pH 7.9; 12.5 mM MgCl 2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl) was resuspended 20 seconds on ice in And then ultrasonicated twice. The supernatant was removed by centrifugation and the supernatant was diluted 50-fold in loading buffer (50 mM phosphoric acid, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni ²⁺ -NTA agarose column (purchased from Quiagen) was prepared in a 5 ml fill volume, washed with 25 ml water and equilibrated with 25 ml loading buffer. The filtered cell extract was loaded onto the column at 0.5 ml per minute. At the start of point fraction recovery, the column was washed with loading buffer to the A ₂₈₀ baseline. Next, the column was washed with secondary washing buffer (50 mM phosphoric acid; 300 mM NaCl, 10% glycerol, pH 6.0) to elute nonspecifically bound proteins. When A ₂₈₀ reached the baseline again, the column was developed with a gradient from 0 to 500 mM imidazole in the secondary wash buffer. 1 ml fractions were collected and analyzed by SDS-PAGE and Western blotting with Ni ²⁺ -NTA (Qiagen) coupled to silver stain or alkaline phosphatase. Fractions containing eluted His ₁₀ tagged CHEPO were pooled and dialyzed against loading buffer.

Alternatively, purification of IgG tagged (or Fc-tagged) CHEPO was performed using known chromatographic techniques, such as protein A or protein G column chromatography.

Example 7: Preparation of antibody binding to CHEPO

This embodiment is a method for producing a monoclonal antibody capable of specifically binding to CHEPO.

Techniques for producing monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that may be used include purified CHEPO of the present invention, fusion proteins comprising CHEPO and cells expressing recombinant CHEPO on the cell surface. Experts can choose an immunogen without unnecessary experimentation.

A mouse, for example Balb / c, was immunized by subcutaneous or intraperitoneal injection of 1 to 100 [mu] g of CHEPO immunogen emulsified in complete adjuvant solution. Alternatively, immunizations were emulsified in MPL-TDM supplement (Ribi Immunochemical Research, Hamilton, MT) and injected into the hindpaw of the animal to be immunized. The immunized mice were then boosted with additional immunogens emulsified in the selected adjuvant 10-12 days later. After that, mice can be boosted with additional vaccines for several weeks. Serum samples were routinely collected from mice by reverse-bleeding and ELISA assays were performed to detect anti-CHEPO polypeptide antibodies.

Once the appropriate antibody titer was detected, CHEPO was injected intravenously into the animal " positive " for the antibody. After 3 to 4 days, mice are sacrificed and spleen cells are recovered. The spleen cells were then fused (using 35% polyethylene glycol) with the selected mouse myeloma cell line, for example P3X63AgU.1, available from ATCC number CRL 1597. Fusion produces hybridoma cells that can be plated in 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium that inhibits the proliferation of non-fused cells, myeloma hybrids and spleen cell hybrids Respectively.

Hybridoma cells can be screened by ELISA for reactivity to CHEPO. Methods for determining " positive " hybridoma cells that secrete a desired monoclonal antibody against a CHEPO polypeptide are well known to those skilled in the art.

Positive hybridoma cells can be injected intraperitoneally to produce ascites containing Balb / c mice containing anti-CHEPO monoclonal antibodies. Alternatively, the hybridoma cells can be cultured in tissue culture flasks or roller bottles. Monoclonal antibodies produced in the swollen can be purified using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on protein A or protein G binding of the antibody may be used.

Example 8 Purification of CHEPO Polypeptides Using Specific Antibodies

Natural or recombinant CHEPO polypeptides can be purified by a variety of standard techniques in the field of protein purification. For example, pro (CHEPO) -CHEPO polypeptides, mature CHEPO polypeptides or pre-CHEPO polypeptides are purified by immunoaffinity chromatography using antibodies specific for the desired CHEPO polypeptide. Generally, an immunoaffinity column is made by covalently coupling an anti-CHEPO polypeptide antibody to an activated chromatographic resin.

Polyclonal immunoglobulins were prepared from immune sera by ammonium sulfate precipitation or purification on Immobilized Protein A (Pharmacia LKB Biotechnology, Fitzgerald, FJ). Similarly, monoclonal antibodies were prepared from mouse remnants by ammonium sulfate precipitation or by chromatography on immobilized Protein A. Partially purified immunoglobulins were covalently attached to chromatographic resins such as CnBr-activated SEPHAROSE (TM) (Pharmacia LKB Biotechnology). The antibody was coupled to the resin, the resin was blocked, and the derived resin was washed according to the manufacturer's instructions.

Such immunoaffinity columns are used in the purification of CHEPO polypeptides by preparing fractions from cells containing the CHEPO polypeptide in soluble form. The agent is induced by solubilization of the whole cell obtained by detergent addition or by other methods well known in the art or solubilization of the subcellular fraction obtained through differential centrifugation. Alternatively, soluble CHEPO polypeptides comprising the signal sequence may be secreted in a useful amount into the medium in which the cells grow.

The soluble CHEPO polypeptide containing preparation is passed over the immunoaffinity column and the column is washed under conditions permitting different absorbance of the CHEPO polypeptide (e.g., high ionic strength buffer in the presence of detergent). The column is then eluted under conditions that disrupt antibody / CHEPO polypeptide binding (e. G., A low pH buffer such as about pH 2 to 3 or a high concentration of chaotrope such as urea or thiocyanate ions) The CHEPO polypeptide was recovered.

Example 9: Drug screening

The present invention is particularly useful for screening compounds with any of a variety of drug screening techniques using CHEPO polypeptides or binding fragments thereof. The CHEPO polypeptide or fragment used in this test may be free in solution, adhered to a solid support, maintained at the cell surface, or located within the cell. One method of drug screening utilizes stably transformed eukaryotic or prokaryotic host cells using recombinant nucleic acids expressing CHEPO polypeptides or fragments. The drug was screened against the transformed cells by competitive binding assay. The cells, either viable or fixed, can be used for standard binding assays. For example, complex formation between the CHEPO polypeptide or fragment and the active agent to be tested can be measured. Alternatively, a decrease in complex formation between the CHEPO polypeptide induced by the active agent to be tested and its target cell or target receptor can be examined.

Accordingly, the present invention provides a method for screening for a drug or any other active agent capable of affecting a CHEPO polypeptide-related disease or disorder. These methods include contacting such an activator with a CHEPO polypeptide or fragment thereof and isolating the complex between (i) the presence of a complex between the active agent and the CHEPO polypeptide or fragment or (ii) the complex between the CHEPO polypeptide or fragment and the cell And analyzing the presence. In this competitive binding assay, CHEPO polypeptides or fragments are usually labeled. After appropriate incubation, the free CHEPO polypeptide or fragment is separated from its binding form, and the amount of free or uncomplexed label is a measure of the ability of a particular active agent to bind to the CHEPO polypeptide or to inhibit the CHEPO polypeptide / cell complex .

Another drug screening technique provides high throughput screening for compounds with binding affinities suitable for polypeptides and is described in detail in WO84 / 03564 (published Mar. 9, 1984). Briefly stated, a number of different small peptide test compounds are synthesized on a solid substrate, such as a plastic pin or some other surface. CHEPO polypeptide, the peptide test compound is reacted with the CHEPO polypeptide and washed. The bound CHEPO polypeptide is detected by methods well known in the art. Purified CHEPO polypeptides can also be coated directly on a flat plate for use in the above-described drug screening techniques. In addition, non-digested antibodies can be used to capture the peptide and immobilize it on a solid support.

The present invention also contemplates the use of competitive drug screening assays in which a neutralizing antibody capable of binding to a CHEPO polypeptide specifically competes with a test compound in binding to a CHEPO polypeptide or fragment thereof. In this manner, the antibody can be used to detect the presence of any peptide that shares one or more antigenic determinants with a CHEPO polypeptide.

Example 10: Rational drug design

The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest (i. E., CHEPO polypeptides) or small molecules that interact with them, for example, agonists, antagonists or inhibitors. Any of these examples can be used to form drugs that are more active or more stable forms of the CHEPO polypeptide, or drugs that enhance or inhibit the function of the CHEPO polypeptide in vivo (Hodgson, Bio / Technology , 9 : 19-21 (1991)).

In one study, the three-dimensional structure of a CHEPO polypeptide or a CHEPO polypeptide-inhibitor complex is determined by x-ray crystallography, computer modeling or most typically by a combination of the two methods. Both the morphology and charge of the CHEPO polypeptide must be identified to reveal the structure of the molecule and determine the active site (s). Although less frequently, useful information about the structure of CHEPO polypeptides can be obtained by modeling based on the structure of the homologous protein. In both cases, the relevant structural information is used to design a homologous CHEPO polypeptide-like molecule or identify an effective inhibitor. Useful examples of rational drug design are molecules that have enhanced activity or stability as shown in Braxton and Wells, Biochemistry, 31 : 7796-7801 (1992) ), Et al. , J. Biochem. , &Lt; / RTI &gt; 113 : 742-746 (1993)], which act as inhibitors, agonists or antagonists of natural peptides.

It is also possible to isolate the target-specific antibody selected by functional analysis as described above and then analyze its crystal structure. This method in principle provides a pharmacore on which subsequent drug design can be a benchmark. It is also possible to omit the protein crystallization method by producing an anti-genotype antibody (anti-id) against a functional pharmacologically active antibody. As a mirror image, the binding site of the anti-id will be expected to be the homolog of the original receptor. The peptides may then be identified and isolated from the chemically or biologically produced peptide banks using anti-id. The isolated peptide will then serve as a perming core.

With the present invention, a sufficient amount of CHEPO polypeptide can be made available to perform analytical studies such as x-ray crystallization. In addition, the CHEPO polypeptide amino acid sequence information provided herein will provide guidance for using computer modeling techniques in addition to or in addition to x-ray crystallization.

The foregoing specification is believed to be sufficient to enable those skilled in the art to practice the claimed invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing detailed description, and these variations are within the scope of the appended claims.

Claims (26)

An isolated nucleotide molecule comprising 1 or about 82 to about 579 nucleotides of Figure 2 (SEQ ID NO: 3). An isolated nucleic acid molecule comprising the nucleotide sequence of Figure 2 (SEQ ID NO: 3). An isolated nucleic acid molecule comprising a nucleotide sequence encoding a sequence of about 1 or about 28 to about 193 amino acid residues in Figure 2 (SEQ ID NO: 2). A vector comprising the nucleic acid molecule of any one of claims 1 to 3, 23 and 26. 5. The vector of claim 4, wherein said nucleic acid molecule is operably linked to a regulatory sequence recognized by a host cell transformed with said vector. A host cell comprising the vector of claim 4. 7. The host cell according to claim 6, which is a CHO cell. 7. The host cell of claim 6, wherein the host cell is E. coli . The host cell according to claim 6, which is a yeast cell. Culturing the host cell of claim 6 under conditions suitable for expression of the polypeptide, and recovering the polypeptide from the cell culture. An isolated polypeptide comprising 1 amino acid residue or about 28 to about 193 of Figure 2 (SEQ ID NO: 2). A chimeric molecule comprising a polypeptide according to any one of claims 11, 24 and 25 fused to a heterologous amino acid sequence. 13. The chimeric molecule of claim 12, wherein the heterologous amino acid sequence is an epitope tag sequence. 13. The chimeric molecule of claim 12, wherein the heterologous amino acid sequence is an Fc region of an immunoglobulin. 26. An antibody which specifically binds to a polypeptide according to any one of claims 11, 24 and 25. 15. The antibody of claim 14, which is a monoclonal antibody. 15. The antibody of claim 14, which is a humanized antibody. &Lt; / RTI &gt; wherein X is any amino acid except proline. 19. The method of claim 18, &Lt; / RTI &gt; wherein X is any amino acid except proline. A chimeric molecule comprising a polypeptide of claim 18 or 19 fused to a heterologous amino acid sequence. 21. The chimeric molecule of claim 20, wherein the heterologous amino acid sequence is an epitope tag sequence. 21. The chimeric molecule of claim 20, wherein the heterologous amino acid sequence is an Fc region of an immunoglobulin. An isolated nucleic acid molecule consisting of the nucleotide sequence of Figure 2 (SEQ ID NO: 3). 1 or an amino acid residue of about 28 to about 193 of Figure 2 (SEQ ID NO: 2). An isolated polypeptide encoded by nucleotide 1 or from about 82 to about 579 of Figure 2 (SEQ ID NO: 3). An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric molecule of any one of claims 12 to 14 and 20 to 22.