JP2003525611A - Cordin-like-2 molecules and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention provides a Cordin-Like-2 (CHL2) polypeptide and a nucleic acid molecule encoding the same. The invention also provides selective binding agents, vectors, host cells, and methods for producing a CHL2 polypeptide. The present invention further provides pharmaceutical compositions for the diagnosis, treatment, amelioration, and / or prevention of diseases, disorders, and conditions associated with CHL2 polypeptide. The invention further provides methods for diagnosing, treating, ameliorating, and / or preventing a disease, disorder, and condition associated with a CHL2 polypeptide.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to Chordin-like-2 (CHL2) polypeptides and nucleic acid molecules that encode the same. The invention also relates to selective binding agents, vectors, host cells and methods for producing CHL2 polypeptides. The invention further relates to pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and / or prevention of diseases, disorders, and conditions associated with CHL2 polypeptides.

BACKGROUND OF THE INVENTION Technological advances in the identification, cloning, expression, and manipulation of nucleic acid molecules and decoding of the human genome have greatly accelerated the discovery of novel therapeutic agents. Currently, rapid nucleic acid sequencing techniques can generate sequence information at unprecedented rates and, combined with computer analysis, allow the assembly of overlapping sequences and identification of polypeptide coding regions over part and all of the genome. To Comparison of the deduced amino acid sequence to a database compilation of known amino acid sequences enables the degree of homology to previously identified sequence and / or structural landmarks to be determined. Cloning and expression of the polypeptide coding region of a nucleic acid molecule provides the polypeptide product for structural and functional analysis. Manipulation of nucleic acid molecules and encoded polypeptides can be accomplished by
It may provide advantageous properties to the product for use as a therapeutic.

Despite significant technological advances in genomic research over the last decade, the potential for the development of new therapeutic agents based on the human genome is not yet widely understood. Many genes encoding potentially beneficial polypeptide therapeutics or the polypeptides they encode, which may serve as "targets" for therapeutic molecules, have not yet been identified.

Accordingly, it is an object of the present invention to identify new polypeptides and nucleic acid molecules encoding them that have diagnostic or therapeutic advantages.

CHL2 is a bone morphogenetic protein (BM) known as cordin (CHD).
P) inhibitors (Sasai et al., 1994, Cell 79: 779-90).
, Or short gastrulation (SOG; Francois et al., 1994, Genes).
Dev. 8: 2602-16). CHL2 gene is CHD
/ Considered to be a member of the SOG family.

Bone morphogenetic protein (BMP) is a member of the transforming growth factor β family, which is, by nature, a cartridge implant (cartr).
was identified as a factor that promotes osteogenesis from the image implant (Wo
zney et al., 1988, Science 242: 1528-34; Celes.
te et al., 1990, Proc. Natl. Acad. Sci. USA 87: 9
843-47). BMPs are also known to play essential roles during early embryogenesis in frogs, flies, and in mammals. The exact concentration of active BMPs appears to be important for the specificity of specific cell types (Dale et al., 1992,
Development 115: 573-85; Dosch et al., 1997, D.
development 124: 2325-34). A BMP2 / 4 activity gradient was observed, for example, in Xenopus embryos, where the lowest expression was detected at the dorsal tip and the highest expression was observed at the ventral tip, and in the embryos, the dorsoventral Establish lateral axis determination. In another example, BM at specific sites of tissue development
Regulation of P concentration suggests a role for BMP in organogenesis. Regulation of BMP expression is by localized expression of the BMP gene product or by the BMP inhibitor cordin (CHD) (Sasai et al., 1994, Cell 79: 7.
79-90) -or short gastrulation (SOG) (Francois et al., 1994).
, Genes Dev. 8: 2602-16).

[0007] CHD / SOG is a Spemann-former (master-con for the dorsal-ventral axis specification at the gastrulation stage of Xenopus embryogenesis).
It is a large secretory protein produced from the control region). CHD
/ SOG is Noggin (Smith and Harland, 1992, Ce.
ll 70: 829-40), which is also secreted by the organizer, functions as a backing factor. Drosophila SOG has a transmembrane domain at its amino terminus, suggesting that it may be a type II transmembrane protein (Francois et al., 1994, Genes Dev. 8:26).
02-16). It was proposed that the carboxyl terminal side (extracellular domain) of Drosophila SOG was cleaved. However, Xenopus CHD (
Sasai et al., 1994, Cell 79: 779-90), zebrafish CHD (Schulte-Merker et al., 1997, Nature 387 :).
862-63) and mouse CHD (Pappano et al., 1998, Genom.
ics 52: 236-39) does not contain a transmembrane domain. Instead, these proteins have a signal peptide and are secreted directly. CHD /
The SOG polypeptide contains four repeats of the cysteine-rich domain (CR1
-4), this domain is also found in various extracellular matrix proteins such as collagen and thrombospondin.

CHD / SOG is known to bind to BMP4, one of the ventrifying factors (Piccolo et al., 1996, Cell 86: 589-98). BM
P4 has been shown to be essential for embryonic development of the posterior ventral mesoderm in mice (Winnier et al., 1995, Genes Dev. 9: 2105-).
16). Binding of CHD / SOG to BMP4 inhibits BMP4 activity by preventing BMP4 from binding to its receptor (Piccolo et al., 1996, Cell 86: 589-98). In this respect, CHD / SO
The functional relationship between G and BMP4 is similar to that between OPG and OPGL, but CHD / SOG is structurally unrelated to the BMP receptor. CH
The binding affinity of D / SOG to BMP4 was specific and close (Kd = 3 × 1).
0 ^-10 M (Piccolo et al., 1996, Cell 86: 589-98) )
, And appears to require proteolysis to achieve release of bound BMP4. This proteolysis is associated with certain metalloproteases (Tolloi).
d (TLD) or BMP1), which cleaves CHD / SOG to release either the first CR motif (CR1) and the last CR motif (CR4), or both (Piccolo et al. , 1997, Ce
11: 407-16). Whether CHD / SOG has other or independent functions by its own receptor should still be determined.

One of the most important roles of CHD / SOG is to establish the BMP4 morphogen gradient (Jones and Smith, 1998, Dev. Biol.
194: 12-17). BMP4 itself appears to act essentially autonomously on cells, only traveling short distances (Jones et al., 1996, C).
urr. Biol. 6: 1468-75). In contrast, the BMP4 inhibitor N
Oggin and CHD / SOG appear to exert far-reaching effects, thereby forming a BMP4 activity gradient.

BMPs also play important roles in addition to early embryogenesis, for example, in organogenesis of the lung, intestine, kidney, skin, heart and teeth, and in later stages of embryogenesis (Hogan, 1996, Genes Dev. 10: 1580-94). Some BMPs are expressed in a highly localized form, while others are widely expressed in tissues. The importance of the localized effects of BMPs on organogenesis was supported by transgenic mouse experiments with constructs, whereby BMP concentrations are artificially enhanced in target tissues. In the case of the lung, BMP4 is expressed at the distal end of the developing lung epithelium and, when overexpressed by the surfactant protein C promoter, causes severe structural organization (ie branching). The development of collapsed small lungs is observed (Bellusci et al., 1996, Development 122: 1693-702). Estimated B
Since MP activity gradients can also be disrupted by transgene expression, tissue-widely expressed BMPs may also play a role in determining the structural organization of tissues.

Noggin is another BMP2 / 4 inhibitor secreted by Spemann-formers (Zimmerman et al. 1996, Cell 86: 599-.
606). The biological role of Noggin and its mode of action is Xenopus
Similar to CHD / SOG in. The most prominent function of Noggin is CHD
/ SOG, which is dorsalized but has a null Noggin (null-
mutant) mice exhibited a bone phenotype (chondrocyte hyperplasia) instead of the early embryonic phenotype (McMahon et al., 1998, Genes Dev. 12:14).
38-52; Brunet et al., 1998, Science 280: 1455-.
57). This suggests that CHL2 or even CHD may have the required function in late stages of embryogenesis.

SUMMARY OF THE INVENTION The present invention relates to novel CHL2 nucleic acid molecules and encoded polypeptides.

The present invention provides an isolated nucleic acid molecule, which nucleic acid molecule has the following: (a) the nucleotide sequence set forth in either SEQ ID NO: 1 or SEQ ID NO: 4; (b) ATCC Deposit Number PTA. Nucleotide sequence of the DNA insert in either -1479 or PTA-1480; (c) a nucleotide sequence encoding the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; (d) (a)-(c) A nucleotide sequence that hybridizes to the complement of any of 1) under moderately or highly stringent conditions; and (e) a nucleotide sequence complementary to any of (a) to (c), Includes a nucleotide sequence that is more selected.

The present invention also provides an isolated nucleic acid molecule, wherein the nucleic acid molecule is a minority of the polypeptide shown in either (a) SEQ ID NO: 2 or SEQ ID NO: 5.
A nucleotide sequence encoding a polypeptide which is at least about 70% identical,
, Wherein the encoded polypeptide is either SEQ ID NO: 2 or SEQ ID NO: 5.
A nucleotide sequence having the activity of the polypeptide shown therein; (b) the nucleotide sequence shown in either SEQ ID NO: 1 or SEQ ID NO: 4.
, D in either ATCC Deposit Number PTA-1479 or PTA-1480
The nucleotide sequence of the NA insert, or (a) an allelic variant or
A nucleotide sequence encoding a splice variant; (c) encodes a polypeptide fragment of at least about 25 amino acid residues
The nucleotide sequence of either SEQ ID NO: 1 or SEQ ID NO: 4,
DNA inserts in either Accession Number PTA-1479 or PTA-1480
Region of (a) or (b), wherein said polypeptide fragment
Is the encoded polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5.
A region having the activity of tide or being antigenic; (d) comprising a fragment of at least about 16 nucleotides, SEQ ID NO: 1
Or the nucleotide sequence of any of SEQ ID NO: 4, ATCC Deposit No. PTA-14
DNA insert in either 79 or PTA-1480, or (a)-
(C) any region; (e) under moderately or highly stringent conditions, (a)-(d)
A nucleotide sequence that hybridizes to any complement thereof; and (f) a nucleotide sequence complementary to any of (a) to (d), a nucleotide sequence selected from the group consisting of:

The invention further provides an isolated nucleic acid molecule, which is shown below: (a) set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 having at least one conservative amino acid substitution. A nucleotide sequence encoding a polypeptide, wherein the encoded polypeptide has the activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; (b) at least 1 A nucleotide sequence encoding the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 having a single amino acid insertion, wherein the encoded polypeptide is either SEQ ID NO: 2 or SEQ ID NO: 5. A nucleotide sequence having the activity of the polypeptide shown in (c) at least one amino acid deletion A nucleotide sequence encoding the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, wherein the encoded polypeptide is the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. A nucleotide sequence having the activity of: (d) a nucleotide sequence encoding the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5 having a C-terminal truncation and / or an N-terminal truncation, wherein: The encoded polypeptide has a nucleotide sequence having the activity of the polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 5; (e) amino acid substitution, amino acid insertion, amino acid deletion, C-terminal truncation, and N-terminal Either SEQ ID NO: 2 or SEQ ID NO: 5 having at least one modification selected from the group consisting of truncation (F) a nucleotide sequence encoding a polypeptide that is encoded by a polypeptide, wherein the encoded polypeptide has the activity of the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5; A nucleotide sequence of any of (a)-(e) comprising a fragment of at least about 16 nucleotides; (g) a complement of any of (a)-(f) under moderately or highly stringent conditions. A nucleotide sequence that hybridizes to: and (h) a nucleotide sequence complementary to any of (a) to (e), and a nucleotide sequence selected from the group consisting of:

The present invention provides an isolated polypeptide, which polypeptide comprises: (a) the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; and (b) ATCC Deposit Number. An amino acid sequence encoded by the DNA insert in either PTA-1479 or PTA-1480, the amino acid sequence being selected from the group consisting of:

The present invention also provides an isolated polypeptide, which polypeptide is set forth in either of the following: (a) as set forth in either SEQ ID NO: 3 or SEQ ID NO: 6, optionally at the amino terminus. An amino acid sequence further comprising methionine; (b) an amino acid sequence for the ortholog of either SEQ ID NO: 2 or SEQ ID NO: 5; (c) at least about 70% identical to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5. An amino acid sequence, wherein the polypeptide has the activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; (d) a sequence comprising at least about 25 amino acid residues A fragment of the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, wherein the fragment is SEQ ID NO: 2 or SEQ ID NO: 2. The polypeptide or having the activity of a set or an antigenic fragment shown in any of SEQ ID NO: 5; amino acid sequence shown in any one of and (e) SEQ ID NO: 2 or SEQ ID NO: 5, A
DN in either TCC Deposit Number PTA-1479 or PTA-1480
An amino acid sequence encoded by the A insert, or an amino acid sequence of an allelic variant or splice variant of the amino acid sequence described in any of (a) to (c), Isolated polypeptide.

The present invention further provides an isolated polypeptide, which polypeptide is set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, which has: (a) at least one conservative amino acid substitution. An amino acid sequence, wherein the polypeptide is
An amino acid sequence having the activity of the polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 5; (b) an amino acid sequence shown in either SEQ ID NO: 2 or SEQ ID NO: 5 having at least one amino acid insertion Here, the polypeptide has an amino acid sequence having the activity of the polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 5; (c) SEQ ID NO: 2 or SEQ ID NO: having at least one amino acid deletion Amino acid sequence shown in any of 5, wherein the polypeptide has the activity of the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5; (d) C-terminal truncation And / or an amino acid sequence as set forth in either SEQ ID NO: 2 or N-terminal truncations, wherein the po Peptides,
An amino acid sequence having the activity of the polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 5; and (e) selected from the group consisting of amino acid substitution, amino acid insertion, amino acid deletion, C-terminal truncation, and N-terminal truncation Amino acid sequence shown in either SEQ ID NO: 2 or SEQ ID NO: 5 having at least one modification, wherein said polypeptide is a polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5. An amino acid sequence selected from the group consisting of:

The present invention further provides an isolated polypeptide, which polypeptide comprises: leucine or methionine at position 2; methionine at position 5; lysine at position 6; alanine at position 7; Isoleucine; phenylalanine at position 14; leucine at position 15;
Threonine at position 23; Leucine at position 25; Valine at position 27; Glutamic acid at position 30; Tyrosine at position 32; Methionine at position 34; Glutamine at position 36; Lysine at position 39; Alanine at position 41; Alanine at position 41; Threonine at position 45; 55th Valine; 59th Valine; 60th Asparagine; 66th Proline; 68th Asparagine; 72th Serine or Threonine; 74th Valine; 75th Arginine; 94th Arginine; 99th Arginine Asparagine; Serine at position 100; Lysine at position 105; 10
6th Valine; 113th Tyrosine; 116th Serine; 118th Serine; 1
Arginine at position 20; Leucine at position 123; Alanine at position 125; Alanine at position 129; Alanine at position 139; Threonine at position 142; Serine at position 144; 14
7th asparagine; 148th valine; 149th serine; 159th alanine; 160th alanine; 161st alanine; 164th valine; 166th valine; 173rd valine; 175th arginine; Aspartic acid at position 177; Alanine at position 190; Phenylalanine at position 191; Arginine at position 192;
Leucine at position 194; Asparagine at position 196; Leucine at position 205; Alanine at position 210; Alanine at position 212; Serine at position 213; Alanine at position 216; 2
Serine at position 17; Alanine at position 218; Isoleucine at position 219; Alanine at position 222; Leucine at position 225; Phenylalanine at position 226; Leucine at position 230; Glutamine or arginine at position 233; Glutamine at position 241; Glutamine at position 242; Leucine; isoleucine at position 244; glutamine or asparagine at position 245; glutamine at position 249; leucine or valine at position 251; alanine at position 256; asparagine at position 257; serine at position 259; alanine at position 260; 26
Glutamine at position 1; phenylalanine at position 265; valine at position 268; leucine at position 269; leucine at position 272; valine at position 275; valine at position 278; 28
Glutamic acid at position 4; Glutamic acid at position 288; Alanine or isoleucine at position 292; Serine at position 300; Isoleucine at position 306; Valine at position 313; 3
Serine at position 14; Leucine at position 319; Glutamine at position 323; Threonine at position 324; Alanine at position 326; Alanine at position 327; Serine at position 329; 331
Serine at position 334; Leucine at position 334; Asparagine at position 337; Valine at position 339; Leucine at position 340; Serine at position 342; Phenylalanine at position 344; 34
Glutamic acid at position 9; isoleucine or valine at position 354; methionine at position 356; valine at position 366; methionine or valine at position 368; isoleucine at position 371; leucine at position 375; leucine at position 376; glutamine at position 377;
Phenylalanine at position 381; Asparagine at position 383; Isoleucine at position 384; Leucine at position 393; Arginine at position 395; Valine at position 398; Alanine or valine at position 399; Tyrosine at position 403; Asparagine at position 406; 40
An isolation comprising the amino acid sequence set forth in SEQ ID NO: 5 having at least one conservative amino acid substitution selected from the group consisting of isoleucine at position 9; alanine or valine at position 415; isoleucine at position 417; and leucine at position 421. A polypeptide which has the activity of the polypeptide shown in SEQ ID NO: 5.

[0020]   Fusion polypeptides comprising the CHL2 amino acid sequence are also provided.

The present invention also includes an expression vector comprising an isolated nucleic acid molecule as set forth herein, a recombinant host cell comprising a recombinant nucleic acid molecule as set forth herein, and CHL2. A method of producing a polypeptide is provided, the method comprising culturing the host cell, optionally isolating the polypeptide so produced.

Also included in the invention are transgenic non-human animals containing a nucleic acid molecule encoding a CHL2 polypeptide. The CHL2 nucleic acid molecule is introduced into an animal in a manner that allows expression of the CHL2 polypeptide and increased levels of CHL2 polypeptide, which may include increased circulating levels. Alternatively, CHL2
The nucleic acid molecule is introduced into the animal in a manner that inhibits expression of the endogenous CHL2 polypeptide (ie, creates a transgenic animal carrying a CHL2 polypeptide gene knockout). The transgenic non-human animal is preferably a mammal, more preferably a rodent (eg rat or mouse).

[0023]   Derivatives of CHL2 polypeptides of the invention are also provided.

Further provided are selective binding agents (eg, antibodies and peptides) capable of specifically binding the CHL2 polypeptides of the present invention. Such antibodies and peptides may be agonistic or antagonistic.

Also included by the invention is a pharmaceutical composition comprising a nucleotide, polypeptide, or selective binding agent of the invention and one or more pharmaceutically acceptable formulation agents. The pharmaceutical composition is used to provide a therapeutically effective amount of a nucleotide or polypeptide of the invention. The invention also provides the polypeptide, nucleic acid molecule,
And methods of using selective binding agents.

The CHL2 polypeptides and CHL2 nucleic acid molecules of the present invention can be used in diseases and disorders (
(Including those listed herein) can be used to treat, prevent, ameliorate, and / or detect.

The present invention also provides for identifying a test molecule that binds to a CHL2 polypeptide,
A method of assaying a test molecule is provided. The method involves contacting a CHL2 polypeptide with a test molecule and determining the extent of binding of the test molecule to the polypeptide. The method further comprises the use of such a test molecule for CHL2
Determining whether the polypeptide is an agonist or antagonist. The invention further provides a method of testing the effect of a molecule on CHL2 polypeptide expression or CHL2 polypeptide activity.

Also included by the invention are methods of modulating CHL2 polypeptide expression and methods of modulating (ie, increasing or decreasing) CHL2 polypeptide levels. One method involves administering a nucleic acid molecule encoding a CHL2 polypeptide to an animal. In another method, nucleic acid molecules containing elements that modulate or regulate the expression of CHL2 polypeptides can be administered. Examples of these methods include gene therapy, cell therapy, and antisense therapy, as described further herein.

In another aspect of the invention, the CHL2 polypeptide has its receptor (“C
HL2 polypeptide receptor "). Various forms of "expression cloning" are widely used to clone receptors for protein ligands. For example, Simonsen and Lod
ish, 1994, Trends Pharmacol. Sci. 15: 437
-41 and Tartaglia et al., 1995, Cell 83: 1263-.
See 71. Isolation of CHL2 polypeptide receptors is useful for identifying or developing novel agonists and antagonists of CHL2 polypeptide signaling pathway. Such agonists and antagonists include soluble CHL2 polypeptide receptors, anti-CHL2 polypeptide receptor selective binding agents (eg, antibodies or derivatives thereof), small molecules, and antisense oligonucleotides, any of which Or may be used for the treatment of one or more diseases or disorders, including those disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. . All references listed in this application are expressly incorporated herein by reference.

Definitions The term “CHL2 gene” or “CHL2 nucleic acid molecule” or “CHL2 polynucleotide” refers to a nucleotide sequence shown in either SEQ ID NO: 1 or SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 5. Nucleotide sequence encoding any of the polypeptides, ATCC Deposit No. PTA-1479 or PTA
A nucleotide sequence of a DNA insert in any of -1480, and a nucleic acid molecule that comprises or consists of a nucleic acid molecule as defined herein.

The term “CHL2 polypeptide allelic variant” refers to any of several possible naturally occurring alternative forms of a gene that occupy a defined locus on the chromosome of an organism or population of organisms. One of the.

The term “CHL2 polypeptide splice variant” is generated by the selective processing of intron sequences in the RNA transcript of the CHL2 polypeptide amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, A nucleic acid molecule (usually RNA).

The term “isolated nucleic acid molecule” refers to (1) at least a protein, lipid, carbohydrate, or other substance with which total nucleic acid is naturally found when isolated from a source cell. A nucleic acid molecule of the present invention isolated from about 50 percent, (2)
A nucleic acid molecule of the invention that is not linked to all or part of a polynucleotide to which the "isolated nucleic acid molecule" is naturally linked; (3) The invention operably linked to a polynucleotide that is not naturally linked. Or a nucleic acid molecule of the present invention that does not naturally occur as part of a larger polynucleotide sequence than (4). Preferably, the isolated nucleic acid molecule of the present invention is any other contaminating nucleic acid molecule, or other contaminant found in its natural environment, either for its use in producing a polypeptide, or for its therapeutic use. , Interfering with diagnostic, prophylactic, or research use).

The term “nucleic acid sequence” or “nucleic acid molecule” refers to a DNA or RNA sequence. The term includes molecules formed from any of the known base analogs of DNA and RNA, including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinyl-. Cytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1 -Methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5 -Methoxyamino-methyl-2-thiouracil, β-D-mannosylkyuosin (
β-D-mannosylqueosine), 5′-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxoxo Xybutoxine, pseudouracil,
Queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, queucin, 2 -Thiocytosine, and 2,6-diaminopurine.

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

The term “expression vector” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing (if an intron is present).

The term “operably linked” is used herein to refer to the arrangement of flanking sequences, where the flanking sequences so described perform their normal function. Configured or constructed as. Thus, a flanking sequence operably linked to a coding sequence may be capable of replicating, transcribing and / or translating the coding sequence. For example, a coding sequence is operably linked to a promoter if it is capable of directing the transcription of the coding sequence. The flanking sequences need not be contiguous with the coding sequence, as long as it functions correctly. Thus, for example, an intervening, non-translated, but transcribed sequence may be present between the promoter and coding sequences, and the promoter sequence still "operably linked" to the coding sequence. Can be considered to be

The term “host cell” has been or can be transformed with a nucleic acid sequence,
It is then used to refer to cells capable of expressing the selected gene of interest. The term includes the progeny of the parent cell, whether the progeny is identical in morphology or genetic structure to the original parent, so long as the selected gene is present.

The term “CHL2 polypeptide” refers to a polypeptide comprising the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5, and related polypeptides. Related polypeptides include CHL2 polypeptide fragments, CHL2 polypeptide orthologs, CHL2 polypeptide variants, and CHL2 polypeptide derivatives, which are of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. It possesses at least one activity. CHL2 polypeptides can be mature polypeptides as defined herein, and may or may not have an amino terminal methionine residue, depending on the method by which they are prepared.

The term “CHL2 polypeptide fragment” refers to a truncation of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 at the amino terminus (with or without a leader sequence) and / or Refers to a polypeptide that includes a truncation at the carboxyl terminus. The term "CHL2 polypeptide fragment" also refers to CH
L2 polypeptide orthologs, CHL2 polypeptide derivatives, or amino-terminal and / or carboxyl-terminal truncations of CHL2 polypeptide variants, or amino acids of CHL2 polypeptide allelic variants or polypeptides encoded by CHL2 polypeptide splice variants Refers to terminal and / or carboxyl terminal truncation. The CHL2 polypeptide fragment is a selective RNA
It can result from splicing, or in vivo protease activity. Membrane-bound forms of CHL2 polypeptides are also contemplated by the present invention. In preferred embodiments, the truncation and / or deletion comprises about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids. The polypeptide fragment thus produced is about 25 contiguous amino acids, or about 50 amino acids, or about 7 amino acids.
5 amino acids, or about 100 amino acids, or about 150 amino acids, or about 20
Contains 0 amino acids, or more than about 200 amino acids. Such CHL2 polypeptide fragments may optionally include an amino terminal methionine residue. It is understood that such fragments can be used, for example, to raise antibodies to the CHL2 polypeptide.

The term “CHL2 polypeptide ortholog” refers to a polypeptide from another species that corresponds to the CHL2 polypeptide amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. For example, the mouse and human CHL2 polypeptides are
They are considered to be orthologs of each other.

The term “CHL2 polypeptide variant” refers to one or more compared to a CHL2 polypeptide amino acid sequence (with or without a leader sequence) set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. CHL2 polypeptide comprising an amino acid sequence having an amino acid sequence substitution, deletion (eg, internal deletion and / or CHL2 polypeptide fragment), and / or addition (eg, internal addition and / or CHL2 fusion polypeptide). Say. Variants can be naturally occurring (eg, CHL2 polypeptide allelic variants, CHL2 polypeptide orthologs, and CHL2 polypeptide splice variants) or can be artificially constructed. Such CHL2 polypeptide variants can be prepared from the corresponding nucleic acid molecule having a DNA sequence that varies from the DNA sequence shown in either SEQ ID NO: 1 or SEQ ID NO: 4. In a preferred embodiment, this variant is 1 to 3.
, Or 1-5, or 1-10, or 1-15, or 1-20, or 1
-25, or 1-50, or 1-75, or 1-100, or more than 100 amino acid substitutions, insertions, additions, and / or deletions, wherein the substitutions are conservative, Or it may be non-conservative, or any combination thereof.

The term “CHL2 polypeptide derivative” refers to a polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, a CHL2 polypeptide fragment, a CHL2 polypeptide ortholog, or a CHL2 polypeptide as defined herein. Refers to peptide variants, which have been chemically modified. The term "CHL2 polypeptide derivative" also refers to a polypeptide encoded by a CHL2 polypeptide allelic variant or CHL2 polypeptide splice variant as defined herein, which is chemically modified. .

The term “mature CHL2 polypeptide” refers to a CHL2 polypeptide lacking the leader sequence. The mature CHL2 polypeptide also has other modifications (eg, amino-terminal (with or without leader sequences) and / or carboxy-terminal proteolytic processing, cleavage of a smaller polypeptide from a larger precursor, N-linked and / or O-linked glycosylation). An exemplary mature CHL2 polypeptide is represented by the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 6.

The term “CHL2 fusion polypeptide” refers to SEQ ID NO: 2, as defined herein.
Alternatively, the polypeptide shown in any of SEQ ID NO: 5, CHL2 polypeptide fragment, CHL2 polypeptide ortholog, CHL2 polypeptide variant,
Alternatively, it refers to a fusion of one or more amino acids (eg, a heterologous protein or peptide) at the amino or carboxy terminus of a CHL2 derivative. The term "CHL2
A "fusion polypeptide" also includes one or more amino acids at the amino or carboxyl termini of a polypeptide encoded by a CHL2 polypeptide allelic variant or CHL2 polypeptide splice variant as defined herein. Refers to fusion.

The term “biologically active CHL2 polypeptide” refers to a CHL2 polypeptide having at least one activity characteristic of a polypeptide comprising the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5. Further, the CHL2 polypeptide is
It may be active as an immunogen; that is, the CHL2 polypeptide comprises at least one epitope against which an antibody may be raised.

The term “isolated polypeptide” refers to (1) at least about 50% of a polypeptide, lipid, carbohydrate, or other substance found together in nature when isolated from a source cell. A polypeptide of the invention isolated from (2) an "isolated polypeptide" is not linked to all or part of the polypeptide to which it is naturally linked (
A polypeptide of the invention (by covalent or non-covalent interaction), (
3) a polypeptide of the invention that is operably linked (by covalent or non-covalent interactions) to a polypeptide that is not naturally linked, or (4) a polypeptide of the invention that does not occur in nature. Say. Preferably, the isolated polypeptide is any other contaminating polypeptide or other contaminant found in its natural environment, which has therapeutic, diagnostic, prophylactic, or research uses. (Preventing use) is substantially not included.

The term “identity” refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences, as is known in the art. In the art, "identity" also refers to the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between two or more nucleotide or two or more amino acid sequences. Means "Identity" is the match between the smaller of two or more sequences and the gap alignment (if any) focused by a particular mathematical model or computer program (ie, "algorithm"). Evaluate the same percentage of.

The term “similarity” relates to a concept, but in contrast to “identity”, “similarity” refers to a measure of relatedness that includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids and the rest are all non-conservative substitutions, then the percent identity and percent similarity are both 50%. In the same example, if there are five more positions that are conservative substitutions, the percent identity remains 50%, but the percent similarity is 7.
It is 5% (15/20). Thus, if there are conservative substitutions, the percent similarity between the two polypeptides will be higher than the percent identity between these two polypeptides.

The terms “naturally occurring” or “native” are found in nature when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, etc. Material that has not been manipulated by. Similarly, "non-naturally occurring" or "non-native" as used herein refers to
Refers to materials that are not found in nature or have been structurally modified or synthesized by humans.

The terms “effective amount” and “therapeutically effective amount” each refer to CHL as indicated herein.
2 refers to the amount of CHL2 polypeptide or CHL2 nucleic acid molecule used to support an observable level of one or more biological activities of a polypeptide.

The term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refers to a CHL2 polypeptide, CHL2 nucleic acid molecule, or pharmaceutical composition as a pharmaceutical composition. Refers to one or more formulation materials suitable for achieving or enhancing delivery of a CHL2 selective binding agent.

The term “antigen” is a molecule or a molecule that can be bound by a selective binding agent (eg, an antibody) and is further used in an animal to produce an antibody capable of binding an epitope of that antigen. Part. An antigen can have one or more epitopes.

The term “selective binding agent” refers to a molecule (with specificity for a CHL2 polypeptide (
Singular or plural). As used herein, the terms "specific" and "specificity" refer to the ability of a selective binding agent to bind a human CHL2 polypeptide and not a human non-CHL2 polypeptide. However, the selective binding agent may also be an ortholog of the polypeptide as described in either SEQ ID NO: 2 or SEQ ID NO: 5 (ie an interspecies variation thereof (eg mouse CHL2 polypeptide and rat CHL2 polypeptide)). It is understood that

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

The term “transfection” is used to refer to the uptake of heterologous or exogenous DNA by a cell, and the cell is “transfected” if the exogenous DNA has been introduced inside the cell membrane. . Many transfection techniques are well known in the art and disclosed herein. For example, Grah
am et al., 1973, Virology 52: 456; Sambrook et al., M.
olecular Cloning, A Laboratory Manual
(Cold Spring Harbor Laboratories, 19
89); Davis et al., Basic Methods in Molecular.
r Biology (Elsevier, 1986); and Chu et al., 198.
1, Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

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

Relatedness of Nucleic Acid Molecules and / or Polypeptides Related nucleic acid molecules include allelic or splice variants of the nucleic acid molecule of either SEQ ID NO: 1 or SEQ ID NO: 4, and above. It is understood to include sequences that are complementary to any of the nucleotide sequences of. A related nucleic acid molecule also contains or consists essentially of substitutions, modifications, additions and / or deletions of one or more amino acid residues compared to the polypeptide in either SEQ ID NO: 2 or SEQ ID NO: 5. A nucleotide sequence encoding a polypeptide of Such related CHL2 polypeptides include, for example, the addition and / or deletion of one or more N-linked or O-linked glycosylation sites, or the addition and / or deletion of one or more cysteine residues. obtain.

Related nucleic acid molecules also include CHL2 of either SEQ ID NO: 2 or SEQ ID NO: 5.
At least about 25 consecutive amino acids of the polypeptide, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150
Fragments of the CHL2 nucleic acid molecule encoding a polypeptide of 1 amino acid, or about 200 amino acids, or more than 200 amino acid residues.

Furthermore, a related CHL2 nucleic acid molecule may also be a CHL2 nucleic acid molecule or polypeptide of either SEQ ID NO: 1 or SEQ ID NO: 4 under moderately or highly stringent conditions as defined herein. A polypeptide comprising a molecule comprising a nucleotide sequence that hybridizes to a fully complementary sequence of an encoding molecule, wherein the polypeptide is SEQ ID NO: 2 or SEQ ID NO: 5, or a nucleic acid fragment as defined herein, or herein. It includes the amino acid sequence set forth in any of the nucleic acid fragments encoding the polypeptide defined therein. The hybridization probe is
It can be prepared using the CHL2 sequences provided herein for screening a cDNA, genomic DNA library or synthetic DNA library for related sequences. Regions of the CHL2 polypeptide DNA and / or amino acid sequence that show significant identity to a known sequence are readily determined using the sequence alignment algorithms described herein, and Regions can be used to design probes for screening.

The term “highly stringent conditions” refers to DNs where the sequences are highly complementary.
Refers to conditions designed to allow hybridization of the A strand and exclude hybridization of significantly mismatched DNA. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents (eg, formamide). An example of "highly stringent conditions" for hybridization and washing is 0.015 at 65-68 ° C.
M Sodium Chloride, 0.0015M Sodium Citrate or 0.015M Sodium Chloride at 42 ° C
CHL2oride), 0.0015M sodium citrate and 50% formamide. Sambrook, Fritsch and Maniatis, M
olecular Cloning: A Laboratory Manual
(2nd edition, Cold Spring Harbor Laboratory, 1
989); Anderson et al., Nucleic Acid Hybrids.
ation: A Practical Approach Chapter 4 (IRLP
See Less Limited).

More stringent conditions (eg higher temperature, lower ionic strength,
Higher formamide or other denaturants) can also be used, but the rate of hybridization is affected. Other agents may be included in the hybridization and wash buffers for the purpose of reducing non-specific and / or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1
% Sodium dodecyl sulfate, NaDodSO ₄ , (SDS), Ficoll (fi
Coll), Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is largely independent of pH.
Anderson et al., Nucleic Acid Hybridization
: A Practical Approach Chapter 4 (IRL Press
Limited).

Factors affecting the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by those of skill in the art to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation: T _m (° C.) = 81.5 + 16.6 (log [Na +]) + 0.41 (% G + C)
) -600 / N-0.72 (% formamide) where N is the length of the duplex formed and [Na +] is the molar concentration of sodium ion in the hybridization or wash solution. And% G +
C is the percentage of (guanine + cytosine) bases in the hybrid. For imperfectly matched hybrids, the melting temperature drops by about 1 ° C. for every 1% mismatch.

The term “moderately stringent conditions” refers to conditions under which a DNA duplex having a higher degree of base pair mismatch than can occur under “highly stringent conditions” can be formed. An example of a typical “moderately stringent condition” is 5
0.015M sodium chloride (sodium CHL2orid at 0-65 ° C)
e), 0.0015M sodium citrate or 0.015M at 37-50 ° C
Sodium CHL2oride, 0.0015M sodium citrate and 20% formamide. By way of illustration, "moderately stringent conditions" of 0.015 M sodium ion at 50 ° C allow a discrepancy of about 21%.

It will be appreciated by those skilled in the art that there is no absolute distinction between “highly stringent conditions” and “moderately stringent conditions”. For example, 0
． For 015M sodium ion (without formamide), a long, perfectly matched D
The melting temperature of NA is about 71 ° C. With a wash at 65 ° C. (at the same ionic strength), this allows a mismatch of about 6%. To capture more distantly related sequences, one of ordinary skill in the art can simply lower the temperature or increase the ionic strength.

A good estimate of the melting temperature in 1M NaCl ^* for oligonucleotide probes up to about 20 nt is given by: Tm = 2 ° C. per AT base pair + 4 ° C. per GC base pair ^* The sodium ion concentration in 6 × sodium citrate salt (SSC) is 1M. Suggs et al., Developmental Biology Usin.
g Purified Genes 683 (Brown and Fox, eds., 19)
81).

High stringency wash conditions for oligonucleotides are typically 6 ×.
SSC, 0 ° C to 5 than Tm of the oligonucleotide in 0.1% SDS
It is at a temperature lower by ℃.

In another embodiment, a related nucleic acid molecule comprises or consists of a nucleotide sequence that is at least about 70 percent identical to the nucleotide sequence set forth in either SEQ ID NO: 1 or SEQ ID NO: 4, Alternatively, it comprises or consists essentially of a nucleotide sequence that encodes a polypeptide that is at least about 70 percent identical to the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. In a preferred embodiment, the nucleotide sequence is about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95 percent to the nucleotide sequence set forth in either SEQ ID NO: 1 or SEQ ID NO: 4. 96, 97, 98, or 99 percent identical, or the nucleotide sequence is about 75 percent, or about 80 percent, or about 85 percent to the polypeptide sequence shown in either SEQ ID NO: 2 or SEQ ID NO: 5. Percent, or about 90 percent, or about 95
, 96, 97, 98, or 99 percent identical polypeptides. Related nucleic acid molecules encode a polypeptide that retains at least one activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

Differences in the nucleic acid sequences can result in conservative and / or non-conservative modifications of the amino acid sequence as compared to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5.

Conservative modifications to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5 (
And corresponding modifications to the encoding nucleotides) result in a polypeptide having functional and chemical characteristics similar to those of the CHL2 polypeptide. In contrast, substantial modification in the functional and / or chemical characteristics of the CHL2 polypeptide may be achieved by selecting a substitution in the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5, which is Its effect on the maintenance of the following is significantly different: (a) the structure of the molecular scaffold in the substitution region, such as the sheet or helical conformation, (b) the charge of the molecule at the target site or Hydrophobicity, or (c) bulkiness of side chains.

For example, a “conservative amino acid substitution” can include a substitution of a native amino acid residue with a non-native residue that has little or no effect on the polarity or charge of the amino acid residue at that position. In addition, any native residue in the polypeptide can also be replaced by alanine, as previously described for "alanine scanning mutagenesis."

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

Naturally occurring residues can be divided into multiple classes based on common side chain properties: 1) Hydrophobicity: Norleucine, Met, Ala, Val, Leu, Ile; 2) Neutral hydrophilicity. 3) Acidity: Asp, Glu; 4) Basicity: Asn, Gln, His, Lys, Arg; 5) Residues affecting chain orientation: Gly, Pro; and 6). Aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into a region of the human CHL2 polypeptide that is homologous to the non-human CHL2 polypeptide, or in a heterologous region of this molecule.

In making such changes, the hydropathic index of amino acids (hydropat)
hic index) may be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. The hydropathy index is
The following are: isoleucine (+4.5); valine (+4.2); leucine (+3)
． 8); Phenylalanine (+2.8); Cysteine / cystine (+2.5);
Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1) .3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3. 5); lysine (-3.9); and arginine (-4.5)
.

The importance of the hydropathic amino acid index in identifying interactive biological functions for proteins is generally understood in the art (Kyte
Et al., 1982, J. Am. Mol. Biol. 157: 105-31). It is known that certain amino acids can be used in place of other amino acids having a similar hydropathic index or score, and still maintain similar biological activity. In making changes based on the hydropathic index, substitution of amino acids whose hydropathic index is within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. .

Substitution of similar amino acids can be made effectively on the basis of hydrophilicity (especially if the biologically functionally equivalent protein or peptide produced thereby is immunologically as in this case). (When intended for use in embodiments)
It is also understood in the art. The greatest local average hydrophilicity of a protein correlates to its immunogenicity and antigenicity, ie, the biological properties of the protein, when governed by the hydrophilicity of its flanking amino acids.

The following hydrophilicity values were assigned to these amino acid residues: arginine (
+3.0); Lysine (+3.0); Aspartic acid (+ 3.0 ± 1); Glutamic acid (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2) ); Glycine (0); Threonine (-0.4); Proline (-
0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (
-1.0); methionine (-1.3); valine (-1.5); leucine (-1.
8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (
-2.5); and tryptophan (-3.4). When making changes based on similar hydrophilicity values, substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are preferred. , Even more particularly preferred. From the primary amino acid sequence, epitopes can also be identified on the basis of hydrophilicity. These regions are also referred to as "epitope core regions".

Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions may be used to identify key residues in a CHL2 polypeptide or may increase or decrease the affinity of a CHL2 polypeptide described herein. Exemplary amino acid substitutions are listed in Table I.

[0081]

[Table 1] One of ordinary skill in the art can determine suitable variants of the polypeptides set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 using well known techniques. To identify suitable regions of the molecule that can be altered without destroying biological activity, one of skill in the art can target regions that are not considered important for activity. For example, if a similar polypeptide with similar activity is known, from the same species or from another species, one of skill in the art will recognize the amino acid sequence of a CHL2 polypeptide as such similar polypeptide. You can compare. Such comparisons can be used to identify residues and portions of molecules that are conserved between similar polypeptides. It is understood that changes in regions of the CHL2 molecule that are not conserved for such similar polypeptides do not appear to adversely affect the biological activity and / or structure of the CHL2 polypeptide. It It is also known to those of skill in the art that chemically similar amino acids can be substituted for naturally occurring residues while retaining activity (replacement of conservative amino acid residues), even in relatively conserved regions. ). Thus, even regions that may be important for biological activity or structure may be subject to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure. .

In addition, one of skill in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such comparisons, one can predict the importance of amino acid residues in the CHL2 polypeptide that correspond to amino acid residues that are important for activity or structure in similar polypeptides. One of skill in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues in the CHL2 polypeptide.

One of skill in the art can also analyze the three-dimensional structure and amino acid sequence for the three-dimensional structure in similar polypeptides. In view of such information, those skilled in the art
Alignments of amino acid residues in CHL2 polypeptides can be predicted with respect to their three-dimensional structure. One of skill in the art can choose not to make abrupt changes to amino acid residues predicted to be present on the surface of the protein. Because such residues may be involved in important interactions with other molecules. In addition, one of skill in the art can generate test variants that contain a single amino acid substitution at each amino acid residue. These variants can be screened using activity assays known to those of skill in the art. Such variants can be used to gather information about suitable variants. For example, if a change to a particular amino acid residue is found to result in disruption, undesirably diminishing, or undesired activity, then variants with such changes are avoided. In other words, on the basis of information gathered from such routine experimentation, one of skill in the art would appreciate that amino acids for which further substitutions should be avoided either alone or in combination with other mutations. , Can be easily determined.

Numerous scientific publications have been devoted to the prediction of secondary structure. Mault, 199
6, Curr. Opin. Biotechnol. 7: 422-27; Chou.
Et al., 1974, Biochemistry 13: 222-45; Chou et al.
1974, Biochemistry 113: 211-2; Chou et al., 1
978, Adv. Enzymol. Relat. Areas Mol. Biol
． 47: 45-48; Chou et al., 1978, Ann. Rev. Biochem
． 47: 251-276; and Chou et al., 1979, Biophys. J.
26: 367-84. Moreover, computer programs are currently available to assist with predicting secondary structure. One method of estimating secondary structure is based on homology modeling. For example, two polypeptides or proteins with greater than 30% sequence identity or greater than 40% similarity often have similar structural topologies. Protein structure database (PDB)
The recent growth of A. has provided enhanced predictability of secondary structure, including the number of potential folds in the structure of a polypeptide or protein. Holm et al., 199
9, Nucleic Acids Res. 27: 244-47.
It has been suggested that there is a limited number of folds in a given polypeptide or protein, and that once a significant number of structures have been analyzed, structure estimation becomes dramatically more accurate (Brenner. Et al., 1997, Curr. O.
pin. Struct. Biol. 7: 369-76).

A further method of estimating secondary structure is by “threading”.
) ”(Jones, 1997, Curr. Opin. Struct. Biol.
7: 377-87; Sippl et al., 1996, Structure 4: 15-.
19), "Profile Analysis" (Bowie et al., 1991, Science, 2).
53: 164-70; Gribskov et al., 1990, Methods Enz.
ymol. 183: 146-59; Gribskov et al., 1987, Proc.
Nat. Acad. Sci. U. S. A. 84: 4355-58), and "Evolutionary Linkages" (see Holm et al., Supra, and Brenner et al., Supra).

Preferred CHL2 polypeptide variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Is mentioned. In one embodiment, the CHL2 polypeptide variant comprises a greater or lesser number of N-linked glycosylation sites than the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. The N-linked glycosylation site is the sequence Asn-X-Ser or Asn-X-.
The amino acid residue characterized by Thr, where X is indicated, can be any amino acid residue other than proline. Substitution of amino acid residues to create this sequence provides a potential new site for the addition of N-linked carbohydrate chains. Alternatively, substitutions that eliminate this sequence remove the existing N-linked carbohydrate chain. 1
Rearrangements of N-linked carbohydrate chains also eliminate one or more N-linked glycosylation sites (typically the naturally occurring glycosylation sites) and create one or more new N-linked sites. Provided. Further preferred CHL2 variants include:
Cysteine, in which one or more cysteine residues are deleted as compared to the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or substituted with another amino acid (eg, serine). Examples include modified forms. The cysteine variant is C
It is useful when the HL2 polypeptide must be refolded into a biologically active conformation, eg, after isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have the same number of cysteine residues, minimizing interactions that result from unpaired cysteines.

In another embodiment, a related nucleic acid molecule has at least one amino acid insertion and the polypeptide has the activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. 2 or SEQ ID NO: 5 or comprising, or consisting of, a nucleotide sequence that encodes a polypeptide as set forth in SEQ ID NO: 2 or a sequence. It comprises or consists of a nucleotide sequence coding for the polypeptide according to either SEQ ID NO: 2 or SEQ ID NO: 5, which has the activity of the polypeptide according to any of SEQ ID NO: 5. A related nucleic acid molecule is also one in which the polypeptide has a carboxyl-terminal and / or amino-terminal truncation, and further wherein the polypeptide has an activity of the polypeptide according to either SEQ ID NO: 2 or SEQ ID NO: 5. Comprising or consisting of a nucleotide sequence that encodes the polypeptide of either SEQ ID NO: 2 or SEQ ID NO: 5. A related nucleic acid molecule also includes at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, carboxyl terminal truncations, and amino terminal truncations, wherein the polypeptide is SEQ ID NO: 2 or It comprises or consists of a nucleotide sequence coding for the polypeptide according to either SEQ ID NO: 2 or SEQ ID NO: 5, which has the activity of the polypeptide according to SEQ ID NO: 5.

In addition, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, or any of the other CHL2 polypeptides, can be fused to a homologous polypeptide to form a homodimer, or to a heterologous polypeptide. Fused to form a heterodimer. Heterologous peptides and polypeptides include, but are not limited to, epitopes that allow detection and / or isolation of CHL2 fusion polypeptides; transmembrane receptor proteins or portions thereof (
Extracellular domains or transmembrane and intracellular domains); ligands or parts thereof that bind to transmembrane receptor proteins; enzymes or parts thereof that are catalytically active; polypeptides or peptides that promote oligomerization ( For example, a leucine zipper domain); a polypeptide or peptide that increases stability (eg, an immunoglobulin constant region); and a polypeptide comprising the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or other C
A polypeptide having a therapeutic activity different from the HL2 polypeptide.

Fusions can be made at either the amino or carboxyl termini of polypeptides that include the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or other CHL2 polypeptides. The fusion may be a direct fusion without a linker or adapter molecule or may be via a linker or adapter molecule. The linker or adapter molecule can be one or more amino acid residues, typically about 20 to about 50 amino acid residues. Linker or adapter molecules may also be designed with cleavage sites for DNA restriction endonucleases or proteases to allow separation of fused moieties. It is understood that, once constructed, the fusion polypeptide can be derivatized according to the methods described herein.

In a further embodiment of the invention, a polypeptide comprising the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5, or another CHL2 polypeptide is fused to one or more domains of the Fc region of human IgG. To do. Antibodies include two functionally independent moieties: the variable domain known as "Fab" that binds antigens and is involved in effector functions such as complement activation and attack by phagocytic cells. A constant domain known as "Fc". Fc has a long serum half-life, while Fab has a short life span. Capon et al., 1989, Nature.
337: 525-31. When assembled with a therapeutic protein, the Fc domain may provide a longer half-life, or may perform functions such as Fc receptor binding, protein A binding, complement fixation, and perhaps placental transfer. Can be incorporated. Ibid. Table II summarizes the use of certain Fc fusions known in the art.

[0091]

[Table 2] In one example, the human IgG hinge region, CH2 region, and CH3 region are:
Fusions can be made at either the amino or carboxyl termini of the CHL2 polypeptides using methods known to those of skill in the art. In another example, the hinge, CH2, and CH3 regions of human IgG can be fused at either the amino or carboxyl termini of a CHL2 polypeptide fragment (eg, the putative extracellular portion of a CHL2 polypeptide).

The resulting CHL2 fusion polypeptide can be purified by use of a Protein A affinity column. Peptides and proteins fused to the Fc region were found to exhibit a substantially longer half-life in vivo than their unfused counterparts. Also, fusion to the Fc region allows for dimerization / multimerization of the fusion polypeptide. The Fc region can be a naturally occurring Fc region or can be modified to improve certain qualities such as therapeutic quality, circulation time, or reduced aggregation.

Identity and similarity of related nucleic acid molecules and polypeptides can be readily calculated by known methods. One such method is Computation
al Molecular Biology (AM Lesk ed., Oxford)
d University Press 1988); Biocomputin
g: Informations and Genome Projects (D.
W. Smith, Academic Press 1993); Comput
er Analysis of Sequence Data (Part 1,
A. M. Griffin and H.C. G. Griffin, Humana Pr
ess 1994); von Heinle, Sequence Anal
ysis in Molecular Biology (Academic P
ress 1987); Sequence Analysis Primer (
M. Gribskov and J.M. Devereux, M .; Stockton
Press 1991); and Carillo et al., 1988, SIAM J.
． Applied Math. , 48: 1073, but are not limited thereto.

Preferred methods to determine identity and / or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are described in publicly available computer programs. A preferred computer program method for determining identity and similarity between two sequences is the GCG program package (GAP (Dever
eux et al., 1984, Nucleic Acids Res. 12: 387; G
genetics Computer Group, University of
Wisconsin, Madison, WI), BLASTP, BLASTN
, And FASTA (Altschul et al., 1990, J. Mol. Biol.
215: 403-10)), but is not limited thereto. BL
The ASTX program is the National Center for Biote
chnology Information (NCBI) and other sources (A
ltschul et al., BLAST Manual (NCB NLM NIH, Be.
thesda, MD); Altschul et al., 1990, supra). The well-known Smith Waterman algorithm can also be used to determine identity.

A particular alignment scheme for aligning two amino acid sequences may result in a fit of only the short regions of these two sequences, and this small alignment region is 2
Very high sequence identity can be achieved even when there is no significant association between the two full length sequences. Thus, in a preferred embodiment, the selected alignment method (GAP program) results in an alignment over at least 50 consecutive amino acids of the claimed polypeptide.

For example, the computer algorithm GAP (Genetics Comput) is used.
er Group, University of Wisconsin, Mad
ison, WI), the two polypeptides whose percent sequence identities are to be determined are aligned for optimal fit of their respective amino acids (the "fit span" determined by the algorithm). To be done. Gap opening penalty (this is calculated as 3 times the mean diagonal: "mean diagonal" means the comparison matrix (comp) used.
arison matrix) diagonal mean; “diagonal” is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and gap extension penalties (gap extensio).
n penalty) (this is typically a cap opening penalty of 0.
1 ×), as well as comparison matrices such as PAM 250 or BLOSUM 62 are used in combination with this algorithm. A standard comparison matrix is also used by this algorithm (Dayhoff et al., 5 Atlas).
of Protein Sequence and Structure (Appendix 3 1978) (PAM250 comparison matrix); Henikoff et al., 1992, P.
roc. Natl. Acad. Sci USA 89: 10915-19 (BL
OSUM 62 comparison matrix)).

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, 1970.
J. Mol. Biol. 48: 443-53; Comparison matrix: BLOSUM 62 (Henikof).
f et al., supra); Gap Penalty: 12 Gap Length Penalty: 4 Threshold of Similarity: 0 This GAP program is useful with the above parameters. The above parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisons include the following: Algorithm: Needleman and Wunsch, supra; Comparison matrix: matches = + 10, misma.
tch = 0 Gap Penalty: 50 Gap Length Penalty: 3 This GAP program is also useful with the above parameters. The above parameters are the default parameters for nucleic acid molecule comparison.

Program Manual, Wisconsin Package, Ve
Other exemplary algorithms, gap opening penalties, ga, including those described in region 9, September, 1997.
p extension penalty, comparison matri
x, and threshold of similarity can be used.
The particular choices to make will be apparent to those of skill in the art, and the particular comparisons to be made (eg, DNA to DNA, protein to protein, protein to DNA).
); And additionally, the comparison is between a given pair of sequences (where GAP or BestFit are generally preferred) or between one sequence and a large database sequence (in this case, FASTA or BLASTA is preferred)
Depends on

Nucleic Acid Molecules Nucleic acid molecules encoding polypeptides comprising the amino acid sequence of CHL2 polypeptides can be expressed in various ways (chemical synthesis, cDNA or genomic library screening, expression library screening, and / or cDNA PCR
Amplification, but is not limited thereto).

Recombinant DNA methods used herein generally refer to Sambrook.
Et al., Molecular Cloning: A Laboratory Man.
ual (Cold Spring Harbor Laboratory Pr
ess, 1989) and / or Current Protocols in
Molecular Biology (Edited by Ausubel et al., Green P)
ublishers Inc. And Wiley and Sons 1994
)It is described in. The present invention provides nucleic acid molecules as described herein, and methods for obtaining such molecules.

When the gene encoding the amino acid sequence of CHL2 polypeptide is identified from one species, all or part of this gene may be used as a probe to identify orthologs or related genes from the same species. Can be used. This probe or primer can be used to screen cDNA from various tissue sources that are suspected of expressing the CHL2 polypeptide. In addition, part or all of the nucleic acid molecule having a sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4 is used to screen a genomic library to identify the gene encoding the amino acid sequence of the CHL2 polypeptide. Can be identified and isolated. Typically, moderate or high stringency conditions are used for the screen to minimize the number of false positives obtained from this screen.

Nucleic acid molecules encoding the amino acid sequence of CHL2 polypeptides can also be identified by expression cloning using detection of positive clones based on the characteristics of the expressed protein. Typically, nucleic acid libraries are screened by binding an antibody or other binding partner (eg, receptor or ligand) to the cloned protein expressed and displayed on the host cell surface. The antibody or binding partner is modified with a detectable label to identify cells that express the desired clone.

These polynucleotides can be produced and the encoded polypeptides expressed according to recombinant expression techniques performed according to the instructions set forth below.
For example, one can readily generate large amounts of the desired nucleotide sequence by inserting the nucleic acid sequence encoding the amino acid sequence of CHL2 polypeptide into a suitable vector. These sequences can then be used to generate detection probes or amplification primers. Alternatively, the polynucleotide encoding the amino acid sequence of CHL2 polypeptide can be inserted into an expression vector. By introducing the expression vector into a suitable host, the encoded CHL2 polypeptide can be produced in large quantities.

Another method for obtaining the appropriate nucleic acid sequence is the polymerase chain reaction (PCR). In this method, the cDNA is converted into poly (
A) + RNA or total RNA. Two primers, typically complementary to two distinct regions of the cDNA encoding the amino acid sequence of the CHL2 polypeptide, were then added to this cDNA along with a polymerase such as Taq polymerase, The polymerase then amplifies the region of the cDNA between these two primers.

Another means of preparing nucleic acid molecules encoding the amino acid sequence of CHL2 polypeptide is described by Engels et al., 1989, Angew. Chem. Intl. Chapter. 28
: 716-34, chemical synthesis using methods well known to those skilled in the art. These methods include phosphotriester, phosphoramidite, and H-phosphonate methods for nucleic acid synthesis, among others. The preferred method for such chemical synthesis is polymer-supported synthesis using standard phosphoramidite chemistry. Typically, the DNA encoding the amino acid sequence of CHL2 polypeptide is several hundred nucleotides long. Nucleic acids longer than about 100 nucleotides can be synthesized as several fragments using these methods. These fragments can then be ligated together to form the full length nucleotide sequence of the CHL2 gene. Usually, the DNA fragment encoding the amino terminus of this polypeptide has ATG, which
Encodes a methionine residue. This methionine, depending on whether the polypeptide produced in the host cell is designed to be secreted from that cell, CH
It may or may not be present in the mature form of the L2 polypeptide. Other methods known to those of skill in the art may be used as well.

In certain embodiments, the nucleic acid variants contain codons that have been altered for optimal expression of the CHL2 polypeptide in a given host cell. The particular codon change depends on the CHL2 polypeptide and host cell selected for expression. Such "codon optimization" can be performed by a variety of methods, such as by selecting preferred codons for use in the highly expressed gene in a given host cell. For codon preference of highly expressed bacterial genes
Eco_high. Computer algorithms that incorporate codon frequency tables, such as "Cod", can be used, and University of Wisconsi
n Package Version 9.0 (Genetics Compu
ter Group, Madison, WI). Other useful codon frequency tables include "Celegans_high.cod", "Cele
gans_low. cod "," Drosophila_high.cod ",
"Human_high.cod", "Maize_high.cod", and "Yeast_high.cod."

In some cases, it may be desirable to prepare a nucleic acid molecule that encodes a CHL2 polypeptide variant. Nucleic acid molecules encoding the variants can be generated using site-directed mutagenesis, in which the primer has the desired point mutation, PCR amplification, or other suitable method (see Sambrook for a description of mutagenesis techniques).
Et al., Supra, and Ausubel et al., Supra). Chemical synthesis using the methods described by Engels et al., Supra, can also be used to prepare such variants. Other methods known to those of skill in the art may be used as well.

Vectors and Host Cells Nucleic acid molecules encoding the amino acid sequence of CHL2 polypeptide are inserted into an appropriate expression vector using standard ligation techniques. Vectors are typically chosen to be functional in the particular host cell used (ie, the vector is compatible with the host cell machinery, so that gene amplification and / or gene expression may occur). Sex). A nucleic acid molecule encoding the amino acid sequence of a CHL2 polypeptide can be amplified / expressed in a prokaryotic host cell, a yeast host cell, an insect (baculovirus system) host cell and / or a eukaryotic host cell. The choice of host cell depends in part on whether the CHL2 polypeptide is post-translationally modified (eg, glycosylated and / or phosphorylated). If so, a yeast host cell,
Insect host cells or mammalian host cells are preferred. For a review of expression vectors, see Meth. Enz. , Volume 185 (D.V. Goeddel, edited, Acad
See emric Press 1990).

[0110] Typically, expression vectors used in any host cell will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences (collectively referred to as "flanking sequences"), in certain embodiments, typically include one or more of the following nucleotide sequences: promoter,
One or more enhancer sequences, origins of replication, transcription termination sequences, complete intron sequences including donor and acceptor splice sites, sequences encoding leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, expressed A polylinker region for inserting a nucleic acid encoding a polypeptide, and a selectable marker element. Each of these sequences is discussed below.

Optionally, the vector may include a "tag" coding sequence (ie, an oligonucleotide molecule located at the 5'or 3'ends of the CHL2 polypeptide coding sequence); the oligonucleotide sequence may be polyHis. (For example, hexa
His), or another “tag” (eg, FLAG, HA (hemagglutinin (he
maglutinin) influenza virus)) or myc for which commercially available antibodies are present. This tag is typically fused to the polypeptide upon expression of the polypeptide and can serve as a means for affinity purification of CHL2 polypeptide from host cells. Affinity purification can be achieved, for example, by column chromatography using an antibody against the tag as an affinity matrix. If desired, the tag is then removed from the purified CHL2 polypeptide by various means, such as by using a particular peptidase for cleavage.

The flanking sequences can be homologous (ie, from the same species and / or strain as the host cell), heterologous (ie, from a species other than the host cell species or strain), or a hybrid (ie, Can be a combination of flanking sequences from more than one source) or can be synthetic, or the flanking sequences can be native sequences that function normally to regulate CHL2 polypeptide expression. .
Thus, the source of flanking sequences can be any prokaryotic or eukaryotic, any vertebrate or invertebrate, or any plant, provided the flanking sequences are functional in the host cell machinery. And can be activated by the host cell machinery.

The flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein, other than the CHL2 gene flanking sequences, have been previously identified by mapping and / or restriction endonuclease digestion, and thus using appropriate restriction endonucleases. , May be isolated from a suitable tissue source. In some cases, the full length nucleotide sequence of the flanking sequences may be known. Here, flanking sequences can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

If all or only part of the flanking sequences are known, PCR is used and / or a genomic library is constructed using appropriate oligonucleotides and / or flanking sequence fragments from the same or another species. It can be obtained by screening. If the flanking sequence is not known, a fragment of DNA containing the flanking sequence may be, for example, a large piece of DNA that may contain the coding sequence or another gene.
Can be isolated from Isolation includes restriction endonuclease digestion to generate the appropriate DNA fragment, followed by isolation using agarose gel purification, Qiagen
® column chromatography (Chatsworth, CA), or other methods known to those of skill in the art. Selection of the appropriate enzyme to achieve this end will be readily apparent to the skilled person.

The origin of replication is typically part of a commercially available prokaryotic expression vector, and this origin serves for amplification of the vector in host cells. Amplification of the vector to a particular copy number may be important for optimal expression of the CHL2 polypeptide in some cases. If the vector of choice does not contain an origin of replication site, it can be chemically synthesized based on known sequences and ligated into the vector. For example, the plasmid pBR322 (New England Biola).
The origin of replication from Bs, Beverly, MA) is suitable for most Gram-negative bacteria, and of various origins (eg SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or HPV or BPV). Such papillomaviruses) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is a mammalian expression vector (eg, SV4
The zero origin is often not needed because it is used only because it contains the early promoter).

A transcription termination sequence will typically be located 3 ′ to the termini of the polypeptide coding region and serves to terminate transcription. Usually, the transcription termination sequence in prokaryotic cells is a GC rich fragment followed by a poly-T sequence. This sequence can be easily cloned from a library or purchased commercially as part of a vector, which can be readily synthesized using methods for nucleic acid synthesis as described herein above. obtain.

Selectable marker genetic elements encode proteins necessary for the survival and growth of host cells grown in a selective culture medium. An exemplary selectable marker gene confers (a) resistance to a prokaryotic host cell to antibiotics or other toxins (eg, ampicillin, tetracycline, or kanamycin); (b) cell auxotrophy. Complement the deficiency; or (c) encode a protein that supplies important nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, ampicillin resistance gene, and tetracycline resistance gene. The neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells.

Other selection genes can be used to amplify the expressed gene. Amplification is the process by which genes, which are largely required for the production of proteins important for growth, are tandemly repeated within the chromosome of continuous production of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DH
FR) and thymidine kinase. Mammalian cell transformants are placed under selection pressure, where only the transformants are uniquely adapted to survive by virtue of the selection gene present in the vector. Selection pressure is the culturing of transformed cells under conditions in which the concentration of the selection factor in the medium changes continuously, thereby leading to the amplification of both the selection gene and the DNA encoding the CHL2 polypeptide. Imposed by. As a result, increased amounts of CHL2 polypeptide are synthesized from the amplified DNA.

The ribosome binding site is normally required for translation initiation of mRNA, and Sh
ine-Dalgarno sequence (prokaryote) or Kozak sequence (eukaryote)
Characterized by This element is typically located 3'to the promoter of the expressed CHL2 polypeptide and 5'to the coding sequence. Sh
The ine-Dalgarno sequence is variable, but is typically polypurine (ie, has a high AG content). Many Shine-Dalgar
No sequences have been identified, each of which can be readily synthesized using the methods described herein and used in prokaryotic vectors.

A leader sequence, or signal sequence, can be used to direct the CHL2 polypeptide from the host cell. Typically, the nucleotide sequence encoding the signal sequence is located in the coding region of the CHL2 nucleic acid molecule or is directly CHL2.
It is located 5'to the 2 polypeptide coding region. Many signal sequences have been identified, and any signal sequence that is functional in the host cell of choice may be used in combination with the CHL2 nucleic acid molecule. Therefore, the signal sequence is C
It may be homologous (natural) or heterologous to the HL2 nucleic acid molecule. Additionally, the signal sequence can be chemically synthesized using the methods described herein. In large part, secretion of the CHL2 polypeptide from the host cell due to the presence of the signal peptide results in removal of the signal peptide from the secreted CHL2 polypeptide. The signal sequence can be a component of the vector, or it can be part of the CHL2 nucleic acid molecule inserted into the vector.

Use of either a nucleotide sequence encoding a native CHL2 polypeptide signal sequence linked to a CHL2 polypeptide coding region or a nucleotide sequence encoding a heterologous signal sequence linked to a CHL2 polypeptide coding region is used in the invention. Within the range of. The heterologous signal sequence selected should be one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native CHL2 polypeptide signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, or thermostable enterotoxin II leaders. To be done. For yeast secretion, the native CHL2 polypeptide signal sequence may be replaced by the yeast invertase, alpha factor, or acid phosphatase leader. For mammalian cell expression, the native signal sequence is satisfactory, but other mammalian signal sequences may be suitable.

In some cases, where glycosylation is desired in eukaryotic host cell expression systems, various signal peptides (p
resource) can be manipulated. For example, the peptidase cleavage site of a particular signal peptide may be altered or pro-sequenced.
) Can be added, which can also affect glycosylation. The final protein product may have one or more additional amino acids at position 1 (relative to the first amino acid of the mature protein) associated with expression, which may not be completely removed. For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site attached to the amino terminus. Alternatively, some enzyme cleavage sites may result in a slightly truncated form of the desired CHL2 polypeptide if the enzyme is cleaved at such regions within the mature polypeptide.

In many cases, transcription of the nucleic acid molecule is increased by the presence of one or more introns within the vector; which results in the polypeptide being produced in a eukaryotic host cell, particularly a mammalian host cell. This is especially true when The intron used may be naturally present in the CHL2 gene, especially if the gene used is the full-length genomic sequence or a fragment thereof. If the intron is not naturally present in the gene (for most cDNAs), the intron may be obtained from another source. The position of the intron with respect to flanking sequences and the CHL2 gene is generally important as the intron must be transcribed effectively. Therefore,
When the CHL2 cDNA molecule is transcribed, the preferred position of the intron is 3'to the transcription start site and 5'to the poly-A transcription termination sequence. Preferably,
Introns are placed on one or the other side of the cDNA (ie, 5'or 3 ') so as not to interfere with the coding sequence. The invention can be practiced with any intron from any source, including viruses, prokaryotes and eukaryotes (plants or animals), provided that the intron is present in the host cell into which it is inserted. It is compatible. Synthetic introns are also included herein. If desired, more than one intron can be used in the vector.

Expression and cloning vectors of the invention will typically contain a promoter that is recognized by the host organism and is operably linked to the molecule encoding the CHL2 polypeptide. A promoter is a non-transcribed sequence located upstream (ie, 5 ') to the start codon of a structural gene (generally about 100 to 1000 bp) that controls the transcription of the structural gene. Promoters are usually grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some changes in culture conditions (eg, the presence or absence of nutrients, or changes in temperature). On the other hand, constitutive promoters initiate continuous gene product production; that is, little or no regulation of the gene for gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to the DNA encoding the CHL2 polypeptide by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector. The native CHL2 promoter sequence is used to amplify and / or amplify a CHL2 nucleic acid molecule.
Or it can be used to direct expression. However, a heterologous promoter is preferred if it allows for higher transcription and higher production of expressed proteins as compared to the native promoter, and is compatible with the host cell line chosen for use.

Suitable promoters for use with prokaryotic hosts include the β-lactamase and lactose promoter systems; alkaline phosphatase; tryptophan (trp).
) Promoter system; and hybrid promoters (eg tac promoter). Other known bacterial promoters are also suitable. These sequences are publicly available so that one of skill in the art will ligate them to the desired DNA sequence using linkers or adapters required to supply any useful restriction sites. You can

Suitable promoters for use with yeast hosts are also well known in the art.
Yeast enhancers are advantageously used for use with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, polyoma virus, fowlpox virus, adenovirus (eg, Adenovirus 2),
Bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferred simian virus 40 (SV40
) Such as a promoter obtained from the genome of a virus. Other suitable mammalian promoters include heterologous mammalian promoters (eg, heat shock promoter and actin promoter).

Additional promoters that may be of interest in controlling CHL2 gene expression include, but are not limited to: SV40 early promoter region (Bern).
oist and Chamber, 1981, Nature 290: 304.
-10); CMV promoter; promoter contained in the 3'long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787).
-97); herpes thymidine kinase promoter (Wagner et al., 1981).
, Proc. Natl. Acad. Sci. U. S. A. 78: 1444-45
); Regulatory sequences of the metallothionein gene (Brinster et al., 1982, Na
296: 39-42); a prokaryotic expression vector such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. N.
atl. Acad. Sci. U. S. A. , 75: 3727-31); or t
ac promoter (DeBoer et al., 1983, Proc. Natl. Acad.
Sci. U. S. A. , 80: 21-25). Also of interest are the following animal transcriptional regulatory regions that exhibit tissue specificity and have been utilized in transgenic animals: Elastase I gene regulatory region active in pancreatic acinar cells (Swift).
Et al., 1984, Cell 38: 639-46; Ornitz et al., 1986, C.
old Spring Harbor Symp. Quant. Biol. Fifty
: 399-409 (1986); MacDonald, 1987, Hepato.
7: 425-515); an insulin gene regulatory region active in pancreatic β cells (Hanahan, 1985, Nature 315: 115-22).
); Immunoglobulin gene regulatory region active in lymphocytes (Grossche)
dl et al., 1984, Cell 38: 647-58; Adamesra, 198.
5, Nature 318: 533-38; Alexander et al., 1987 ,.
Mol. Cell. Biol. , 7: 1436-44); mouse mammalian tumor virus regulatory region (Led) active in testis, breast, lymphocytes, and mast cells.
er et al., 1986, Cell 45: 485-95); the albumin gene regulatory region active in liver (Pinkert et al., 1987, Genes and D).
evel. 1: 268-76); the α-fetoprotein gene regulatory region active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.,
5: 1639-48; Hammer et al., 1987, Science 235: 5.
3-58); α1-antitrypsin gene regulatory region active in liver (Kels
ey et al., 1987, Genes and Devel. 1: 161-71); β-globin gene regulatory region active in myeloid cells (Mogram et al., 198).
5, Nature 315: 338-40; Kollias et al., 1986, Ce.
ll 46: 89-94); a myelin-based protein gene regulatory region active in oligodendrocytes of the brain (Readhead et al., 1987, Cell 4).
8: 703-12); myosin light chain-2 gene regulatory region active in skeletal muscle (
Sani, 1985, Nature 314: 283-86); and the gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mason et al.,
1986, Science 234: 1372-78).

Enhancer sequences may be inserted into the vector to increase transcription of the CHL2 polypeptide-encoding DNA of the present invention by higher eukaryotes. Enhancers act on promoters to increase transcription, usually about 10-3.
It is a cis-acting element of 00 bp long DNA. The enhancer is
Independent of relative orientation and position. These were found 5'and 3'to the transcription unit. Several enhancer sequences available from mammalian genes are known (eg, globin, elastase, albumin, α-fetoprotein and insulin). However, typically viral-derived enhancers are used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer are exemplary enhancing elements for activation of eukaryotic promoters. Enhancers can be spliced into the vector at the 5'or 3'positions with respect to the CHL2 nucleic acid molecule, but are typically located 5'to the promoter.

The expression vector of the present invention may be constructed from a starting vector such as a commercially available vector. Such a vector may or may not contain all desired flanking sequences. If one or more of the flanking sequences described herein are not already in the vector, then they can be obtained individually and ligated into the vector. The methods used to obtain each of the flanking sequences are well known to those of skill in the art.

Preferred vectors for carrying out the present invention include bacterial host cells, insect host cells,
And a vector compatible with mammalian host cells. Such vectors include, among others, pCRII, pCR3, and pcDNA3.1 (Invitrog
en, San Diego, CA), pBSII (Stratagene, La)
Jolla, CA), pET15 (Novagen, Madison, WI)
, PGEX (Pharmacia Biotech, Piscataway, N
J), pEGFP-N2 (Clontech, Palo Alto, CA), p
ETL (BlueBacII, Invitrogen), pDSR-alpha
(PCT Publication No. WO 90/14363) and pFastBacDual
(Gibco-BRL, Grand Island, NY).

[0131]   Further suitable vectors include, but are not limited to, cosmids, plasmids, or
Or modified viruses, these vector systems can be used with selected host cells.
It is understood that and must be compatible. Such vectors include:
Without limitation, a Bluescript® plasmid derivative (high copy
Number ColEl-based phagemid, Stratagene Cloning
  Systems, La Jolla CA), Taq amplification PCR product
PCR cloning plasmids designed to ^TM   TA Cloning (registered trademark) Kit, PCR2.1 (registered trademark)
Rasmid derivatives, Invitrogen, Carlsbad, CA), and
Mammalian vector such as baculovirus expression system, yeast vector or virus
Svector (pBacPAK plasmid derivative, Clontech, Palo
Alto, CA).

After the vector has been constructed and the nucleic acid molecule encoding the CHL2 polypeptide has been inserted into the vector at an appropriate site, the complete vector can be inserted into a host cell suitable for amplification and / or polypeptide expression. . Transformation of expression vectors for CHL2 polypeptides into selected host cells can be accomplished by methods such as transfection, infection, calcium chloride, electroporation, microinjection, lipofectin, DEAE-dextran, or other known techniques. Can be achieved by known methods including. The method selected will be, in part, a function of the type of host cell used. These and other suitable methods are well known to those of skill in the art and are described, for example, in Sambrook et al., Supra.

The host cell can be a prokaryotic host cell (eg, E. coli) or a eukaryotic host cell (eg, yeast cell, insect cell, or vertebrate cell). The host cell, when cultured under suitable conditions, synthesizes the CHL2 polypeptide, which is subsequently from the culture medium (when the host cell secretes the polypeptide into the medium).
It can be harvested or directly from the host cell producing the polypeptide (if the polypeptide is not secreted). Selection of the appropriate host cell will depend on various factors such as desired expression levels, polypeptide modifications desired or necessary for activity (eg, glycosylation or phosphorylation), and ease of folding into biologically active molecules. Depends on factors.

Many suitable host cells are known in the art, many of which are American.
an Type Culture Collection (ATCC), Man
available from Assass, VA. Examples include, but are not limited to, Chinese hamster ovary cells (CHO), CHO DHFR (−) cells (Urlaub).
Et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 97: 4
216-20), human embryonic kidney (HEK) 293 or 293T cells, or mammalian cells such as 3T3 cells. Methods for selection and transformation, culture, amplification, screening, product production, and purification of suitable mammalian host cells are known in the art. Other suitable mammalian cell lines are monkey COS
-1 and COS-7 cell lines, and CV-1 cell line. Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines derived from in vitro cultures of primary tissues, as well as primary explants are also suitable. Candidate cells are
The selection gene may be genotypically deficient or may include a preferentially acting selection gene. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, Swiss-derived 3
Examples include T3 strain, Balb-c or NIH mouse, BHK or Hak hamster cell line. Each of these cell lines is known and available to those of skill in the art of protein expression.

Similarly, bacterial cells are useful as suitable host cells for the present invention. For example, E
． coli (eg, HB101, DH5α, DH10, and MC1061)
Various strains of S. alba are well known as host cells in the field of biotechnology. B. su
btilis, Pseudomonas spp. , Other Bacillus
pp. , Streptomyces spp. Various strains such as can also be used in the method.

Many strains of yeast cells known to those of skill in the art are also available as host cells for expression of the polypeptides of the invention. Examples of preferable yeast cells include Sa
ccharomyces cerevisiae and Pichia pasto
ris is mentioned.

Furthermore, if desired, insect cell lines can be utilized in the methods of the invention.
Such systems are described, for example, in Kitts et al., 1993, Biotechnique.
s, 14: 810-17; Lucklow, 1993, Curr. Opin. B
iotechnol. 4: 564-72; and Lucklow et al., 1993,
J. Virol. , 67: 4566-79. Preferred insect cells are
Sf-9 and Hi5 (Invitrogen).

Transgenic animals can also be used to express glycosylated CHL2 polypeptides. For example, transgenic milk-producing animals (eg, cows or goats) can be used to obtain the glucosylated polypeptides of the invention in animal milk. Plants may also be used to produce CHL2 polypeptides, but in general, the glycosylation that occurs in plants is different from that produced in mammalian cells and is not suitable for human therapy. Can occur.

Polypeptide Production Host cells containing the CHL2 polypeptide expression vector can be cultured using standard media well known to those of skill in the art. This medium usually contains all the nutrients necessary for cell growth and survival. E. Suitable media for culturing E. coli cells include, for example, Luria Broth (LB) and / or Terrific
Broth (TB) is mentioned. Suitable media for culturing eukaryotic cells include the Roswell Park Memorial Institute.
medium 1640 (RPMI 1640), Minimal Ese
ntial Medium (MEM) and / or Dulbecco's M
modified Eagle Medium (DMEM), all of which can be supplemented with serum and / or growth factors required for the particular cell line being cultured. Appropriate media for insect cells are yeast rate (y
Eatate), lactalbumin hydrolysates, and / or Grace's medium supplemented with fetal bovine serum.

Typically, antibiotics or other compounds useful for the selective growth of transfected or transformed cells are added to the medium as a supplement.
The compound used is detectable by the selectable marker element present on the plasmid with which the host cell is transformed. For example, if the selectable marker element is kanamycin resistance, the compound added to the culture medium is kanamycin. Other compounds for selective growth include ampicillin, tetracycline, and neomycin.

The amount of CHL2 polypeptide produced by a host cell can be evaluated using standard methods known in the art. Such methods include, but are not limited to, Western blot analysis, SDS-polyacrylamide gel electrophoresis,
Activity assays such as non-denaturing gel electrophoresis, high performance liquid chromatography (HPLC) separations, immunoprecipitation, and / or DNA binding gel migration assays are included.

If the CHL2 polypeptide is designed to be secreted from the host cell, the majority of the polypeptide may be found in cell culture medium. However, if the CHL2 polypeptide is not secreted from the host cell, it is present in the cytoplasm and / or nucleus (for eukaryotic host cells) or in the cytosol (for Gram-negative bacterial host cells).

For CHL2 polypeptides located in the host cell cytoplasm and / or nucleus (for eukaryotic host cells) or in the cytosol (for bacterial host cells), intracellular material (inclusion bodies for Gram-negative bacteria). Can be extracted from the host cells using any standard technique known to those of skill in the art. For example, host cells can be lysed to release their periplasm / cytoplasmic content by French press, homogenization, and / or sonication followed by centrifugation.

When the CHL2 polypeptide forms inclusion bodies within the cytosol, the inclusion bodies can often be associated with inner and / or outer cell membranes and are therefore primarily found in pellet material after centrifugation. The pellet material can then be treated at pH extremes or at an alkaline pH in the presence of a reducing agent such as dithiothreitol or at an acidic pH in the presence of triscarboxyethylphosphine.
Can be treated with a chaotropic agent, such as a detergent, guanidine, a guanidine derivative, urea, or a urea derivative, to release, cleave, and dissolve the inclusion bodies. The lysed CHL2 polypeptide can then be analyzed using gel electrophoresis, immunoprecipitation and the like. When it is desired to isolate a CHL2 polypeptide, isolation is described herein and in Marston et al., 1990, Meth. En
z. , 182: 264-75, can be achieved using standard methods.

In some cases, the CHL2 polypeptide may not be biologically active upon isolation. "Refolding," that is, a variety of methods for converting a polypeptide into its tertiary structure and creating disulfide linkages, can be used to duplicate biological activity. Such methods include the step of exposing the solubilized polypeptide to a pH (usually above 7) and the presence of a particular concentration of chaotropic agent. The choice of chaotropic agent is very similar to the options used for inclusion body lysis, but usually chaotropic agents are used at low concentrations and are not necessarily the same as the chaotropic agents used for lysis. Often, the refolding / oxidation solution also contains a reducing agent, or a specific ratio of reducing agent and its oxidized form, to generate a specific redox potential, resulting in disulfide shuffling that results in the formation of cysteine bridges in the protein. To enable. Some commonly used redox couples include cysteine / cystamine, glutathione (GSH) / dithiobisGS
H, copper (II) chloride, dithiothreitol (DTT) / dithian DTT, and 2,2-mercaptoethanol (bME) / dithio-b (ME).
In many cases, cosolvents may be used or may be necessary to increase the efficiency of refolding, and a more common reagent used for this purpose is glycerol, of various molecular weights. Examples thereof include polyethylene glycol and arginine.

When inclusion bodies are not formed to a significant extent in the expression of CHL2 polypeptide, the polypeptide is found primarily in the supernatant after centrifugation of cell homogenates. The polypeptide can be further isolated from the supernatant using methods as described herein.

Purification of CHL2 polypeptides from solution can be accomplished using a variety of techniques. The polypeptide is hexahistidine (CHL2 polypeptide / hexaHi
s) or other small peptides such as FLAG (Eastma).
n Kodak Co. , New Haven, CT) or myc (Invi
trogen, Carlsbad, Calif.)) was synthesized in one step by passing the solution through an affinity column having a high affinity for the tag when the column matrix was synthesized to include either at its carboxy or amino terminus. .

For example, polyhistidine binds to nickel with great affinity and specificity. Therefore, nickel affinity columns (eg Qiage
n (R) nickel column) can be used for the purification of CHL2 polypeptide / polyHis. For example, Current Protocols i
n Molecular Biology § 10.11.8 (Ausube
l et al., eds. , Green Publishers Inc. and Wi
See ley and Sons 1993).

In addition, CHL2 polypeptides can be purified by the use of monoclonal antibodies that can specifically recognize and bind the CHL2 polypeptide.

Other suitable means for purification include, but are not limited to, affinity chromatography, immunoaffinity chromatography, ion exchange chromatography, molecular sieve chromatography, HPLC, electrophoresis (native gel electrophoresis). , Followed by gel elution and preparative isoelectric focusing (“Isoprime” machine / technology, Hoefer Scientific).
c, San Francisco, CA). In some cases, two or more purification techniques may be combined to achieve increased purity.

CHL2 polypeptides have also been described by Merrifield et al., 1963, J. Am. Am
． Chem. Soc. 85: 2149; Houghten et al., 1985, Pro.
c Natl Acad. Sci. USA 82: 5132; and Stewa.
rt and Young, Solid Phase Peptide Synth
chemistry (Pierce Chemical Co. 1984), using techniques known in the art, such as chemical synthesis methods (eg, solid phase peptide synthesis). Such polypeptides can be synthesized with or without a methionine at the amino terminus. Chemically synthesized CHL2 polypeptides can be oxidized using the methods described in these references to form disulfide bridges. A chemically synthesized CHL2 polypeptide is expected to have comparable biological activity to the corresponding CHL2 polypeptide that is recombinantly produced or purified from natural sources, and thus recombinant. Alternatively it can be used interchangeably with the native CHL2 polypeptide.

Another means of obtaining CHL2 polypeptides is by purification from biological samples such as tissue and / or fluids of the source in which the CHL2 polypeptides are naturally found. Such purification can be performed using methods for protein purification as described herein. During purification, the presence of CHL2 polypeptides can be monitored, for example, using antibodies prepared against recombinantly produced CHL2 polypeptides or peptide fragments thereof.

Many additional methods for producing nucleic acids and polypeptides are known in the art, and this method can be used to produce polypeptides with specificity for CHL2 polypeptides. For example, Roberts et al., 19
97, Proc. Natl. Acad. Sci. U. S. A. 94: 12297
See -303. This describes the production of fusion proteins between mRNA and its encoded peptide. Roberts, 1999, Curr. Op
in. Chem. See also Biol 3: 268-73. In addition, US Pat. No. 5,824,469 describes methods for obtaining oligonucleotides capable of performing particular biological functions. This procedure involves generating a heterogeneous pool of oligonucleotides, each having a 5'randomized sequence, a central preselected sequence, and a 3'randomized sequence. The resulting heterologous pool is introduced into a population of cells that do not exhibit the desired biological activity. Subpopulations of cells are then screened for those exhibiting the desired biological function. From that subpopulation, oligonucleotides that can perform the desired biological function are isolated.

US Pat. Nos. 5,763,192; 5,814,476; 5,72.
3,323; and 5,817,483 describe processes for producing peptides or polypeptides. This is accomplished by producing stochastic genes or fragments thereof and then introducing those genes into host cells that produce one or more proteins encoded by the stochastic genes. The host cell is then screened to identify those clones that produce the peptide or polypeptide with the desired activity.

Another method for producing a peptide or polypeptide is Athersys
, Inc. PCT / US98 / 20094 (WO99 / 15
650) ("Random Activation of Gene Expr
(known as "session for Gene Discovery" (known as RAGE-GD)), which involves activation of endogenous gene expression or gene overexpression by in situ recombination methods. For example, expression of an endogenous gene is activated or increased by incorporating regulatory sequences into target cells that may activate expression of the gene by heterologous or aberrant recombination. This target DNA is first subjected to radiation and then inserted into the gene promoter. This promoter is finally placed in front of the gene and initiates transcription of the gene. This results from the expression of the desired peptide or polypeptide.

These methods can be used to generate a comprehensive CHL2 polypeptide expression library, which can be followed by a variety of assays (eg, biochemical, cellular and whole organism assays (eg. It is understood that it can be used for high throughput phenotypic screening, eg in plants, mice etc.)).

(Synthesis) It will be appreciated by those of skill in the art that the nucleic acid and polypeptide molecules described herein can be produced by recombinant and other means.

Selective Binding Agent The term “selective binding agent” refers to a molecule that has specificity for one or more CHL2 polypeptides. Suitable selective binding agents include, but are not limited to, antibodies and their derivatives, polypeptides, and small molecules. Suitable selective binding agents can be prepared using methods known in the art. Exemplary CHL2 polypeptide selective binding agents of the present invention are capable of binding a particular portion of a CHL2 polypeptide, thereby inhibiting the binding of the polypeptide to the CHL2 polypeptide receptor.

Selective binding agents such as antibodies and antibody fragments that bind CHL2 polypeptides are within the scope of the invention. This antibody is polyclonal, including monospecific polyclonal; monoclonal (MAb); recombinant; chimeric; humanized (
For example, CDR grafting); human; single chain; and / or bispecific; and fragments; variants; or derivatives thereof. Antibody fragments include those portions of antibodies that bind to an epitope on the CHL2 polypeptide. Examples of such fragments include Fab and F (ab ′) 2 fragments produced by enzymatic cleavage of full length antibodies. Other binding fragments include fragments produced by recombinant DNA techniques (eg, expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions).

Polyclonal antibodies specific for CHL2 polypeptides are generally produced in animals (eg, rabbits or mice) by multiple subcutaneous or intraperitoneal injections of CHL2 polypeptide and adjuvant. It may be useful to couple the CHL2 polypeptide to a carrier protein, which protein
It is immunogenic in the species immunized, such as keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregating agents such as alum are used to enhance the immune response. Following immunization, the animals are bled and the serum is assayed for anti-CHL2 antibody titer.

Monoclonal antibodies specific for CHL2 polypeptides are produced using any method provided to produce antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies are the hybridoma method of Kohler et al., 1975, Nature 256: 495-97, and the human B cell hybridoma method (Kozbor, 1984, J. Immunol.
133: 3001; Brodeur et al., Monoclonal Antibod.
y Production Techniques and Applicat
ions 51-63 (Marcel Dekker, Inc., 1987)).
Is mentioned. Hybridoma cell lines that produce monoclonal antibodies that react with CHL2 polypeptides are also provided by the invention.

The monoclonal antibodies of the invention can be modified for use as therapeutic agents. One embodiment is a “chimeric” antibody, the heavy chain (H) and / or light chain (
Part of L) is derived from a particular species or is identical or homologous to the corresponding sequence in an antibody of a particular antibody class or subclass, while the rest of the chain Is derived from another species or is identical or homologous to the corresponding sequence in an antibody belonging to a particular antibody class or subclass. Also included are fragments of these antibodies so long as the fragments of the antibody exhibit the desired biological activity. U.S. Pat. No. 4,816,567; Morriso
n et al., 1985, Proc. Natl. Acad. Sci. 81: 6851-5
See 5.

In another embodiment, the monoclonal antibodies of the invention are "humanized" antibodies. Methods for humanizing non-human antibodies are well known in the art. See U.S. Patents 5,585,089 and 5,693,762. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Humanization has been described, for example, in the art (Jones et al., 1986, Nature 3
21: 522-25; Riechmann et al., 1998, Nature 332.
: 323-27; Verhoeyen et al., 1988, Science 239:
1534-36), using the method described in 1534-36).
By substituting at least a portion of a corresponding region of the human antibody.

Human antibodies that bind CHL2 polypeptides are also encompassed by the invention. Using a transgenic animal (eg, a mouse) that is capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin products, such an antibody is used to generate a CHL2 polypeptide antigen (ie, at least 6). Individual contiguous amino acids), and optionally conjugated to a carrier. For example, Jakobovits et al., 1993, Proc. Natl
． Acad Sci. 90: 2551-55; Jakobovits et al., 199.
3, Nature 362: 255-58; Bruggermann et al., 199.
3, Year in Immuno. See 7:33. In one method, such transgenic animals disable the endogenous loci encoding heavy and light chain immunoglobulins herein and insert human heavy and light chain proteins into their genome. Is produced by The partially modified animal, which has less than the full complement of variants, is then crossbreaded to obtain all of the desired variants of the immune system. When the immunogen is administered, these transgenic animals have an antibody with a human (eg, non-mouse) amino acid sequence, which amino acid sequence contains modified regions that are immunospecific for these antigens. To produce. For example, PCT application number PCT / US96
/ 05928 and PCT / US93 / 06926. Further methods are described in US Pat. No. 5,545,807, PCT Application No. PCT / US91 / 245.
And PCT / GB89 / 01207, and EP 546073B1 and 546073A1. Human antibodies can also be used to engineer recombinant DN in host cells.
It can be produced by expression of A or expression in hybridoma cells as described herein.

In an alternative embodiment, human antibodies can also be produced from phage display libraries (Hoogenboom et al., 1991, J. Mol. Biol.
． 227: 381; Marks et al., 1991, J. Am. Mol. Biol. 222:
581). These handle mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by binding to selected antigens. One such technique is PCT application number PCT
/ US98 / 17364, which describes the isolation of high affinity and functional antagonist antibodies for MPL- and msk-receptors using such an approach.

Chimeric, CDR-grafted, and humanized antibodies are typically produced by recombinant methods. Nucleic acid encoding the antibody is introduced into a host cell and expressed using the materials and procedures described herein. In a preferred embodiment, the antibody is treated in mammalian host cells (eg CHO cells). Monoclonal (eg, human) antibodies are as described herein.
It can be produced by expression of recombinant DNA in host cells or expression in hybridoma cells.

The anti-CHL2 antibodies of the invention can be used in any known assay method (eg, competitive binding assay, direct and indirect sandwich assay, and immunoprecipitation assay) for detection and quantification of CHL2 polypeptides. Can be used (S
ola, Monoclonal Antibodies: A Manual o
f Technologies 147-158 (CRC Press, Inc.,
1987)). This antibody binds the CHL2 polypeptide with an affinity that is appropriate for the assay method used.

[0168]   For diagnostic applications, in certain embodiments, the anti-CHL2 antibody is detectable
Can be labeled with different moieties. This detectable moiety is either direct or indirect
, And can be any moiety capable of producing a detectable signal. For example, detectable
This part is a radioactive isotope (for example,^ThreeH,¹⁴C,³²P,³⁵S,¹²⁵I, ⁹⁹ Tc,¹¹¹In, or⁶⁷Ga); fluorescent compound or chemiluminescent compound
(Fluorescein isothiocyanate, rhodamine, or luciferin),
Or enzymes (alkaline phosphatase, β-galactosidase, or hose
Radish peroxidase) (Bayer et al., 1990, Meth
． Enz. 184: 138-63).

Competitive binding assays rely on the ability of labeled standards (eg, CHL2 polypeptides, or immunologically active portions thereof) to test samples for binding to limited amounts of anti-CHL2 antibody. Compete with the sample (CHL2 peptide). The amount of CHL2 polypeptide in the test sample is inversely proportional to the amount of standard that binds the antibody. To facilitate determining the amount of standard that binds, the antibody is typically immunized before or after competition, and the standards and analytes that bind the antibody are separated from those that remain unbound. It can be conveniently separated.

The sandwich assay typically involves the use of two antibodies, each capable of binding to a different immune portion or epitope of the protein to be determined and / or quantified. In a sandwich assay, the test sample analyte is typically bound by a primary antibody that is immunized on a solid support, and then a secondary antibody is bound to the analyte to form a soluble three-part complex. Form. For example, US Patent No. 4
, 376, 110. The secondary antibody itself may be labeled with a detectable moiety (direct sandwich assay) or anti-labeled with a detectable moiety.
It can be measured using immunoglobulin antibodies (indirect sandwich assay). For example, one type of sandwich assay is the enzyme linked immunosorbent assay (ELISA), where the detectable moiety is an enzyme.

Selective binding agents, including anti-CHL2 antibodies, are also useful for in vivo imaging. An antibody labeled with a detectable moiety can be administered to an animal, preferably the bloodstream, and the presence and location of the labeled antibody in the host assayed. The antibody may be labeled with any moiety that is detectable in the animal, either by nuclear magnetic resonance, radiology, or other detection means known in the art.

Selective binding agents of the invention, including antibodies, can be used as therapeutic agents. These therapeutic agents are generally agonists or antagonists, which either enhance or decrease the biological activity of at least one CHL2 polypeptide, respectively. In one embodiment, an antagonist antibody of the invention specifically binds a CHL2 polypeptide and specifically binds a CHL2 polypeptide that is capable of inhibiting or eliminating the functional activity of a CHL2 polypeptide in vivo or in vitro. Antibody or binding fragment thereof. In a preferred embodiment, the selective binding agent (eg, antagonist antibody) is at least about 50%, and preferably at least about 80% CH.
Inhibits the functional activity of the L2 polypeptide. In another embodiment, the selective binding assay comprises a CHL2 polypeptide binding partner (ligand or receptor).
Is an anti-CHL2 polypeptide antibody capable of interacting with and thereby inhibiting or eliminating CHL2 polypeptide activity in vitro or in vivo. Selective binding agents, including agonist and antagonist antibody-CHL2 polypeptide antibodies, are identified by screening assays well known in the art.

The present invention also relates to kits containing CHL2 selective binding agents (eg, antibodies) and other reagents useful for detecting CHL2 polypeptide levels in biological samples. Such reagents may also include detectable labels that block serum, positive and negative control samples, and detection reagents.

Microarray It is understood that DNA microarray technology can be utilized in accordance with the present invention. DNA microarrays are small, high-density arrays of nucleic acids arranged on a solid support (eg glass). Each cell or element in the array contains multiple copies of a single nucleic acid species, which acts as a label for hybridizing with complementary nucleic acid sequences (mRNA). In expression profiles using DNA microarray technology, mRNA is first extracted from a cell or tissue sample and then converted to enzymatically fluorescently labeled cDNA. This material is hybridized to the microarray and unbound cDNA is removed by washing. The expression of the individual genes shown on this array is then visualized by quantifying the amount of labeled cDNA that is specifically bound to each target the nucleic acid molecule. In this way, expression of thousands of genes can be quantified in a high-throughput, parallel fashion from a single sample of biological material.

This high-throughput expression profile has widespread application in the context of the CHL2 molecules of the invention, including but not limited to: CHL2 disease as a target for treatment. Identification and confirmation of related genes; related CH
Molecular toxicity of the L2 molecule and its inhibitors; population and production stratification of surrogate markers for clinical trials; and aid in identifying selected compounds in high-throughput screens to identify relevant CHL2 polypeptide small molecule drug discoveries. To strengthen.

Chemical Derivatives Chemically modified derivatives of CHL2 polypeptides can be prepared by one of skill in the art, as this disclosure is described herein. CHL2 polypeptide derivatives are modified in different ways, either in the type or position of the molecules naturally attached to the polypeptide. Derivatives may include molecules formed by the removal of one or more naturally attached chemical groups. SEQ ID NO: 2, SEQ ID NO: 5, or other CHL
Polypeptides containing the amino acid sequence of any of the two polypeptides can be modified by covalent attachment of one or more polymers. For example, the selected polymers are typically water soluble so that the bound proteins do not precipitate in an aqueous environment (eg, physiological environment). Mixtures of polymers are included within the scope of suitable polymers. Preferably, for therapeutic use in the preparation of the desired product, the polymer is
It is pharmaceutically acceptable.

Each of the polymers can have any molecular weight and can be branched or unbranched. Each of the polymers typically has an average molecular weight of between about 2 kDa and about 100 kDa (the term "about" is used in the preparation of water-soluble polymers, with some higher than the above molecular weight). It has less weight). The average molecular weight of each polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 12 kDa and about 40 kDa, and most preferably about 20 kDa.
It is between Da and about 35 Da.

Suitable water-soluble polymers or mixtures thereof include, but are not limited to: N-linked or O-linked carbohydrates, sugars, phosphates, polyethylene glycol (PEG), which is mono-. (Including C ₁ -C ₁₀ ), alkoxy-, or aryloxy-polyethylene glycol, including forms of PEG used to derive proteins), monomethoxy-polyethylene glycol, dextran (eg, low molecular weight (eg, , Dextran) of about 6 kD), cellulose, or other carbohydrate-based polymers, poly- (N-vinylpyrrolidine) polyethylene glycols, propylene glycol homopolymers, polypropyleneoxy / ethylene oxide copolymers, polyoxyethylated polyols (eg Glycerol), and polyvinyl alcohol. Also included in the invention are bifunctional cross-linking molecules that can be used to prepare covalently linked CHL2 polypeptide multimers.

In general, chemical induction can be carried out under any suitable conditions used to react proteins with activated polymer molecules. A method for preparing a chemical derivative of a polypeptide comprises the following steps: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or any other CHL2 polypeptide, Reacting the activated polymer molecule (eg, a reactive ester or aldehyde derivative of the polymer molecule) with the polypeptide under conditions that bind to one or more polymer molecules, and (b) obtaining a reaction product. Optimal reaction conditions are determined based on known parameters and desired results. For example, the greater the ratio of polymer molecules to protein, the greater the percentage of polymer molecules bound. In one embodiment, the CHL2 polypeptide derivative has an amino-terminated single polymer molecular moiety. For example, US Pat. No. 5,234,784.
See issue.

Pegylation of a polypeptide can be specifically performed using any pegylation reaction known in the art. Such reactions include, for example,
It is described in the following references: Francis et al., 1992, Focus.
on Growth Factors 3: 4-10; European Patent 015431.
6 and 0401384; and U.S. Pat. No. 4,179,337. For example, pegylation can be carried out via an acylation or alkylation reaction with a reactive polyethylene glycol molecule (or similar reactive water-soluble polymer), as described herein. For the acylation reaction, the polymer selected has a single reactive ester group. For reductive alkylation, the polymer selected has a single reactive aldehyde group. For example, a reactive aldehyde, polyethylene glycol propionaldehyde, which is water soluble, or a mono C ₁ -C ₁₀ alkoxy or aryloxy derivative thereof (see US Pat. No. 5,252,714).

In another embodiment, the CHL2 polypeptide can be chemically linked to biotin. The biotin / CHL2 polypeptide molecule can then be bound to avidin, resulting in a tetravalent avidin / biotin / CHL2 polypeptide molecule. The CHL2 polypeptide may also be covalently linked to dinitrophenol (DNP) or chrysylnitrophenol (TNP), and the resulting conjugate may be precipitated with anti-DNP or anti-TNP-IgM and depleted at 10 valency. Form a body join.

Generally, conditions that can be alleviated or modulated by administration of a CHL2 polypeptide derivative of the invention include those described herein for CHL2 polypeptides. However, the CHL2 polypeptide derivatives disclosed herein have further activity, enhanced or decreased biological activity, when compared to underivatized molecules,
Or it may have other properties, such as increased or decreased half-life.

Genetically Engineered Non-Human Animals Non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other farm animals are further included within the scope of the invention, Where natural CH
The gene encoding the L2 polypeptide is disrupted (ie, "knocked out") and the expression level of the CHL2 polypeptide is significantly reduced or abolished. Such animals can be prepared using the techniques and methods as described in US Pat. No. 5,557,032.

The invention further includes non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other farm animals, in which the natural form or heterologous form of the CHL2 gene of the animal. Either sex CHL2 gene is overexpressed by the animal, thereby creating a "transgenic" animal. Such transgenic animals are described in US Pat. No. 5,489,743 and PCT Publication No. W.
It can be prepared using well known methods such as those described in O94 / 28122.

The invention further includes non-human animals, wherein the promoters of one or more CHL2 polypeptides of the invention are activated (eg, by using homologous recombination methods). , Or not activated, alters the level of expression of one or more native CHL2 polypeptides.

These non-human animals can be used for drug candidate screening. In such a screen, the effect of the drug candidate on the animal can be measured.
For example, the drug candidate may reduce or increase expression of the CHL2 gene. In certain embodiments, the amount of CHL2 polypeptide produced can be measured after exposing the animal to the drug candidate. Moreover, in certain embodiments, the actual effect of the drug candidate on the animal may be detected. For example, due to overexpression of certain genes,
Causes or is associated with a disease or pathological condition. In such cases, the ability of the drug candidate to reduce gene expression or the ability to prevent or inhibit a pathological condition may be tested. In another example, the production of a particular metabolite, such as a fragment of a polypeptide, results in or is associated with a disease or pathological condition. In such cases, the ability of the drug candidate to reduce the production of such metabolites or the ability to prevent or inhibit a pathological condition may be tested.

Assays for Other Modulators of CHL2 Polypeptide Activity In some situations, it may be desirable to identify molecules (eg, agonists or antagonists) that are modulators of CHL2 polypeptide activity.
Natural or synthetic molecules that modulate a CHL2 polypeptide can be identified using one or more screening assays, as described herein. Such molecules can be administered by injection, or oral delivery, implantation devices, etc., in either an in vitro or ex vivo manner.

A “test molecule” refers to a molecule that evaluates for its ability to modulate (ie, increase or decrease) the activity of a CHL2 polypeptide. Most commonly, the test molecule interacts directly with the CHL2 polypeptide. However, the test molecule can also be used, for example, by affecting CHL2 gene expression or in CHL2.
CHL2 polypeptide activity may be indirectly regulated by binding to two polypeptide binding partners, such as receptors or ligands. In one embodiment, the test molecule is at least about 10 ⁻⁶ M, preferably about 10 ⁻⁸ M,
More preferably, it binds to the CHL2 polypeptide with an affinity constant of about 10 ⁻⁹ M, and even more preferably about 10 ⁻¹⁰ M.

Methods for identifying compounds that interact with a CHL2 polypeptide are encompassed by the present invention. In certain embodiments, the CHL2 polypeptide is incubated with the test molecule under conditions that allow the test molecule to interact with the CHL2 polypeptide and the extent of interaction measured. Test molecules can be screened in a substantially purified form or crude mixture.

In certain embodiments, the CHL2 polypeptide agonist or antagonist can be a protein, peptide, carbohydrate, lipid, or low molecular weight molecule, which interacts with the CHL2 polypeptide to modulate its activity. To do. CH
A molecule that regulates expression of an L2 polypeptide is complementary to a nucleic acid encoding a CHL2 polypeptide, or a nucleic acid molecule that is complementary to a nucleic acid sequence that directs or controls expression of the CHL2 polypeptide, and expression Nucleic acid molecules that act as antisense regulators of.

Once a test compound has been identified once it interacts with the CHL2 polypeptide, the molecule can be further evaluated for its ability to increase or decrease CHL2 polypeptide activity. Measuring the interaction of a test molecule with a CHL2 polypeptide can be performed in several forms, including cell-based binding assays, membrane binding assays, solution phase assays, and immunoassays. Generally, the test molecule is incubated with the CHL2 polypeptide for a specified period of time and C
HL2 polypeptide activity is determined by one or more assays to measure biological activity.

Interactions between test molecules and CHL2 polypeptides can also be directly assayed using polyclonal or monoclonal antibodies in immunoassays. Alternatively, modified forms of CHL2 polypeptides that include an epitope tag as described herein can be used in solution and immunoassays.

In cases where a CHL2 polypeptide exhibits biological activity via interaction with a binding partner (eg, receptor or ligand), various in vitro assays have shown that the corresponding binding partner (selective binding agent, selective binding agent, It can be used to measure the binding of CHL2 polypeptides to receptors (or ligands). These assays can be used to screen test molecules to increase or decrease the rate and / or rate of binding of CHL2 polypeptides to binding partners. In one assay, CHL2 polypeptide is immobilized in the wells of a microtiter plate. Then radiolabeled CHL
The two polypeptide binding partners (eg, iodinated CHL2 polypeptide binding partner) and the test compound can be added to this well one at a time (in either order) or simultaneously. After incubation, the wells are washed and the radioactivity is counted using a scintillation counter to determine the extent to which the binding partner binds the CHL2 polypeptide. Typically, the molecule is
A series of control wells tested over a range of concentrations and lacking one or more of the test assays can be used for the accuracy of evaluation of the results. An alternative to this method is to reverse the "position" of the protein (ie immobilize the CHL2 polypeptide binding partner to the microtiter plate wells, incubate the test molecule and radiolabeled CHL2 polypeptide, and CHL2 Determining the extent of polypeptide binding). For example, Current
Protocols in Molecular Biology, Chapter 18 (
Ausubel et al., Ed. Green Publishers Inc. And W
See iley and Sons 1995).

As an alternative to radiolabelling, the CHL2 polypeptide or its binding partner can be conjugated with biotin and the presence of the biotinylated protein then detected by an enzyme such as wasabi peroxidase (HRP) or alkaline phosphatase. (AP)), which are detected colorimetrically, can be detected using streptavidin or can be detected by fluorescent tagging of streptavidin. Antibodies to CHL2 polypeptides or CHL2 polypeptide binding partners, which are bound to biotin, may also be used for detection purposes following incubation of the complex with enzyme-linked streptavidin linked to AP or HRP. Can be used for.

CHL2 polypeptides or CHL2 polypeptide binding partners can be immobilized by attachment to agarose beads, acrylic beads, or other types of such inert solid phase substrates. The substrate-protein complex can be placed in a solution containing the complementary protein and the test compound. After incubation, the beads can be pelleted by centrifugation and the amount of binding between the CHL2 polypeptide and its binding partner can be assessed using the methods described herein. Alternatively, the substrate-protein complex can be immobilized within the column with the test molecule and complementary protein passing through the column. The formation of complexes between the CHL2 polypeptide and its binding partner can then be assessed using any of the techniques described herein (eg, radiolabel or antibody binding).

Another in vitro assay useful for identifying test molecules that increase or decrease the formation of a complex between a CHL2 polypeptide binding protein and a CHL2 polypeptide binding partner is a surface plasmon resonance detector system (eg, , BI
Acore assay system (Pharmacia, Piscataway, N
J)). The BIAcore system is utilized as specified by the manufacturer. This assay is essentially a CHL2 polypeptide or CHL2
Covalent attachment of any of the polypeptide binding partners to the dextran coated sensor chip, which is present on the detector. The test compound and other complementary protein are then either simultaneously or sequentially
It can be injected into the chamber containing the sensor chip. The amount of complementary protein that binds can be assessed based on the change in mass of a molecule physically associated with the dextran-coated side of the sensor chip, which change in mass is measured by a detector system.

In some cases, it may be desirable to evaluate two or more test compounds together for their ability to increase or decrease the formation of a complex between a CHL2 polypeptide and a CHL2 polypeptide binding partner. Can be done. In these cases, the assays described herein can be readily modified by the addition of such additional test compounds, either concomitantly with or subsequent to the first test compound. The rest of the steps in this assay are described herein.

In vitro assays, such as those described herein, are used to screen large numbers of compounds for their effect on the formation of complexes between CHL2 polypeptides and CHL2 polypeptide binding partners. It can be used to advantage. These assays can be automated to screen compounds produced in phage display, synthetic peptides, and chemically synthesized libraries.

A compound that increases or decreases the formation of a complex between a CHL2 polypeptide and a CHL2 polypeptide binding partner may also be a CHL2 polypeptide or CH
Cells and cell lines expressing any of the L2 polypeptide binding partners F can be used to screen in cell culture. The cells and cell lines can be obtained from any mammal, but are preferably derived from humans or other primate, canine or rodent sources. The binding of the CHL2 polypeptide to cells expressing the CHL2 polypeptide binding partner at its surface is assessed in the presence or absence of the test compound and the degree of binding is determined, for example, to the CHL2 polypeptide binding partner. It can be determined by flow cytometry using a biotinylated antibody. Cell culture assays can be advantageously used to further evaluate compounds that score positive in the protein binding assays described herein.

Cell cultures can also be used to screen the effects of drug candidates. For example, the drug candidate may reduce or increase expression of the CHL2 gene. In certain embodiments, the amount of CHL2 polypeptide or CHL2 polypeptide fragment produced can be measured after exposure of the cell culture to the drug candidate.
In certain embodiments, the substantial effect of the drug candidate on cell culture can be detected. For example, overexpression of a particular gene can have a particular effect on cell culture. In such cases, the drug candidate's ability to increase or decrease gene expression, or its ability to prevent or inhibit a particular effect on cell culture, can be tested. In other examples, the production of particular metabolites (eg, fragments of polypeptides, etc.) can result in or be associated with a disease or pathological condition.
In such cases, the ability of drug candidates to reduce the production of such metabolites in cell culture can be tested.

Internalization Proteins The tat protein sequence (from HIV) can be used to internalize proteins into cells. For example, Falwell et al., 1994, Proc. N
atl. Acad. Sci. U. S. A. 91: 664-68. For example, the 11-amino acid sequence of the HIV tat protein (Y-G-R-K-K-
R-R-Q-R-R-R; SEQ ID NO: 10) ("protein transduction domain",
Or TAT PDT) has been described as mediating delivery across the cytoplasmic and nuclear membranes of cells. Schwarze et al., 1999, Scien.
285: 1569-72; and Nagahara et al., 1998, Nat.
． Med. 4: 1449-52. In these procedures, FITC
Construct (FITC labeled G-G-G-G-Y-G-R-K-K-R-R-Q-R-
RR; SEQ ID NO: 11), which penetrates tissue following intraperitoneal administration, was prepared and binding of such constructs to cells was determined by fluorescence cytometric separation (luor).
(escape activated cell sorting; FACS)
Detected by analysis. Cells treated with the tat-β-gal fusion protein show β-gal activity. Following injection, expression of such constructs can be detected in many tissues (liver, kidney, lung, heart and brain tissues). It is believed that such a construct may undergo some modification to enter the cell and may itself require refolding following import into the cell.

Thus, it is understood that tat protein sequences can be used to internalize a desired polypeptide into a cell. For example, using the tat protein sequence, a CHL2 antagonist (eg, anti-IL-lra-R selective binding agent,
Small molecule, soluble receptor, or antisense oligonucleotide)
It can be administered intracellularly to inhibit the activity of the L2 molecule. As used herein, the term “CHL2 molecule” refers to both CHL2 nucleic acid molecules and CHL2 polypeptides, as defined herein. If desired, the CHL2 protein itself can also be administered intracellularly using these procedures. Stra
See also us, 1999, Science 285: 1466-67.

Identification of Cell Sources Using CHL2 Polypeptides In accordance with certain embodiments of the invention, it is useful to be able to determine the source of particular cell types associated with CHL2 polypeptides. obtain. For example, it may be useful to determine the origin of a disease or pathological condition as an aid in selecting an appropriate treatment. In particular embodiments, nucleic acids encoding CHL2 polypeptides can be used as probes to identify the cells described herein by screening cellular nucleic acids with such probes. In other embodiments, anti-CHL2 polypeptide antibodies are used to detect CHL2 in cells.
One can test for the presence of the polypeptide, and thus determine whether such cells are of the type described herein.

CHL2 Polypeptide Compositions and Administration Therapeutic compositions are within the scope of this invention. Such a CHL2 polypeptide pharmaceutical composition comprises a therapeutically effective amount of a CHL2 polypeptide or CHL2 nucleic acid molecule with a pharmaceutically or physiologically acceptable prescription agent selected to be compatible with the mode of administration. It may be included in the mixture. The pharmaceutical composition comprises a therapeutically effective amount of one or more CHL.
The two polypeptide selective binding agents may be included in admixture with a pharmaceutically or physiologically acceptable formulation agent selected to be compatible with the mode of administration.

Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed.

A pharmaceutical composition may, for example, modify pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissociation or release, adsorption, or penetration of the composition. ,
It may include formulation materials for maintenance or storage. Suitable formulation materials include:
These include, but are not limited to: amino acids (eg, glycine, glutamine, asparagine, arginine, or lysine), antibacterial agents, antioxidants (eg, ascorbic acid, sodium sulfite, or sodium bisulfite (so.
), buffer (eg borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acid).
, Bulking agents (eg mannitol or glycine), chelating agents (ethylenediaminetetraacetic acid (EDTA)), complexing agents (eg caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin). , Fillers, monosaccharides, disaccharides, and other carbohydrates (eg glucose, mannose or dextrin), proteins (eg serum albumin, gelatin, or immunoglobulins), colorants, flavoring and diluents, emulsifiers, hydrophilic Polymers (eg polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (eg sodium), preservatives (eg benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propyiene. Luparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (eg, glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (eg, mannitol or sorbitol)
, Suspending agents, surface-active agents or wetting agents (eg pluronic
); PEG; sorbitan ester; polysorbate (for example, polysor
bate 20 or polysorbate 80); triton; tromethamine; lecithin; cholesterol or tyloxapol).
, Stability enhancers (eg sucrose or sorbitol), tonicity enhancers (eg alkali metal halides (preferably sodium chloride or potassium chloride)
, Or mannitol, sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Remington's Pharmaceu
Tal Sciences (18th Edition, AR Gennaro, Ed., Ma
See ck Publishing Company 1990.

Optimal pharmaceutical compositions will be determined by those of skill in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. For example, Remington
See's Pharmaceutical Sciences, supra.
Such compositions may affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the CHL2 molecule.

The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection is water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. There is. Neutral buffered saline or saline mixed with serum albumin is a further exemplary vehicle. Other exemplary pharmaceutical compositions have a Tr of about pH 7.0-8.5.
is buffer or acetate buffer at about pH 4.0-5.5, which further comprises
It may include sorbitol or a suitable substitute. In one embodiment of the invention, the CHL2 polypeptide composition provides a selected composition having the desired degree of purity in any prescription drug (Remington's Pharmaceutical).
It can be prepared for storage in the form of a lyophilized cake or an aqueous solution by mixing with Sciences, supra). Further, the CHL2 polypeptide product can be formulated as a lyophilizate using appropriate excipients such as sucrose.

The CHL2 polypeptide pharmaceutical composition may be selected for parenteral delivery. Alternatively, these compositions may be selected for inhalation or delivery via the gastrointestinal tract (eg, orally). Preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation components are present in concentrations that are acceptable at the site of administration. For example, buffers are used to maintain the composition at physiological pH or slightly lower pH (typically within the pH range of about 5 to about 8).

When intended for parenteral administration, therapeutic compositions for use in the present invention are pyrogen-free, parenterally acceptable containing a desired CHL2 molecule in a pharmaceutically acceptable vehicle. It can be in the form of an aqueous solution. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the CHL2 molecule is formulated as a sterile, isotonic solution, and stored properly. Yet another preparation is the desired molecule and drug (eg, injectable microspheres, bio-erodible drugs, polymeric compounds (polylactic or polyglycolic acid), beads or liposomes).
With a formulation that provides a controlled or sustained release of the product that can then be delivered via depot injection. Hyaluronic acid can also be used,
And this can have the effect of promoting a sustained duration in the circulation.
Other suitable means for introduction of the desired molecule include implantable drug delivery devices.

[0212] In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, the CHL2 polypeptide can be formulated as a dry powder for inhalation. CH
Inhalation solutions of L2 polypeptides or nucleic acid molecules can also be formulated with a nebulizer for aerosol delivery. In yet another embodiment, the solution may be nebulized.
Pulmonary administration is further described in PCT Publication No. WO94 / 20069, which describes pulmonary delivery of chemically modified proteins.

It is also contemplated that certain formulations may be administered orally. In one embodiment of the invention, the CHL2 polypeptide administered in this manner is formulated with or without carriers conventionally used in the formulation of solid dosage forms (eg tablets or capsules). obtain. For example, capsules may be designed to release the active portion of the formulation at a point within the gastrointestinal tract when bioavailability is maximized and presystemic degradation is minimized. Further drugs are CHL
It may be included to facilitate absorption of the two polypeptides. Diluents, seasonings, low melting waxes, vegetable oils, lubricants, suspensions, tablet disintegrating agents, and binders are also
Can be used.

Another pharmaceutical composition may include an effective amount of CHL2 polypeptide in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents (calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate); or binders (eg starch, gelatin or acacia). ); Or a lubricant (eg magnesium stearate, stearic acid or talc)
.

Additional CHL2 polypeptide pharmaceutical compositions will be apparent to those of skill in the art and include formulations that include CHL2 polypeptides in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible microspheres or porous beads and depot injections, are also provided.
It is known to those skilled in the art. See, eg, PCT / US93 / 00829, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions.

Further examples of sustained release formulations include semipermeable polymer matrices in the form of shaped articles, eg films, or microcapsules. Sustained-release matrices include polyesters, hydrogels, polylactides (US Pat. No. 3,773,919 and EP 058481), copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., 1983, Bi.
Polymers 22: 547-56), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater.
Res. 15: 167-277 and Langer, 1982, Chem. Te
ch. 12: 98-105), ethylene vinyl acetate (Langer et al., Supra) or poly-D (-)-3-hydroxybutyric acid (European Patent No. 133988).
. Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. For example, Eppstein et al., 19
85, Proc. Natl. Acad. Sci. USA 82: 3688-92.
European Patent Nos. 0366676, 088046, and 1439;
See No. 49.

CHL2 pharmaceutical compositions used for in vivo administration typically must be sterile. This can be accomplished by filtration through a sterile filtration membrane. Where the composition is lyophilized, sterilization using this method can be performed either before, or subsequent to lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in solution. In addition, parenteral compositions are generally placed in a container having a sterile access port such as an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form that requires reconstitution prior to administration (lyophilized form).

In a particular embodiment, the present invention relates to a kit for producing a single dose dosage unit. The kit may each include both a first container having a dry protein and a second container having an aqueous formulation. Also included within the scope of the invention are kits including single and multi-chambered pre-filled syringes (eg, liquid syringes and lyosyringes).

The effective amount of the pharmaceutical composition of CHL2 used therapeutically depends, for example, on the therapeutic content and purpose. Those of ordinary skill in the art will appreciate that the appropriate dosage level for treatment will be
Depends in part on the molecule delivered, the indication that the CHL2 molecule is used, the route of administration, and the size (body weight, body surface, or organ size) and condition (age and general health) of the patient. And understand that it changes. Thus, the clinician may titer the dosage and modify the route of administration in order to obtain the optimal therapeutic effect. A typical dosage will range from about 0.1 μg / kg to depending on the factors mentioned above.
It can range up to about 100 mg / kg or more. In other embodiments, the dosage is from 0.1 μg / kg to about 100 mg / kg; or 1 μg / kg to about 10
It can range up to 0 mg / kg; or up to 5 μg / kg up to about 100 mg / kg.

The frequency of dosing will depend on the pharmacokinetic parameters of the CHL2 molecule in the formulation used. Typically, the clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition, and therefore the composition, as a single dose over time,
It can be administered as two or more doses, which may or may not contain the same amount of the desired molecule, or as a continuous infusion through an implantation device or catheter. Further refinement of the appropriate dosage is well within the scope of the problems routinely made and carried out by those skilled in the art. The proper dosage is
It can be confirmed through the use of appropriate dose-response data.

The route of administration of the pharmaceutical composition is consistent with known methods as follows: orally; intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular. , Via an intraarterial, intraportal, or intralesional route of injection; by a sustained release system; or by an implantable device. If desired, these compositions can be administered by bolus injection, or continuously by infusion, or by implantation device.

Alternatively or additionally, the composition may be administered locally via implantation of a membrane, sponge, or other suitable material onto which the desired molecule is absorbed or encapsulated. If an implant device is used, the device can be implanted in any suitable tissue or organ and delivery of the desired molecule can be via diffusion, timed release bolus or continuous administration.

In some cases, it may be desirable to use pharmaceutical compositions of CHL2 polypeptides in an ex vivo manner. In such instances, cells, tissues, or organs removed from the patient are exposed to the CHL2 polypeptide after the cells, tissues, or organs have been subsequently transplanted back into the patient.

In other cases, the CHL2 polypeptide is genetically engineered to transplant specific cells expressing and secreting the CHL2 polypeptide using the methods described herein. Can be delivered. Such cells can be animal or human cells, and can be autologous, xenogeneic, or exogenous cells. If desired, the cells can be immortalized. To reduce the chance of an immunological response, cells can be encapsulated to avoid infiltration of surrounding tissue. The encapsulating material is typically a biocompatible, semi-permeable polymeric enclosure or membrane that allows the release of the protein product, but which is otherwise free of the patient's immune system or surrounding tissues. Prevents destruction of cells by harmful factors.

As discussed herein, treating an isolated cell population (eg, stem cells, lymphocytes, red blood cells, chondrocytes, neurons, etc.) with one or more CHL2 polypeptides comprises: May be desirable. This can be accomplished by exposing the isolated cells directly to the polypeptide when it is in a permeable form to the cell membrane.

Further embodiments of the invention include cells and methods (eg, homologous recombination and / or homologous recombination and / or) for both in vitro production of therapeutic polypeptides and production and delivery of therapeutic polypeptides by gene therapy or cell therapy. Or other recombinant production methods). Homologous recombination and other methods of recombination can be used to modify cells that normally contain a CHL2 gene that is silent for transcription (ie, an underexpressed gene), thereby producing a therapeutically effective amount of CHL2. Cells can be produced which express the polypeptide.

Homologous recombination was originally a technique developed to target genes to induce or correct mutations in transcriptionally active genes. Kucherl
apati, 1989, Prog. in Nucl. Acid Res. & M
ol. Biol. 36: 301. Because basic techniques introduce specific mutations into specific regions of the mammalian genome (Thomas et al., 1986, Cell 44: 4.
19-28; Thomas and Capecchi, 1987, Cell 51.
: 503-12; Doetschman et al., 1988, Proc. Natl. A
cad. Sci. U. S. A. 85: 8583-87), or to correct specific mutations in the defective gene (Doetschman et al., 1987, Natu.
Re 330: 576-78). Exemplary homologous recombination techniques are described in US Pat. Nos. 5,272,071; European Patents 9193051 and 505500; PCT / US90 / 07642, and PCT Publication No. WO 91/09955). .

By homologous recombination, the DNA sequence to be inserted into the genome is
By attaching to A, it can be directed to a specific region of the gene of interest. This target DNA is a nucleotide sequence that is complementary (homologous) to a region of genomic DNA. A small piece of target DNA complementary to a particular region of the genome is a DNA
During the replication process, it is contacted with the parent strand. This is a general property of hybridized DNA inserted into cells and, therefore, recombines with other fragments of endogenous DNA via shared homologous regions. If this complementary strand is attached to an oligonucleotide containing a mutation or a different sequence or additional nucleotides, it will also be incorporated into the newly synthesized strand as a result of this recombination.
As a result of the proofreading function, new sequences of DNA can serve as templates. Therefore, the transferred DNA is integrated into the genome.

DNA capable of interacting with CHL2 polypeptide or controlling its expression
Regions (eg, flanking sequences) attach to these fragments of the target DNA. For example, a promoter / enhancer element, suppressor, or exogenous transcriptional regulatory element is sufficient to affect the transcription of this DNA in the vicinity of the DNA encoding the desired CHL2 polypeptide, in the genome of the intended host cell. With a proper orientation,
Is inserted. The control elements control the portion of DNA present in the host cell genome. Thus, expression of the desired CHL2 polypeptide is not due to transfection of the DNA encoding the CHL2 gene itself, but rather CHL2.
This can be accomplished by the use of target DNA (including regions of homology with the endogenous gene of interest) linked to a DNA regulatory segment that provides the endogenous gene sequence with a recognizable signal for transcription of the two genes.

In an exemplary method, expression of a desired target gene (ie, a desired endogenous cellular gene) in a cell is achieved through at least regulatory sequences via homologous recombination into the cell genome at a preselected site, It is modified by the introduction of DNA containing exons and splice donor sites. These components are introduced into the chromosomal (genomic) DNA in such a way that they actually result in the production of new transcription units (where the regulatory sequences, exons, and splice donor sites present in the DNA construct are Operably linked to an endogenous gene). The introduction of these components into chromosomal DNA results in altered expression of the desired endogenous gene.

As described herein, altered gene expression can result in activation (or expression) of a gene that is normally silent (unexpressed) in cells that have been obtained, as well as It involves increased expression of genes that are not expressed at physiologically significant levels in intact cells. This embodiment further includes the step of altering the pattern of regulation or induction so that it differs from the pattern of regulation or induction that occurs in the as-obtained cells and in the as-obtained cells. Decrease (including eliminate) expression of the expressed gene.

One way in which phase-identifying recombination can be used to increase or cause the production of CHL2 polypeptide from the endogenous CHL2 gene of a cell is to first use homologous recombination. , A recombination sequence derived from a site-specific recombination system (eg,
Cre / loxP, FLP / FRT) (Sauer, 1994, Curr. Op.
in. Biotechnol. , 5: 521-2; Sauer, 1993, Me.
methods Enzymol. , 225: 890-900) to the endogenous C of cells.
Locating upstream of the HL2 polypeptide coding region (ie 5 '). A plasmid containing a recombination site homologous to a region located immediately upstream of the genomic CHL2 polypeptide coding region is introduced into the modified cell line along with the appropriate recombinase enzyme. This recombinase allows the plasmid to integrate through the recombination site of the plasmid into the recombination site located immediately upstream of the genomic CHL2 polypeptide coding region of the cell line (Bauboni).
s and Sauer, 1993, Nucleic Acids Res. 21:
2025-29; O'Garman et al., 1991, Science 251: 1.
351-55). Any flanking sequences known to increase transcription (eg, enhancers / promoters, introns, translational enhancers), when properly placed in this plasmid, will not be transferred to the endogenous CHL2 gene of the cell. Alternatively, they are combined in such a way as to create a new or modified transcription unit that results in increased CHL2 polypeptide production.

A further method using a cell line in which the site-specific recombination sequences are located immediately upstream of the cell's endogenous genomic CHL2 polypeptide coding region is a second recombination at another location in the genome of the cell line. The use of homologous recombination to introduce the site. The appropriate recombinant enzyme is then introduced into the two recombination site cell line to reduce recombination (
Deletion, conversion, and metastasis) (Sauer, 1994, Curr. Opin
． Biotechnol. , 5: 521-27; Sauer, 1993, Met.
hods Enxymol. , 225: 890-900), which creates new or modified transcription units that result in novel or increased CHL2 polypeptide production from the endogenous CHL2 gene of the cell.

A further approach to increasing or causing expression of a CHL2 polypeptide from the endogenous CHL2 gene of the cell is a modality that results in the production of new or increased CHL2 polypeptide from the endogenous CHL2 gene of the cell. And / or increasing the expression of the gene (s) (eg transcription factors) and / or decreasing the expression of the gene (s) (eg transcription repressors). This method uses non-naturally occurring polypeptides (
(Eg, a polypeptide containing a site-specific DNA binding domain fused to a transcription factor domain), a new or increased CHL from the endogenous CHL2 gene of the cell.
Introducing into the cell such that production of the 2 polypeptide occurs.

The present invention further relates to DNA constructs useful in the method of altering the expression of a target gene. In certain embodiments, a representative DNA construct comprises: (a) one or more target sequences, (b) regulatory sequences, (c) exons, and (d).
Unpaired splice donor site. The target sequence in the DNA construct is element (a)-
It directs the integration of (d) into the target gene in the cell so that these elements (b)-(d) are operably linked to the sequence of the endogenous target gene. In another embodiment, the DNA construct comprises: (a) one or more target sequences, (
b) regulatory sequences, (c) exons, (d) splice donor sites, (e) introns, and (f) splice acceptor sites. Here, the target sequence directs the integration of elements (a)-(f), so that these elements (b)-(
f) is operably linked to the endogenous gene. The target sequence is homologous to a preselected site in the cell chromosomal DNA where homologous recombination occurs. In this construct, the exon is generally 3'to the control sequences and the splice donor site is 3'to this exon.

If the sequence of a particular gene (eg, the nucleic acid sequence of a CHL2 polypeptide described herein) is known, the DN complementary to the selected region of the gene
The portion of A may be synthetic or otherwise obtained, such as by appropriate restriction of native DNA at a particular recognition site bound to the region of interest. This part, when inserted into the cell, serves as the target sequence and hybridizes to the homologous region within its genome. If this hybridization occurs during DNA replication, a portion of this DNA, and any additional sequences attached to it, will act as an Okazaki fragment and be incorporated into the newly synthesized daughter strand of the DNA. Thus, the invention includes a nucleotide encoding a CHL2 polypeptide, which nucleotide can be used as a target sequence.

CHL2 polypeptide cell therapy (eg, transplantation of cells that produce CHL2 polypeptide) is also contemplated. This embodiment provides a biologically active form of CHL.
2 transplanting cells capable of synthesizing and secreting the 2 polypeptide. Such a CHL2 polypeptide-producing cell may be a cell that is a natural product of the CHL2 polypeptide, or a recombinant cell in which the ability to produce the CHL2 polypeptide encodes a gene encoding the desired polypeptide. Alternatively, it may be a recombinant cell which has been expanded by transforming with a gene which increases the expression of CHL2 polypeptide. Such modifications can be accomplished by appropriate vectors to deliver the gene and promote its expression and secretion. The natural cells that produce the CHL2 polypeptide are of human origin when produced with the administration of a heterologous polypeptide to minimize a strong immunological response in patients receiving the CHL2 polypeptide, and It is preferred to produce human CHL2 polypeptide. Similarly, recombinant cells producing a CHL2 polypeptide are preferably transformed with an expression vector containing a gene encoding a human CHL2 polypeptide.

Transplanted cells can be encapsulated to avoid penetration of surrounding tissues. Human or non-human animal cells are biocompatible semipermeable polymer inclusions or membranes (which are C
It allows the release of HL2 polypeptides but prevents the destruction of cells by other deleterious factors from the patient's immune system or surrounding tissues). Alternatively, the patient's own cells transformed to produce the CHL2 polypeptide ex vivo can be directly transplanted into the patient without such encapsulation.

Techniques for encapsulating live cells are known in the art, and preparation of encapsulated cells and their implantation into patients can be accomplished routinely. For example, Baetge et al. (PCT Publication WO 95/05452 and PCT / U
S94 / 09299) describes a membrane capsule containing cells that have been genetically engineered for effective delivery of biologically active molecules. The capsule is biocompatible and is easily removable. The capsule is transfected with a recombinant DNA molecule comprising a DNA sequence encoding a biologically active molecule operably linked to a promoter that is not down-regulated in vivo when transplanted into a mammalian host. Encapsulated cells. The device provides delivery of living cell-derived molecules to specific sites within the recipient. Furthermore, US Pat.
, 892,538; 5,011,472; and 5,106,62.
See No. 7. A system for encapsulating live cells is described in PCT Publication WO 91/10425 (Aebischer et al.). PCT release 9
1 / 10-40470 (Aebischer et al.); Winn et al. 1991, Expe.
r. Neurol. 113: 322-29; Aebischer et al., 1991.
Expert. Neurol. 111: 269-75; and Tresco et al., 1
See also 992, ASAIO 38: 17-23.

In vivo and in vitro gene therapy delivery of CHL2 polypeptides is also envisioned. One example of gene therapy technology is the CHL2 gene (genomic D) encoding a CHL2 polypeptide that can be operably linked to a constitutive or inducible promoter.
NA, cDNA and / or synthetic DNA) to form a "gene therapy DNA construct". This promoter is based on the endogenous CHL2
Homozygous or heterozygous for a gene, provided that it is active in the cell or tissue type into which the construct is inserted. Other components of gene therapy DNA constructs include DN, which is optionally designed for site-specific integration.
Identifies A molecules (eg, endogenous sequences useful for homologous recombination), tissue-specific promoters, enhancers or silencers, DNA molecules capable of providing selective dominance over parental cells, transformed cells Useful as a label for
Molecules, negative selection systems, cell-specific binding agents (eg for cell targeting)
Is mentioned. Cell-specific internalization, transcription factors that increase expression from vectors,
And factors that enable the production of the vector.

The gene therapy DNA construct can then be introduced into cells using either viral or non-viral vectors (either ex vivo or in vivo).
One method for introducing the gene therapy DNA construct is by the use of viral vectors as described herein. Certain vectors, such as retroviral vectors, deliver the DNA construct to the chromosomal DNA of the cell, and the gene can be integrated into the chromosomal DNA. Other vectors function as episomes and the gene therapy DNA construct remains cytosolic.

[0243] In yet another embodiment, the regulatory element is CHL2 in the target cell.
May be included for controlled expression of genes. Such elements are stimulated in response to appropriate effectors. In this way, the therapeutic polypeptide can be expressed when desired. One conventional control means is a chimeric protein containing a small molecule binding domain and a domain that can initiate biological processes (eg,
The use of low molecular weight dimerizers or rapalogs that dimerize DNA binding proteins or transcriptionally active proteins) (PCT Publication WO 96/4).
1865, WO 97/31898 and WO 97/31899).
Protein dimerization can be used to initiate transcription of a transgene.

An alternative regulation technique uses the method of storing the protein expressed from the gene of interest inside the cell as aggregates or clusters. The gene of interest is expressed as a fusion protein containing a conditional aggregation domain that results in retention of the aggregation protein in the endogenous reticulum. The stored protein is stable and inactive inside the cell. However, the protein can be released by administering a drug that removes the conditional aggregation domain, such as a small molecule ligand, thereby destroying the aggregate or cluster, which results in the protein being secreted from the cell. Can be done. Aridor et al., 2000, Science 287: 816.
-17 and Rivera et al., 2000, Science 287: 826-3.
See 0.

Other suitable control means or gene switches include, but are not limited to, the systems described herein. Mifepristone (RU4
86) is used as a progesterone antagonist. Binding of the modified progesterone receptor ligand binding domain to the progesterone antagonist activates transcription by forming a dimer of two transcription factors,
It then reaches the nucleus and binds to DNA. This ligand binding domain is modified to eliminate the ability of the receptor to bind the natural ligand. Modified steroid hormone receptor systems are further described in US Pat. No. 5,364,791, and PCT publications WO 96/40911 and WO 97/10337.

Yet another regulatory system binds to the ecdysone receptor (cytoplasmic receptor),
And ecdysone (Drosophila steroid hormone) which activates this is used. This receptor then translocates to the nucleus and binds to specific DNA response elements (promoters from ecdysone-responsive genes). The ecdysone receptor contains a transactivation domain, a DNA binding domain, and a ligand binding domain and initiates transcription. The ecdysone system is further described in US Pat. No. 5,514.
, 578, and PCT publications WO 97/38117, WO 96/37609, and WO 93/03162.

Another control means uses positive tetracycline controllable transactivators. This system consists of a mutated tet repressor protein DNA-binding domain linked to a polypeptide that activates transcription (a mutated tetR-4 amino acid change that resulted in a reverse tetracycline-regulated transactivator protein, ie, Binds to the tet operator in the presence of tetracycline). Such systems are described in US Pat. Nos. 5,464,758, 5,650,298, and 5,654,168.

Additional expression control systems and nucleic acid constructs can be found in US Pat. Nos. 5,741,679 and 5,834,186 (Innovir Laboratories).
Inc. ).

In vivo gene therapy involves replacing the gene encoding the CHL2 polypeptide with CHL2.
It may be achieved by local injection of the two nucleic acid molecules or by introduction into the cells by a suitable viral or non-viral delivery vector. Hefti, 19
94, Neurobiology 25: 1418-35. For example, a nucleic acid molecule encoding a CHL2 polypeptide can be included in an adeno-associated virus (AAV) vector for delivery to target cells (eg, Johnson, PCT Publication W).
See O95 / 34670; PCT application PCT / US95 / 07178.
Recombinant AAV genomes are typically C operably linked to a functional promoter.
It comprises a DNA sequence encoding the HL2 polypeptide and an AAV inverted terminal repeat flanked by polyadenylation sequences.

Alternative suitable viral vectors include retroviruses, adenoviruses,
Herpes simplex virus, lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papillomavirus vectors. US Pat. No. 5,672,344 describes an in vivo virus-mediated gene transfer system containing a recombinant neurotrophic HSV-1 vector. US Pat. No. 5,399,346
The issue provides an example of a process for providing a therapeutic protein to a patient by delivery of human cells treated in vitro to insert a DNA segment encoding the therapeutic protein. Additional methods and materials for practicing gene therapy techniques are described in US Pat. No. 5,631,236 (including adenovirus vectors),
No. 5,672,510 (including retrovirus vector), No. 5,635.
, 399 (including retroviral vectors expressing cytokines).

Non-viral delivery methods include liposome-mediated delivery, naked DNA delivery (direct injection), receptor-mediated delivery (ligand-DNA complex), electroporation, calcium phosphate precipitation, and particulate bombardment (eg, gene gun). )
But is not limited to these. Gene therapy materials and methods also include inducible promoters, tissue-specific enhancer-promoters, DNA sequences designed for site-specific integration, DNA sequences capable of providing selective dominance over parental cells, transformed Labels to identify cells, negative selection and expression control systems (safety indicators), cell-specific binding factors (for cell targeting)
, Cell-specific internalization factors, and transcription factors that enhance expression by the vector and methods of vector production. Such additional methods and materials for practicing gene therapy techniques are described in US Pat. Nos. 4,970,154 (including electroporation techniques), 5,679,559 (liposome for gene delivery). Protein-containing systems), 5,676,954 (including liposomal carriers), 5,593,875 (describing methods for calcium phosphate transfection), and 4, 945,050, ibid. (Describing the process by which biologically active particles are sprayed onto cells at a certain rate, whereby the particles penetrate the surface of the cells and are incorporated inside the cells), and P
CT publication WO 96/40958 (including nuclear ligands).

It is also contemplated that CHL2 gene therapy or cell therapy may further include delivery of one or more additional polypeptides in the same or different cell (s). Such cells can be introduced separately into the patient, the cells can be contained in a single implantable device (eg, the encapsulating membrane described above), or the cells can be separated by a viral vector. It can be modified.

A means of increasing expression of an endogenous CHL2 polypeptide in a cell by gene therapy is to insert one or more enhancer elements in the CHL2 polypeptide promoter, where the enhancer element is the transcriptional activity of the CHL2 gene. Can work to increase. The enhancer elements used are
Selected on the basis of the tissue in which it is desired to activate the gene-enhancer elements are known to confer promoter activation in that the tissue is selected. For example, if the gene encoding the CHL2 polypeptide "turns on" in T cells, the lck promoter enhancer element may be used. Thus, the added functional portion of the transcription element can be inserted into the fragment of DNA containing the CHL2 polypeptide promoter using standard cloning techniques (and, if desired, the vector and / or 5'and / Or inserted in the 3'flanking sequence). This construct, known as the "homologous recombination construct", can then be introduced into the desired cells, either ex vivo or in vivo.

Gene therapy can also be used to increase CHL2 polypeptide expression by modifying the nucleotide sequence of the endogenous promoter. Such modifications are typically accomplished by homologous recombination methods. For example, DNA containing all or part of the promoter of the CHL2 gene selected for inactivation
The molecule can be engineered to remove and / or replace a fragment of a promoter that regulates transcription. For example, the TATA box and / or binding site of the promoter's transcriptional activator can be deleted using standard molecular biology techniques; such deletion can inhibit promoter activity, Can block transcription of the corresponding CHL2 gene. Deletion of the TATA box or transcriptional activator binding site in the promoter results in the CHL2 polypeptide promoter (regulated C
D containing all or related parts (from the same or related species as the HL2 gene)
Can be achieved by generating an NA construct, wherein one or more TATA box and / or nucleotides of the transcriptional activator binding site are mutated by substitutions, deletions, and / or insertions of one or more nucleotides. To be done. As a result, the TATA box and / or activator binding site decreases activity,
Or completely deactivated. This construct, which is also typically the native (endogenous) 5'and 3'DN flanked by the modified promoter segments
Containing at least about 500 bases corresponding to the A sequence), either directly or via a viral vector as described herein, into appropriate cells (either ex vivo or in vivo). Can be introduced. Typically, the integration of this construct into the genomic DNA of the cell is by homologous recombination, where the 5'and 3'DNA sequences of this promoter construct are integrated into the endogenous chromosomal DNA.
By hybridizing to, may serve to help integrate the modified promoter region.

Therapeutic Uses CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof are used to treat many diseases, disorders or conditions, including those listed herein. , May be diagnosed, recovered or prevented.

CHL2 polypeptide agonists and antagonists include molecules that modulate CHL2 polypeptide activity and either increase or decrease the activity of at least one of the mature forms of the CHL2 polypeptide. Agonists or antagonists can be cofactors (eg, proteins, peptides, carbohydrates, lipids or small molecules) that interact with the CHL2 polypeptide and thereby regulate its activity. Potential polypeptide agonists or antagonists include antibodies that react with either soluble or membrane bound forms of CHL2 polypeptides that include some or all of the extracellular domain of the CHL2 protein. Molecules that regulate CHL2 polypeptide expression typically include C, which may act as an antisense regulator of expression.
Included are nucleic acids encoding HL2 polypeptides.

In adults, gene products of the BMP family (especially BMP2 and BMP
One of the major roles of 4) is the regulation of bone weight. BMP1 has been shown to cleave and inactivate CHD, and has been isolated from bone along with BMP2 and BMP3 (Wozney et al., 1988, Science 242).
: 1528-34; Celeste et al., 1990, Proc. Nat. Acad
． Sci. USA 87: 9843-47), CHL2 polypeptides may play important regulatory roles in bone formation. Thus, CHL2 nucleic acid molecules, polypeptides and agonists and antagonists thereof, including but not limited to anti-CHL2 selective binding agents, may be useful in diagnosing or treating diseases and conditions affecting bone density. Examples of such diseases and conditions include, but are not limited to, osteofibrosis and osteoporosis. Other diseases and conditions that affect bone density are included within the scope of this invention.

Direct delivery of BMP4 or other BMP family members to the regenerating bone via the bloodstream appears to be a direct therapeutic concept for the treatment of bone fibrosis.
However, this can be difficult to achieve because BMP4 is known to travel only short distances in vivo (Jones et al., 1996, Curr. Biol. 6: 1468-75). In the case of CHD during embryonic development, BMP and CHLII
Formation of a complex with the polypeptide can result in further migration of BMPs and the formation of a concentration gradient of BMP4 (Jones and Smith, 1998, Dev.
Biol. 194: 12-17).

Based on the spatially tight regulation of CHL2 gene expression on the surface of articular cartilage, where the first signs of cartilage damage are detected during the pathogenesis of osteoarthritis.
Altered expression levels of 2 polypeptides may play a role in the pathogenesis of osteoarthritis or rheumatoid arthritis. Thus, CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof, prevent cartilage fibrosis during the early stages of osteoarthritis and destroy surface areas once destroyed in the late stages of osteoarthritis or rheumatoid arthritis. Can be useful in regenerating or producing an appropriate superficial area (ie, surface) in transplanted cartilage.

BMP polypeptides have been shown to function in organogenesis during late embryogenesis. Organogenesis in embryonic kidney, lung and gastrointestinal tract has been shown to be affected by BMP4 expression (Hogan 1996, Genes D.
ev. 10: 1580-94). The combination of BMP4 and CHL2 polypeptides may be useful for controlling the proliferation and differentiation of progenitor cells in these tissues, thereby allowing the regulation of tissue regeneration or wound healing in vivo. Thus, CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may be useful in promoting tissue regeneration or wound healing.

CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof can also be used in the development and development of hematopoietic stem cells. BMP / CH
It may be possible to use L2 polypeptide complexes to regulate primordial hematopoietic stem cells and thereby regulate repopulating stem cells of adult bone marrow. Alternatively, it may be possible to control the development of stem cells derived from mesodermal stem cells. Thus, the CHL2 polypeptides and nucleic acids of the invention, along with BMPs, may be useful for ex vivo expansion of hematopoietic stem cells and gene therapy carried out through such cells.

BMP4 is an essential factor for generating hematopoietic progenitor cells from mouse ES cells. However, the effective concentration of BMP4 is in a narrow range (0.5 ng / ml to 5 ng / ml).
ml), which is consistent with the idea that differences in the active concentration of BMPs correlate with differences in cell types arising from totipotent ectoderm. No system for reproducible in vitro generation of hematopoietic stem cells from ES cells has yet been disclosed.
However, this can be achieved by precise control of the concentration of BMP4. The CHL2 nucleic acid molecules and polypeptides of the present invention may be useful in optimizing culture conditions for in vitro production of hematopoietic stem cells from ES cells.

Progenitor hematopoietic stem cells have recently been demonstrated in mouse yolk sac (Yode.
r et al., 1997, Proc. Natl. Acad. Sci. USA 94:67.
76-80). This subclass of hematopoietic stem cells does not show bone marrow regrowth activity in adults. However, when exposed to the environment of the neonatal liver, these progenitor stem cells are converted into long-term myelorepopulating stem cells (ie, secondary stem cells). This can also be achieved by culturing primordial stem cells on a particular stromal cell line. The long-term survival of this secondary stem cell in culture and the long-term maintenance of progenitor stem cells that spontaneously differentiate into secondary stem cells have not yet been established. BMPtpC
Interactions with HL2 polypeptides may function in the regulation of secondary stem cells. The recombinant CHL2 polypeptides and CHL2 antibodies of the invention may be useful tools for long-term maintenance of hematopoietic stem cells in vitro and generation of secondary stem cells from primordial stem cells in vitro. Alternatively, BMP4 or putative novel CHL2 interacting molecules may be useful for controlling these processes.

Moreover, primitive hematopoietic stem cells have not yet been well characterized. Progenitor stem cells can be of lymphocytic cell type, but such cells can also be mesoderm precursors, which can give rise to hematopoietic cell types and other mesodermal progeny. In support of this notion, adult bone marrow uses endothelial progenitor cells, cells that regenerate liver (Peterse).
n, et al., 1999, Science 284: 1168-70), as well as common stem cells that have the ability to induce endothelial cells, myocytes and hematopoietic cells in vivo (Ferrari et al., 1998, Science
279: 1528-30). Furthermore, the osteoblast lineage, which consists of bone marrow stroma, is known to be derived from mesodermal stem cells present in the bone marrow. It is also possible that common mesodermal stem cells may be responsible for the generation of both stromal and hematopoietic cells.
It has been previously speculated.

[0265] CHL2 polypeptide expression was detected in skeletal muscle, indicating that CHL2
2 polypeptides may also play a role in skeletal muscle development and function. Thus, CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may be useful in diagnosing or treating diseases and conditions affecting skeletal muscle. Examples of such diseases and conditions include, but are not limited to, cachexia and muscular dystrophy. Other diseases and conditions related to skeletal muscle development and function are included within the scope of this invention.

The expression of CHL2 polypeptide was detected in the heart, indicating that CHL2
Polypeptides may play a role in cardiac development and function. Therefore, C
HL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof, may also be useful in diagnosing or treating diseases and conditions affecting the heart. Examples of such diseases and conditions include, but are not limited to, arrhythmias, angina, hypertension, myocardial infarction and congestive heart failure. Other heart-related diseases and conditions are included within the scope of the present invention.

CHL2 polypeptides may play a role in gastric development and function, as expression of CHL2 polypeptides was detected in the stomach. Therefore, CHL
The 2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof, may also be useful in diagnosing or treating diseases and conditions related to the stomach. Examples of such diseases and conditions include, but are not limited to, gastric cancer and gastric ulcer. Other diseases and conditions associated with the stomach are included within the scope of this invention.

CHL2 polypeptide expression was detected in the liver, indicating that CHL2
Polypeptides may play a role in liver development and function. Therefore, C
HL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof, may also be useful in diagnosing or treating diseases and conditions involving the liver. Examples of such diseases and conditions include, but are not limited to, hepatitis and liver cancer. Other diseases and conditions associated with the liver are included within the scope of this invention.

An agonist or antagonist of CHL2 polypeptide function may be combined (simultaneously or together) with one or more cytokines, growth factors, antibiotics, anti-inflammatory and / or chemotherapeutic agents as appropriate for the condition being treated. Can be used continuously).

Other diseases caused or mediated by undesired levels of CHL2 polypeptides are included within the scope of this invention. Undesired levels include excess levels of CHL2 polypeptide and subnormal levels of CHL2 polypeptide.

Uses of CHL2 Nucleic Acids and Polypeptides Nucleic acid molecules of the present invention, including nucleic acid molecules that do not themselves encode biologically active polypeptides, can be used on a chromosome to integrate the CHL2 gene and related genes. The location of the gene to be mapped can be mapped. Mapping can be performed using techniques known in the art (for example,
PCR amplification and in situ hybridization).

CHL2 nucleic acid molecules, including nucleic acid molecules which themselves do not encode biologically active polypeptides, are qualitatively or quantitatively for the presence of CHL2 nucleic acid molecules in mammalian tissue or body fluid samples. May be useful as a hybridization probe in a diagnostic assay to test either.

Other methods may also be used where it is desired to inhibit the activity of one or more CHL2 polypeptides. Such inhibition may be provided by a nucleic acid molecule that is complementary to and hybridizes to the expression control sequences (triple helix) or CHL2 mRNA. For example, an antisense DNA or RNA molecule having a sequence that is complementary to at least a portion of the CHL2 gene can be introduced into the cell. Antisense probes can be designed by available techniques using the sequences of the CHL2 gene disclosed herein. Typically, each such antisense molecule is complementary to the start site (5 'end) of each selected CHL2 gene. This antisense molecule then forms the corresponding CHL2m
Translation of this mRNA is blocked or reduced when hybridized to RNA. Antisense inhibitors provide information associated with the reduction or absence of CHL2 polypeptides in cells or organisms.

Alternatively, gene therapy may be used to generate dominant negative inhibitors of one or more CHL2 polypeptides. In this context, DNA encoding each selected variant of CHL2 polypeptide polypeptide is prepared and the viral or non-viral method as described herein is used to It can be introduced into cells. Each such variant is typically designed to compete with the endogenous polypeptide in its biological role.

Further, CHL2 polypeptides (whether biologically active or not)
Can be used as an immunogen, ie the peptide contains at least one epitope against which antibodies can be raised. A selective binding agent that binds a CHL2 polypeptide (as described herein) is provided for in vivo and in vitro diagnostic purposes (for detecting the presence of a CHL2 polypeptide in a body fluid or cell sample, Can be used in, but is not limited to, use in labeled form. These antibodies can also be used to prevent, treat or diagnose a number of diseases and disorders, including the diseases and disorders listed herein. These antibodies bind to the CHL2 polypeptide and
At least one activity characteristic of the 2 polypeptide may be reduced or blocked or may be bound to the polypeptide to increase at least one activity characteristic of the CHL2 polypeptide (by increasing the pharmacokinetics of the CHL2 polypeptide. Including that).

The CHL2 polypeptides of the present invention can be used to clone CHL2 polypeptide receptors using expression cloning strategies. Radiolabeled ( ¹²⁵ iodine) CHL2 polypeptides or affinity / activity-tagged CHL2 polypeptides (eg, Fc fusions or alkaline phosphatase fusions) identify cell types or cell lines or tissues that express CHL2 polypeptide receptors. Can be used in a binding assay for RNA isolated from such cells or tissues is converted to cDNA, cloned into a mammalian expression vector and transfected into mammalian cells (eg, COS or 293 cells) to produce an expression library. You can The radiolabeled or tagged CHL2 polypeptide can then be used as an affinity ligand to identify and isolate from this library a subset of cells that express the CHL2 polypeptide receptor on their surface. DNA can then be isolated from these cells and transfected into mammalian cells to create a secondary expression library. The percentage of cells expressing the CHL2 polypeptide receptor in this library is several times higher than in the original library. This enrichment process can be iteratively repeated until a single recombinant clone containing the CHL2 polypeptide receptor is isolated. The isolation of this CHL2 polypeptide receptor is useful for identifying or developing novel agonists and antagonists of the CHL2 polypeptide signaling pathway. Such agonists and antagonists include soluble CHL2 polypeptide receptors, anti-CHL2 polypeptide receptor antibodies, small molecules or antisense oligonucleotides, and these are one or more of the diseases or disorders described herein. The treatment,
It can be used for prevention or diagnosis.

The mouse and human CHL2 nucleic acids of the invention also have their corresponding chromosomal CHL.
It is a useful tool for isolating 2 polypeptide genes. For example, CHL2
Mouse chromosomal DNA containing sequences can be used to construct knockout mice, which allows testing of their role in vivo for CHL2 polypeptides. Human CHL2 genomic DNA can be used to identify genetic tissue degenerative diseases.

Deposits of cDNAs encoding mouse and human CHL2 polypeptides were also subcloned into pSPORT1 (Gibco BRL) and Ameri.
can Type Culture Collection, 10801 Un
diversity Boulevard, Manassas, VA 20110
Accession No. PTA-147 created by Mar. 14, 2000 by Mar. 14, 2000
9 and PTA-1480.

The following examples are intended for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Example 1 Cloning of Mouse CHL2 Polypeptide Genes In general, the materials and methods as described in Sambrook et al. (Supra) are used to clone and analyze the gene encoding the mouse CHL2 polypeptide. Used to

Two mouse placental cDNA libraries were prepared to isolate sequences encoding the mouse CHL2 polypeptide. Total RNA was extracted from mouse placenta and poly-A + RNA was selected using standard extraction and isolation techniques. The random-primed cDNA is then cloned into S
upperscript Plasmid System for cDNA S
was synthesized from poly-A + RNA using synthesis (Gibco-BRL, Rockville, MD). The resulting cDNA was digested with NotI and fractionated on a 0.8% agarose gel. 300-1000bp electrophoresed
CDNA was isolated and ligated into the signal trap vector kFGF7, and a cDNA larger than 1.5 kb was isolated and ligated into pSPORT1. The ligation reaction was transformed into E. coli using standard transformation techniques. E. coli, these transformants were selected on medium containing ampicillin, and these transformants were harvested to make two cDNA libraries.

Clones containing a signal peptide sequence were enriched from a kFGF-based library using the kFGF signal trapping technique (US patent application Ser. No. 09 / 026,959). Plasmid DNA from this cDNA library was prepared from 10 pools of 100,000 colonies each using standard techniques. This DNA was transformed into NIH by calcium phosphate transfection.
Transfected cells were introduced into 3T3 cells and then the transfected cells were grown for 14 days in selection medium supplemented with 0.5% fetal bovine serum. Only transformants containing the plasmid with the signal peptide sequence produced colonies. These colonies were harvested by trypsinization and total RNA from these colonies was collected according to the manufacturer's recommended protocol for TRIzon reagent (Gib.
co-BRL). Poly-A + RNA was added to mRNA Puri
fiction Kit (Amersham Pharmacia Biot
ech, Piscataway, NJ) was used to isolate from this total RNA.

To generate a cDNA library enriched for molecules containing a signal peptide sequence, the first-strand cDNA was ligated with SuperScript ^™ preamplification (pream).
preparation system (Gibco-BRL) was first used to prepare the poly-A + RNA isolated above. This first strand cDNA reaction
1 μg of poly-A + RNA and 2 pmol of primer 16 in a total volume of 15 μL
05-21

[0284]

[Chemical 1] And the template-primer mixture was heated at 70 ° C. for 10 minutes. The template-primer mixture was transferred to 50 ° C. and 2.5 μL 10 × buffer, 2.5 μL 25 mM MgCl ₂ , 1.3 μL
Of 10 mM dNTPs and 2.5 μL of 0.1 M dithiothreitol were added to the premixture. Then, reverse transcriptase (25
0 U) was added and the reaction was incubated at 50 ° C. for 1 hour. The first strand cDNA reaction is stopped by heating at 70 ° C for 15 minutes, and 2
RNA was digested by treatment with U RNAse H for 20 minutes at 37 ° C.

Following first-strand cDNA synthesis, 2 μL of first-strand cDNA, primer 1239-08, at a final concentration of 0.5 μM each in a volume of 100 μL.

[0286]

[Chemical 2] And 1605-22

[0287]

[Chemical 3] Containing 200 μM dNTPs and 2.5 U of Pfu polymerase (PE
Double-stranded cDNA was generated by PCR amplification in reactions containing Biosystems, Norwalk, CT). The PCR reaction was performed at 95 ° C for 1 minute for 1 cycle; 95 ° C for 30 seconds, 66 ° C for 45 seconds, and 72 ° C for 2 minutes for 30 cycles; and 72 ° C for 10 minutes for 1 cycle. Amplification product,
Digested with NotI and SalI and ligated into kFGF7. This ligation reaction was transformed into E. coli using standard transformation techniques. E. coli to generate a signal enriched cDNA library. Plasmid DNA was prepared from 400 selected clones and analyzed by sequencing.

The sequence of one clone (designated ymkz5-00011-c10) is X
Cordin (ch) derived from enopus (GenBank registration number Q91713)
ordin) precursor and was found to share significant homology. This identified clone encodes the N-terminal 115 amino acids of mouse CHL2, 418b.
It was found to contain a p insertion. The full-length cDNA clone was designated as ymkz5-0.
The 0011-c10 clone was used as a probe to isolate from a cDNA library cloned into pSPORT1.

To screen the pSPORT1 library, 1 × 10 ⁶ clones were plated on 150 mm plates at approximately 5 × 10 ⁴ clones per plate, and then these clones were plated from the plates onto nitrocellulose membranes. Filters were prehybridized in ExpressHyb Hybridization Solution (Clontech) for 30 minutes at 68 ° C., then ³² P-dCT.
Hybridization was performed overnight at 68 ° C. in the same hybridization solution containing P-labeled probe. The hybridized filters were washed twice in 2 × SSC and 0.1% SDS for 10 minutes at room temperature and in 0.1 × SSC and 0.1% SDS at 65 ° C. for 30 minutes, 2 Washed twice. After washing, the filters were subjected to autoradiography at -80 ° C overnight in the presence of an intensified screen. Positive clones were screened again. According to the secondary screen, two positive clones were identified and the DNA from these two clones was isolated and sequenced. One clone, designated pSPORTmCHL2, contained an insert of approximately 1.8 kb, which encoded the complete mouse CHL2 polypeptide.

Sequence analysis of the full-length cDNA for mouse CHL2 polypeptide showed that this gene contains a 1278 bp open reading frame encoding a protein of 426 amino acids. 1A-1C show mouse CHL
2 illustrates the nucleotide sequence of two genes (SEQ ID NO: 1) and the predicted amino acid sequence of mouse CHL2 polypeptide (SEQ ID NO: 2). In FIG. 1A, von
The signal peptide sequence predicted by the Heijne algorithm is underlined.

Computer analysis of the mouse CHL2 amino acid sequence revealed that this polypeptide
It has been shown to have three procollagen repeats (CR motifs), such as the CHL (CHL1) and CHLd5 polypeptides (co-pending and co-owned US patent application Ser. No. 09 / 724,915) (in contrast). In addition, four repeats are observed in CHD) (Fig. 5). The amino acid sequence of CHL2 is
Shares 24.4% identity with mouse cordin (or 2 in GAP study)
Share 8% identity). FIG. 2 shows mouse CHL2 polypeptide (mouse CHL2; SEQ ID NO: 2) and mouse cordin (Af069501;
The alignment of the amino acid sequence of SEQ ID NO: 7) is exemplified. The amino acid sequence of mouse CHL2 polypeptide also shares 50% identity with CHL1 and B
It shares structural similarities with MP and SOG, and this similarity is particularly high in the CR domain. The BLAST survey of the Celera Human Genome Database is CHL2
It was shown that the gene shares maximum identity with the CHL1 gene. These results suggest that the CHL2 gene is a novel member of the CHD / SOG gene family.

Example 2 Cloning of Human CHL Polypeptide Genes Generally, the materials and methods as described in Sambrook et al. (Supra) are used to clone and analyze the gene encoding the human CHL2 polypeptide. Used to

To isolate the sequence encoding the human CHL2 polypeptide, human placenta cd
An NA library was prepared. Total RNs using standard extraction and isolation techniques
A was extracted from human placenta and poly-A + RNA was selected. Then, the oligo d (T) -primed (Origo d (T) -primed) cDNA was
Superscript Plasmid System for cDNA
Synthesized from poly-A + RNA using Synthesis (Gibco-BRL). The resulting cDNA was digested with NotI and fractionated on a 0.8% agarose gel. The electrophoresed cDNA larger than 1.5 kb was isolated and
Ligated into RT1. The ligation reaction was transformed into E. coli using standard transformation techniques. co
li, the transformants were selected on medium containing ampicillin, and these transformants were harvested to generate a human placental cDNA library.

A ³² P-dCTP labeled mouse CHL2 cDNA fragment was used to screen a human placental cDNA library. Plasmid DNA,
1 μg from each pool, isolated from 12 pools of 100,000 clones each
Plasmid DNA was digested with NotI and SalI, electrophoresed on a 0.8% agarose gel and then transferred to a nitrocellulose membrane. The filter is placed in an ExpressHyb hybridization solution (C
prehybridized in lontech) and then mouse CHL2 cDNA
Hybridization was performed overnight at 60 ° C. in the same hybridization solution containing the probe. The hybridized filter is 2 × SSC and 0.1% SD
Wash twice in S for 10 minutes at room temperature and 0.5 × SSC and 0.1% S
Washed twice in DS at 60 ° C for 30 minutes. After washing, the filters were subjected to autoradiography at -80 ° C overnight in the presence of an intensified screen. Positive cDNA fragments were identified in 2 out of 12 plasmid pools.

Individual clones containing sequences encoding the human CHL2 polypeptide were cloned into 150
3 × 10 ⁵ clones from each pool were added to the mm plate at about 5 × per plate.
Isolated from the two plasmid pools identified above by plating at 10 ⁴ . These clones were then mounted from the plates onto nitrocellulose filters and the filters analyzed as described above. Two positive clones were identified and each was subjected to sequencing analysis. One clone (pSP
(Named ORThCHL2) contained an approximately 1.5 kb insert encoding the complete human CHL2 polypeptide.

Sequence analysis of the full-length cDNA of human CHL2 polypeptide showed that this gene contains a 1287 bp open reading frame encoding a protein of 429 amino acids. 3A-3C illustrate the nucleotide sequence of the human CHL2 gene (SEQ ID NO: 4) and the deduced amino acid sequence of the human CHL2 polypeptide (SEQ ID NO: 5). The amino acid sequence of CHL2 is similar to that of human cordin 2.
It was found to share 6.7% identity. Figure 4 depicts the amino acid sequence alignment of human VHL2 polypeptide (human CHL2; SEQ ID NO: 5) and human cordin (Af076612; SEQ ID NO: 8).

Example 3 CHL2 mRNA Expression Multiple Human Tissue Northern Blots or Mouse Tissue Northern Blots (Clo)
nCH) is ³² P-dCTP-labeled human or mouse CH, respectively.
It was probed with the L2 cDNA fragment. Northern blot, Ex
Prehybridized at 68C in press Hybridization solution (Clontech) for 30 minutes, then hybridized at 68C overnight in the same solution with labeled probe added. After hybridization, the filters are washed twice in 2x SSC and 0.1% SDS for 10 minutes at room temperature with 0.1x.
Washed twice in SSC and 0.1% SDS for 30 minutes at 68 ° C. After washing, the blot was subjected to audradiography in the presence of an intensifying screen at 80 ° C for 72 hours.

Northern blot analysis of human tissue blots revealed a predominant CHL2 transcript of approximately 2 kb in prostate, testis, uterus (very abundant expression), colon, small intestine, heart, skeletal muscle and smooth muscle. Weak expression was detected in trachea, placenta and bone marrow, and very weak expression was detected in liver. Northern blot analysis of mouse tissue blots revealed a predominant CHL2 transcript of approximately 1.8 kb in liver and kidney. Very weak expression was detected in the heart. Weak expression of a transcript of approximately 2 kb was detected in testis and skeletal muscle. CHL2 expression was not analyzed in mouse stomach tissue. Very weak expression was detected in mouse embryonic tissue of day 7, 11, 15 and 17 embryos.

Northern blot analysis strengthens the relationship between CHD / SOG and CHL2 polypeptides. CHD / SOG is expressed at relatively high levels in E7 embryos and at lower levels in E11, E15 and E17 embryos (Pappan).
et al., 1998, Genomics 52: 236-39). CHL2 mRN
A was similarly detected in E7, E11, E15 and E17 embryos, but CH
The level was lower than that of the D / SOG transcript. CHD / SOG expression was detected in spleen, liver, and kidney (Pappano et al., 1998). CHL2 mR
NA was similarly detected in liver and kidney. The similarity in expression pattern is
Combined with structural similarities, it is suggested that CHL2 and CHD / SOG have similar biological activities, although these proteins may function at different developmental stages.

Expression of CHL2 mRNA was localized by in situ hybridization. In situ hybridization to sections of embryonic and adult mouse tissues was described (Wilcox, 1993, J. Histochem. C.
ytochem. 41: 1725-33). Mouse CH
The L2 probe was first loaded with 1.2 kb NcoI-S from pSPORTmCHL2.
prepared by removing the acI fragment to give pSPmCHL2OOH
Which was then linearized with EcoRI. Furthermore, the HindIII fragment was taken out to obtain pSPmCHL2NH2. Antisense RNA was synthesized using SP6 RNA polymerase. The RNA synthesized from these plasmids contained non-overlapping sequences.

In-situ hybridization analysis revealed that CHL2 mRNA expression was initiated in the sternum at about day 12.5 embryos in mice and persisted in a confined area of adult articular cartilage. CHL2 mRN
A expression was not detected in sections tested in embryos 10.5 days old. In 13.5 day embryos, strong CHL2 mRNA expression was detected throughout chondrocytes in limb bones (FIGS. 6 and 7) and sternum, as well as in perichondral mesenchymal cells adjacent to developing joints. Strong but very limited CHL2 mRNA
Expression was also detected in superficial areas of chondrocytes within the developing articular cartilage of vertebral and long bone epiphyseal cartilage (FIGS. 6-8). Up to 18.5 days embryo and until adulthood, CH
Skeletal expression of L2 mRNA was restricted to chondrocyte monolayers in the epidermal region of articular cartilage. Epiphyseal growth plates show CHL2 m at any time of mouse development.
It showed no RNA expression.

Among non-cartilage tissues, significant CHL2 mRNA expression was found in the ovaries, fallopian tubes and uterus of female mice, and in the testes, epididymis and possibly other adnexal glands of male mice (eg, seminiferous). Sac, coagulation, and prostate). The strong signal observed in the uterine wall was somewhat similar to CHL1 mRNA expression detected in the uterus. Weak expression of CHL2 mRNA was also detected on the colon surface. However, the location of CHL2 expression in the colon is distinctly different from that of CHL1, where CHL2 mRNA separates submucosal and mucosal fibroblasts /
It was detected in connective tissue (Nakayama et al., 2001). In contrast to the CHL1 gene, CHL2 mRNA expression was not detected in the stomach or small intestine. Therefore, among soft tissues, the CHL2 gene was particularly expressed in the reproductive organs.

Example 4: Chromosomal Mapping of Mouse CHL2 Polypeptide Gene Fluorescence in situ hybridization (FISH) analysis is used to determine the chromosomal localization of mouse CHL2 gene (Shi et al., 1997, Geno).
mics 45: 42-47). FISH probe with standard techniques and mouse C
A primer corresponding to the 5'untranslated region of the HL2 gene (5'-TCCTCTCA
TCCTCACCTTAG-3 '; SEQ ID NOS: 15 and 5'-GGAGAAAG
Mouse ES-129 / SvJ II BAC chromosomal DNA library (G) by PCR using TGAGATAAGGACAC-3 ′; SEQ ID NO: 16).
It was prepared from a BAC clone (F1067) isolated from Ename Systems. The mouse CHL2 gene was localized to chromosome 7 using the chromosome 7 centromere-specific P1 clone as a co-hybridization probe (Shi et al., 1997). A total of 80 metaphase cells were analyzed and 10 out of 77 cells that showed the specific label were used for the co-hybridization experiments. As a result, the CHL2 gene is located at a distance of 66% from the heterochromatin-intrinsic chromatin border for chromosome 7 telomeres (bands 7E2-E3.
Corresponding to the region). Therefore, the CHL2 gene is different from the CHL1 gene, ie, the CHL2 gene is autosomal.

Example 5: Production of CHL2 Polypeptides A. Expression of CHL2 Polypeptides in Bacteria PCR is used to amplify a template DNA sequence encoding a CHL2 polypeptide. Primers corresponding to the 5'and 3'ends of this sequence are used for this PCR. The amplified DNA product may be modified to contain restriction enzyme sites to allow insertion into an expression vector. The PCR product is gel purified and inserted into an expression vector using standard recombinant DNA methodologies. pAMG21 (AT containing a gene encoding lux promoter and kanamycin resistance
Representative vectors such as CC No. 98113) are digested with BamHI and NdeI for directional cloning of the inserted DNA. The ligated mixture was electroporated by E. coli. E. coli host strain and transformants are selected for kanamycin resistance. Plasmid DNA from selected colonies is isolated and subjected to DNA sequencing to confirm the presence of insert.

Transformed host cells are incubated at 30 ° C. in 2 × YT medium containing 30 μg / mL kanamycin prior to transfection. Gene expression is induced by addition of N- (3-oxohexanoyl) -dl-homoserine lactone to a final concentration of 30 ng / mL, followed by incubation at 30 ° C or 37 ° C for 6 hours. Expression of CHL2 polypeptides is assessed by culture, resuspension of bacterial pellets and centrifugation of lysates, and analysis of host cell proteins by SDS polyacrylamide gel electrophoresis.

Inclusion bodies containing CHL2 polypeptides are purified as follows. Bacterial cells are pelleted by centrifugation and resuspended in water. The cell suspension is lysed by sonication and pelleted by centrifugation at 195,000 xg for 5-10 minutes. The supernatant is discarded and the pellet washed and transferred to the homogenizer. Add 5 mL of Perc to the pellet until uniformly suspended.
Homogenize in an Oll solution (75% liquid Percoll and 0.15M NaCl), then dilute and centrifuge at 21,600 xg for 30 minutes. Gradient fractions containing inclusion bodies are collected and pooled. The isolated inclusion bodies are analyzed by SDS-PAGE.

E. A single band on the SDS polyacrylamide gel, corresponding to the CHL2 polypeptide produced by E. coli, was excised from the gel and the N-terminal amino acid sequence was determined essentially by Matsudaira et al., 1987, J. Mol. Biol. Chem.
262: 10-35.

(B. Construction of CHL2 Polypeptide Mammalian Expression Vector) Mouse CHL2 was transiently expressed using the pSRαmCHL2 vector, which was prepared as follows. The open reading frame of CHL2 polypeptide was determined using standard techniques, as well as primers 2360-40 (5'-
GCTATCTAGAGCCACCCATGGTTCCCCGGGGTGAGGGAT
CATC-3 '; SEQ ID NO: 17) and 2360-41 (5'-GCTAGTC
It was first amplified by PCR using GACCATTAATGTCTTGGTCACTTTGTCTG-3 '; SEQ ID NO: 18). Amplified PCR product is Xb
Digested with a I and Sal I, and then SRα-based expression plasmid (Takebe et al., 1988, Mol. Cell. Biol. 8: 466-.
72) Insert in. pSRαmCHL2 was generated.

A FLAG-tagged mouse CHL2 polypeptide expression construct was prepared as follows. Using the full-length mouse CHL2 DNA fragment (where the stop codon is replaced with a Sal I site) as a template, the full-length mouse cDNA clone and the primer 5′-GCTAGCGGCCGCGCCACCCATGTGTCC were used.
CGGGGTGAGGATCATC-3 ′; SEQ ID NO: 19) and 5′-GCT
AGTCGAACTAATGTCTTGGTCACTTTGTCTGGGGC-3 '
Was obtained by PCR using SEQ ID NO: 20). The amplified PCR product is N
It was digested with otI and SalI and then inserted into the pFLAG-CMV-5a expression vector (Sigma) with the FLAG sequence linked in-frame with the CHL2 sequence at its carboxy terminus. Obtained mCHL2-FLAG
The expression plasmid was named pFLAGmCHL2.

Expression of mCHL2-FLAG polypeptide was expressed in 5% (w / v) skim milk powder (T.
Rader Joe's, Thousand Oaks CA) at 3% (w /
v) 1 according to manufacturer's recommendations, except used in place of bovine serum albumin
Detected by direct Western blot using 0 μg / ml anti-FLAG mouse monoclonal antibody, M2 (Sigma). Rabbit polyclonal antibody against mouse CHL2 was isolated from Harlow and Lane, Using Ant.
ibodies: A Laboratory Manual (Cold Spr)
ing Harbor Laboratory Press, 1988) with synthetic peptides based on the carboxy-terminal sequence CPEDEAEDDHSEVISTR (SEQ ID NO: 21). The resulting antisera were used directly or subjected to affinity purification with the corresponding peptides.

To generate clones that can stably express mCHL2-FLAG, linear pFLAGmCHL2 was used to transfect 293 cells. Stable transformants were selected and the expression levels of the corresponding FLAG-tagged proteins in both serum-free conditioned medium and cell lysates were compared by Western blot analysis (Nakayama et al., 2001, Dev. Biol. Print. During)
. Conditioned medium was concentrated prior to Western blot and then the protein was visualized. Expression based on transient transformation was performed using 293T cells. The 10-fold concentrated conditioned medium was then analyzed as described above.

For medium-scale preparation, stable clones were cloned into CL 1000 flasks (Inte.
gra Biosciences, Ijamsville, MD), 2
Cultivated in 93 SFM II serum-free medium and conditioned medium was collected every 2-3 days. Expression levels of about 3-4 μg / ml were obtained. FLAG-tagged proteins were collected from 500 ml of collected supernatant by single step affinity chromatography using anti-FLAG M2 affinity gel (Sigma) under high salt conditions (Piccolo et al., 1997, Cell 91: 407-16). Purified.
The protein concentration of each preparation was determined by Western blot analysis with M2 and compared to FLAG bacterial alkaline phosphatase standard (Sigma).

Example 6: Generation of Anti-CHL2 Polypeptide Antibodies Antibodies to CHL2 polypeptides can be obtained by immunizing with purified proteins or CHL2 peptides generated by biological or chemical synthesis. . Suitable procedures for raising antibodies are described by Hudson and Ba.
y, Practical Immunology (Second Edition, Blackwell
The procedures described in Scientific Publication) are included.

In one procedure for antibody production, animals (typically mice or rabbits) were injected with a CHL2 antigen (eg, CHL2 polypeptide) and determined by ELISA for hybridoma production. Animals with sufficient serum titer are selected. The spleen of the immunized animal is harvested and prepared as a single cell suspension from which splenocytes are harvested. Spleen cells were fused to mouse myeloma cells (eg Sp2 / 0-Ag14 cells) and DMEM (containing 200 U / mL penicillin, 200 μg / mL streptomycin sulfate, and 4 mM glutamine).
Incubate first, then in HAT selection medium (hypoxanthine, aminopterin and thymidine). After selection, tissue culture supernatant is removed from each fusion well and tested by ELISA for anti-CHL2 antibody production.

Another procedure may also be used to obtain anti-CHL2 antibodies. This procedure can be used, for example, in the immunization of transgenic mice carrying the human Ig locus for the production of human antibodies, and in the screening of synthetic antibody libraries, such as those produced by mutagenesis of antibody variable domains. is there.

Example 7: Biological activity of mouse CHL2 in Xenopus embryo Cordin forms gastrulation by inhibiting the activity of BMP4 Xe.
It is known to dorsalize nopus embryos. The effect of CHL2 on the development of Xenopus embryos was tested as follows. EcoRI of pSPORTmCHL2
The NotI fragment was first labeled with pCS2 + (Ruppp et al., 1994, Ge.
nes Dev. 8: 1311-23), linearized with Not I, and capped mRNA was synthesized with SP6 polymerase and quantified (Nishinakamura et al., 1999, Dev. Biol. 2).
16: 481-90). This pSPORTmCHL2 plasmid is also called Not
It was linearized with I and transcribed with T7 polymerase. Both constructs induced secondary axis formation. As a negative control, elongation factor 1 (EF1) RNA was synthesized.

Each RNA sample was injected into two ventral blastomeres of 4-cell stage Xenopus embryos. After injection, embryos were cultured for 48 hours in 10% Steinberg solution, and embryos were scored for the ectopic axis (Nishinakamura et al., 1999.
FIG. 9). When blastomeres were injected with 1 pg of mouse CHL2 RNA, axial replication rates ranged from 77-87%, while control embryos and EF that were not injected.
The rate in 1 RNA injected embryos was 0%. Mouse C as a positive control
Experiments with HL1 RNA were also performed. In these experiments, an axial replication rate of 83% was obtained with 10-30 pg of CHL1 RNA. Therefore, mouse CHL2 RNA has the activity of antagonizing the endogenous ventrifying factor (probably BMP4).

Example 8 Inhibition of BMP4 Action by Mouse CHL2 Polypeptide Formation of the cordin-CHL1 complex is known to inhibit BMP4 function. Similar axial replication activity observed for CHL2 polypeptides (Example 7)
Suggested that the CHL2 polypeptide also directly inhibited BMP4 action. CD34 ⁺ / CD31 ^hi lymphocyte hematopoietic progenitor cells, CD34 ⁺ / CD
31 ^lo erythroid-myeloid progenitor cells, and CD45 ⁺ myelomonocytic cells have been shown to be dependent on the presence of 0.15-2 ng / ml BMP4 during the differentiation of mouse embryonic stem cell (ES) cells. (Nakayama et al., 2000, Blood 9
5: 2275: 83).

The ability of CHL2 polypeptides to inhibit the activity of BMP4 was tested directly as follows. Rosa26 ES cells were transferred to fibronectin coated plates, conditioned in serum-free medium for 2 days and then differentiated in serum-free methylcellulose medium in the presence of 0.9 ng / ml BMP4 (Nakayama et al., 200.
0). A BMP4 concentration of 0.9 ng / ml produced near maximal levels. Embryoid bodies (EBs) were harvested on day 7 and separated with coregenase. This cell is called CD3
Staining with monoclonal antibodies for hematopoietic progenitor cell markers such as 1 and CD34. Then, the stained sample was subjected to FACScan (Becton Dickin
Son, San Jose, CA).

When affinity purified mCHL2-FLAG was added at 100 ng / ml or higher, the CD34 ⁻ / CD31 ⁺ cell population and the CD34 ⁺ / CD31 ⁺ cell population had background levels (ie, levels achieved without BMP4;
(See FIG. 10). These results show that FLAG-tagged mCHL
It suggests that the two proteins were able to inhibit the action of BMP4 in vitro.

Example 9 Inhibition of BMP2 and BMP4 Action by Mouse CHL2 Polypeptide Both BMP2 and BMP4 are known to induce alkaline phosphatase expression in C2C12 myoblasts. CHL2 polypeptide inhibition of BMP2 and BMP4 action was demonstrated in the C2C12 alkaline phosphatase assay.

The pre-myoblast cell line C2C12 (ATCC Accession No. CRL-1722) was treated with 5% C at 37 ° C. in DMEM containing 10% fetal bovine serum (FBS) and antibiotics.
Maintained in a humid atmosphere of O ₂ . Receptor sensitivity can be reduced in highly passaged cells, so cells were discarded after 10-15 passages. C2C12 cells contained 100 μl DMEM (containing 2% bovine serum and antibiotics),
In a 96-well microtiter plate at a density of 3 × 10 ⁴ cells / well,
Plated. The outermost wells of the microtiter plate were filled with 200 μl DMEM only to avoid excessive drying. The cells are then incubated at 37 ° C for 5
Incubated overnight in a humid atmosphere of% CO ₂ .

After plating, C2C12 cells were treated with mouse CHL in the presence of either 909 ng / ml (35 nM) BMP2 or 566 ng / ml (22 nM) BMP4.
Serial dilution of 2-FLAG (1.47, 2.93, 5.9, 11.7, 23.4,
46.9, 93.8, 187.5, 375, 750, and 1500 ng / ml
) Was exposed to. The cells were then incubated at 37 ° C. in a humidified atmosphere of 5% CO ₂ for 3 days. After incubation, the medium is removed and the cells are washed with 0.1 M
Rinse with Tris, pH 7.4, and 150 μl glycine buffer (0.1 M glycine, 1 mM MgCl ₂ , pH 10.5) (0.1% IGEP
AL CA-630 (containing Sigma)) was added to the wells. The cells were then frozen at −80 ° C., thawed, 50 μl of cell supernatant removed for use in the Bradford protein assay, and 100 μl of phosphate p-
Nitrophenyl disodium (Sigma; diluted to 4 mg / ml in glycine buffer) was added to the remaining cell supernatant. The mixture was incubated at 37 ° C. for 30 minutes and the reaction was stopped by adding 50 μl of 0.5N NaOH. The plate was then read at 405-410 nm. CHL2-FLAG is
It was found to inhibit both BMP2 and BMP4 in a dose-dependent manner (Figure 11).

Example 10: Direct interaction between CHL2 polypeptide and BMP Mouse CHL1 polypeptide and human BMP4, BMP5, BMP6 and TG
Direct interaction with Fβ2 has been previously demonstrated (Nakayama et al., 20.
01). Except that mCHL2-FLAG protein was used at 600 ng / ml,
Similar immunoprecipitation experiments were performed with mouse CHL2 polypeptide. mCHL
2-FLAG proteins are BMP2, BMP4, BMP5, BMP6, GDF
It was found to co-immunoprecipitate with 5 (BMP14) or activin A (FIG. 12). However, high concentrations of BMP5 were required to show detectable levels of interaction with mCHL2-FLAG. CHL2 appeared to have a weaker affinity for BMP5 than for other BMPs. Similar to mCHL1, activin A (
Another TGFβ superfamily member) is C under any of the set conditions.
There was no evidence of interaction with HL2. However, compared to mouse CHL1, T
No GFβ interaction was observed. BMP2 and BMP4 are BMP
5, BMP6 and BMP7 form separate subfamilies (Celes
te et al., 1990, Proc. Natl. Acad. Sci. U. S. A. 87
: 9843-47). Thus, both CHL1 and CHL2 polypeptides bind to all BMP binding proteins (pan-BMP bind).
ing protein).

Example 11: Expression of CHL2 Polypeptides in Transgenic Mice To assess the biological activity of CHL2 polypeptides, encode CHL2 polypeptide / Fc fusion protein under the control of liver-specific ApoE promoter. To prepare the construct. Delivery of this construct is expected to produce pathological changes that are informative regarding the function of the CHL2 polypeptide. Similarly, a construct containing the full-length CHL2 polypeptide under the control of the β-actin promoter is prepared.
Delivery of this construct is expected to result in ubiquitous expression.

To generate these constructs, PCR is used to amplify the template DNA sequence encoding the CHL2 polypeptide. Primers corresponding to the 5'and 3'ends of the desired sequence are used for this PCR. This primer then incorporates a restriction enzyme site that allows the insertion of the amplification product into the expression vector.
After amplification, the PCR product is gel purified, digested with the appropriate restriction enzymes and ligated into the expression vector using standard recombinant DNA techniques. For example, amplified CHL2
The polypeptide sequence is described in Graham et al., 1997, Nature Geneti.
cs, 17: 272-74 and Ray et al., 1991 Genes Dev. 5
: 2265-73, it can be cloned into an expression vector under the control of the human β-actin promoter.

After ligation, the reaction mixture was used to transform E. E. coli host strains are transformed by electroporation and transformants selected for drug resistance. Plasmid DNA from selected colonies is isolated and subjected to DNA sequencing to confirm the presence of the appropriate insert and the absence of mutations. The CHL2 polypeptide expression vector is purified through two rounds of CsCl density gradient centrifugation, cut with the appropriate restriction enzymes, and the linearized fragment containing the CHL2 polypeptide transgene is purified by gel electrophoresis. The purified fragment was
2 mg / mL in mM Tris (pH 7.4) and 0.2 mM EDTA
Resuspend at a concentration of.

Single cell embryos derived from BDF1 × BDF1 crossed mice were described (PCT Publication No. W
O97 / 23614). Embryos are cultured overnight in a CO ₂ incubator and 15-20 2-cell embryos are transferred into oviducts of pseudopregnant CD1 female mice. The progeny obtained from the implantation of microinjected embryos are screened by PCR amplification of the transgene incorporated into the genomic DNA sample as follows. 20 mL of ear buffer (20 mM Tris, pH 8.0, 10 mM)
Digest in EDTA, 0.5% SDS, and 500 mg / mL proteinase K) at 55 ° C overnight. The sample is then diluted with 200 mL TE and 2 mL ear sample is used in a PCR reaction with appropriate primers.

At 8 weeks of age, transgenic founder animals and control animals are sacrificed for necropsy and pathological analysis. Remove a portion of spleen and remove Total RN
Total cellular RNA is isolated from spleens using the A Extraction Kit (Qiagen) and transgene expression is determined by RT-PCR.
RNA recovered from the spleen was analyzed by SuperScript ^™ Pre as follows.
c using amplification system (Gibco-BRL)
Convert to DNA. Appropriate primers (located in the expression vector sequence and CH
The 3'side of the L2 polypeptide transgene) is used to prime cDNA synthesis from the transgene transcript. 10 mg of total spleen RNA from transgenic founder animals and controls was used for 7 minutes with 1 mM primer.
Incubate at 0 ° C and place on ice. Then, the reaction was supplemented with 10 mM T
ris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl ₂ , 1
0 mM of each dNTP, 0.1 mM DTT, and 200 U of SuperScr
Supplement with iptII reverse transcriptase. After 50 minutes incubation at 42 ° C,
The reaction is stopped by heating at 72 ° C for 15 minutes and digested with 2U RNase H for 20 minutes at 37 ° C. The sample is then amplified by PCR with primers specific for the CHL2 polypeptide.

Example 12: Biological activity of CHL2 polypeptides in transgenic mice Prior to euthanasia, transgenic animals are weighed, anesthetized with isoflurane and bled by cardiac puncture. The sample is subjected to hematology and serum chemistry analysis. Radiographs will be taken after the final exsanguination. The major internal organs are subjected to gravimetric analysis during gross dissection.

Following gross dissection, tissues (ie liver, spleen, pancreas, stomach, whole gastrointestinal tract, kidneys, reproductive organs, skin and mammary glands, bones, brain, heart, lungs, thymus, trachea, esophagus, thyroid gland,
Adrenal glands, bladder, lymph nodes and skeletal muscle) are removed and fixed in 10% buffered Zn-formalin for histological examination. After fixation, the tissue is processed in a paraffin block and 3 mm sections are obtained. All sections are stained with hematoxylin and eosin and then subjected to histological analysis.

Spleens, lymph nodes and Peyer's patches of both transgenic and control mice are subjected to immunohistological analysis for B-cell and T-cell specific antibodies as follows. Formalin fixed paraffin embedded sections are deparaffinized and hydrated in deionized water. The sections were quenched with 3% hydrogen peroxide and then Protein Block (Lipshaw, Pittsbu
rgh, PA) and incubated in rat monoclonal anti-mouse B220 and CD3 (Harlan, Indianapolis, IN). Antibody binding was performed using DAB (BioT) as chromogen.
ek, Santa Barbara, CA) using biotinylated rabbit anti-rat immunoglobulin and peroxidase-conjugated streptavidin (Bio).
Genex, San Ramon, CA). Sections are counterstained with hematoxylin.

After necropsy, MLN and sections of spleen and thymus from transgenic animals and control littermates are removed. 100 mm with flat end of syringe
Nylon Cell Strainer (Becton Dickinson, Frank)
Single cell suspensions are prepared by gently triturating this tissue against the bottom of lin Lakes, NJ). Cells were washed twice and counted, then approximately 1 × 10 ⁶ cells from each tissue were added to 0.5 μg CD16 / 32 (Fc) in a 20 μL volume.
Incubate with γIII / II) Fc block for 10 minutes. The sample was then conjugated to FITC or PE, CD90.2 (Thy-1.2),
A monoclonal antibody against CD45R (B220), CD11b (Mac-1), Gr-1, CD4 or CD8 (PharMingen, San Diego).
o, CA) with 0.5 μg antibody in 100 μL volume of PBS (lacking Ca ⁺ and Mg ⁺ ), 0.1% bovine serum albumin and 0.01% sodium azide for 30 minutes at 2-8 ° C. To dye. After antibody binding, cells are washed and then FAC
Analyze by flow cytometry on a Scan (Becton Dickinson).

Although the present invention has been described in terms of preferred embodiments, it will be appreciated that variations and modifications will occur to those skilled in the art. Therefore, the appended claims are intended to cover all such equivalent modifications that fall within the scope of the present invention.

[Brief description of drawings]

1A-1C depict the nucleotide sequence of the mouse CHL2 gene (SEQ ID NO: 1) and the deduced amino acid sequence of the mouse CHL2 polypeptide (SEQ ID NO: 2). The predicted signal sequence is shown (underlined).

FIG. 2 shows the amino acid sequence alignment of mouse CHL2 polypeptide (mouse CHL2; SEQ ID NO: 2) and mouse cordin (Af069501; SEQ ID NO: 7).

3A-3C depict the nucleotide sequence of the human CHL2 gene (SEQ ID NO: 4) and the deduced amino acid sequence of the human CHL2 polypeptide (SEQ ID NO: 5). The predicted signal sequence is shown (underlined).

FIG. 4 shows human cordin (huCHD; SEQ ID NO: 8), human CHL1 polypeptide (huCHL; SEQ ID NO: 9), and human CHL2 polypeptide (huCH).
L2: shows the amino acid sequence alignment of SEQ ID NO: 5).

FIG. 5 shows a schematic diagram of mouse Chordin, CHL1 polypeptide, and CHL2 polypeptide. Pre-collagen repeat (CR; homologous C
R domain is indicated by a gray box), signal peptide (SP), putative BMP1
/ Tolloid cleavage site ( ^* ) and sites of amino acid sequence variation in CHL1 (dE and d5).

FIG. 6. Detection by in situ hybridization in the E17.5 mouse hip joint (panels A and B) and the rib-chondral joint (panel C; showing signals in articular chondrocytes on both sites of the joint). Mouse CHL2
The expression of mRNA is shown.

FIG. 7: Normal adult spinal joints (panels A, B, and C; in articular cartilage cells on the surface of articular cartilage on either side of the articular process or in the facet joints between adjacent spinal processes). Signal) and the intervertebral disc fibrosis anulus fi
3 shows expression of mouse CHL2 mRNA detected by in situ hybridization in fibrocartilage of brosus (panel D).

FIG. 8 shows mouse CHL2 mRNA expression detected by in situ hybridization in the sternum and placenta of E18.5 mice and in the uterus, colon, and small intestine of normal adult mice.

[Figure 9]   Figure 9 shows the secondary axis forming activity of mouse CHL2 polypeptide.

FIG. 10 shows CD34 ⁺ / CD31 ⁺ erythro-myeloid.
3) shows the effect of CHL2 polypeptide on BMP4-dependent production of progenitor cells (R7) and CD34 ⁻ / CD31 + cells (R3).

FIG. 11 shows the effect of CHL2 polypeptide on the induction of BMP2-dependent alkaline phosphatase in C2C12 myoblasts.

FIG. 12 shows the results of Western blot using mCHL2-FLAG protein. In panel A, mCHL2-FLAG was mixed with BMP5 or activin A, treated with anti-mCHL2 antiserum and precipitated with protein A agarose beads. In panel B, mCHL2-FLAG is
BMP2, BMP4, BMP5, BMP6, GDF5 (BMP14), or activin A were mixed, treated with anti-mCHL2 antiserum, and precipitated with protein A agarose beads.

[Sequence list]

[Procedure amendment]

[Submission date] September 19, 2002 (2002.19.19)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Detailed explanation of the invention

[Correction method] Change

[Contents of correction]

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to Chordin-like-2 (CHL2) polypeptides and nucleic acid molecules that encode the same. The invention also relates to selective binding agents, vectors, host cells and methods for producing CHL2 polypeptides. The invention further relates to pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and / or prevention of diseases, disorders, and conditions associated with CHL2 polypeptides.

BACKGROUND OF THE INVENTION Technological advances in the identification, cloning, expression, and manipulation of nucleic acid molecules and decoding of the human genome have greatly accelerated the discovery of novel therapeutic agents. Currently, rapid nucleic acid sequencing techniques can generate sequence information at unprecedented rates and, combined with computer analysis, allow the assembly of overlapping sequences and identification of polypeptide coding regions over part and all of the genome. To Comparison of the deduced amino acid sequence against a database compilation of known amino acid sequence, it is possible to ing to determine the degree of homology to landmarks identified sequences and / or structures previously. Cloning and expression of the polypeptide coding region of a nucleic acid molecule provides the polypeptide product for structural and functional analysis. Manipulation of nucleic acid molecules and encoded polypeptides may confer advantageous properties on the product for use as a therapeutic.

Despite significant technological advances in genomic research over the last decade, the potential for the development of new therapeutic agents based on the human genome is not yet widely understood. Many genes encoding potentially beneficial polypeptide therapeutics or the polypeptides they encode, which may serve as "targets" for therapeutic molecules, have not yet been identified.

Accordingly, it is an object of the present invention to identify new polypeptides and nucleic acid molecules encoding them that have diagnostic or therapeutic advantages.

CHL2 is a bone morphogenetic protein (BM) known as cordin (CHD).
P) inhibitors (Sasai et al., 1994, Cell 79: 779-90).
, Or short gastrulation (SOG; Francois et al., 1994, Genes).
Dev. 8: 2602-16). CHL2 gene is CHD
/ Considered to be a member of the SOG family.

Bone morphogenetic protein (BMP) is a member of the transforming growth factor β family, which is, by nature, a cartridge implant (cartr).
was identified as a factor that promotes osteogenesis from the image implant (Wo
zney et al., 1988, Science 242: 1528-34; Celes.
te et al., 1990, Proc. Natl. Acad. Sci. USA 87: 9
843-47). BMPs are also known to play essential roles during early embryogenesis in frogs, flies, and in mammals. The exact concentration of active BMPs appears to be important for the specificity of specific cell types (Dale et al., 1992,
Development 115: 573-85; Dosch et al., 1997, D.
development 124: 2325-34). A BMP2 / 4 activity gradient was observed, for example, in Xenopus embryos, where the lowest expression was detected at the dorsal tip and the highest expression was observed at the ventral tip, and in the embryos, the dorsoventral Establish lateral axis determination. In another example, BM at specific sites of tissue development
Regulation of P concentration suggests a role for BMP in organogenesis. Regulation of BMP expression is by localized expression of the BMP gene product or by the BMP inhibitor cordin (CHD) (Sasai et al., 1994, Cell 79: 7.
79-90) -or short gastrulation (SOG) (Francois et al., 1994).
, Genes Dev. 8: 2602-16).

[0007] CHD / SOG is a Spemann-former (master-con for the dorsal-ventral axis specification at the gastrulation stage of Xenopus embryogenesis).
It is a large secretory protein produced from the control region). CHD
/ SOG is Noggin (Smith and Harland, 1992, Ce.
ll 70: 829-40), which is also secreted by the organizer, functions as a backing factor. Drosophila SOG has a transmembrane domain at its amino terminus, suggesting that it may be a type II transmembrane protein (Francois et al., 1994, Genes Dev. 8:26).
02-16). It was proposed that the carboxyl terminal side (extracellular domain) of Drosophila SOG was cleaved. However, Xenopus CHD (
Sasai et al., 1994, Cell 79: 779-90), zebrafish CHD (Schulte-Merker et al., 1997, Nature 387 :).
862-63) and mouse CHD (Pappano et al., 1998, Genom.
ics 52: 236-39) does not contain a transmembrane domain. Instead, these proteins have a signal peptide and are secreted directly. CHD /
The SOG polypeptide contains four repeats of the cysteine-rich domain (CR1
-4), this domain is also found in various extracellular matrix proteins such as collagen and thrombospondin.

CHD / SOG is known to bind to BMP4, one of the ventrifying factors (Piccolo et al., 1996, Cell 86: 589-98). BM
P4 has been shown to be essential for embryonic development of the posterior ventral mesoderm in mice (Winnier et al., 1995, Genes Dev. 9: 2105-).
16). Binding of CHD / SOG to BMP4 inhibits BMP4 activity by preventing BMP4 from binding to its receptor (Piccolo et al., 1996, Cell 86: 589-98). In this respect, CHD / SO
The functional relationship between G and BMP4 is similar to that between OPG and OPGL, but CHD / SOG is structurally unrelated to the BMP receptor. CH
The binding affinity of D / SOG to BMP4 was specific and close (Kd = 3 × 1).
0 ^-10 M (Piccolo et al., 1996, Cell 86: 589-98) )
, And appears to require proteolysis to achieve release of bound BMP4. This proteolysis is associated with certain metalloproteases (Tolloi).
d (TLD) or BMP1), which cleaves CHD / SOG to release either the first CR motif (CR1) and the last CR motif (CR4), or both (Piccolo et al. , 1997, Ce
11: 407-16). CHD / SOG is whether to have the its own other functions by receptors or independent functions, still to be determined.

One of the most important roles of CHD / SOG is to establish the BMP4 morphogen gradient (Jones and Smith, 1998, Dev. Biol.
194: 12-17). BMP4 itself appears to act essentially autonomously on cells, only traveling short distances (Jones et al., 1996, C).
urr. Biol. 6: 1468-75). In contrast, the BMP4 inhibitor N
Oggin and CHD / SOG appear to exert far-reaching effects, thereby forming a BMP4 activity gradient.

BMPs also play important roles in addition to early embryogenesis, for example, in organogenesis of the lung, intestine, kidney, skin, heart and teeth, and in later stages of embryogenesis (Hogan, 1996, Genes Dev. 10: 1580-94). Some BMPs are expressed in a highly localized form, while others are widely expressed in tissues. The importance of the localized effects of BMPs on organogenesis was supported by transgenic mouse experiments with constructs, whereby BMP concentrations are artificially enhanced in target tissues. For lung, BMP4 is expressed in the distal end of the epithelium of the lung that are occurring, and when overexpressed by a surfactant (Surfactant) flop Rotein C promoter, structural organization (i.e., branching) is The development of severely collapsed small lungs is observed (Bellusci et al., 1996, Development 122: 1693-702). Estimated B
Since MP activity gradients can also be disrupted by transgene expression, tissue-widely expressed BMPs may also play a role in determining the structural organization of tissues.

Noggin is another BMP2 / 4 inhibitor secreted by Spemann-formers (Zimmerman et al. 1996, Cell 86: 599-.
606). The biological role of Noggin and its mode of action is Xenopus
Similar to CHD / SOG in. The most prominent function of Noggin is CHD
/ SOG, which is dorsal, but does not express Noggin (null-mu)
tant) mice have a bone phenotype (chondrocyte hyperplasia) instead of the early embryonic phenotype
(McMahon et al., 1998, Genes Dev. 12: 1438).
-52; Brunet et al., 1998, Science 280: 1455-57.
). This suggests that CHL2 or even CHD may have the required function in late stages of embryogenesis.

SUMMARY OF THE INVENTION The present invention relates to novel CHL2 nucleic acid molecules and encoded polypeptides.

The present invention provides an isolated nucleic acid molecule, which nucleic acid molecule has the following: (a) the nucleotide sequence set forth in either SEQ ID NO: 1 or SEQ ID NO: 4; (b) ATCC Deposit Number PTA. Nucleotide sequence of the DNA insert in either -1479 or PTA-1480; (c) a nucleotide sequence encoding the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; (d) (a)-(c) A nucleotide sequence that hybridizes to the complement of any of 1) under moderately or highly stringent conditions; and (e) a nucleotide sequence complementary to any of (a) to (c), Includes a nucleotide sequence that is more selected.

The present invention also provides an isolated nucleic acid molecule, which is at least about 70% identical to a polypeptide set forth in either (a) SEQ ID NO: 2 or SEQ ID NO: 5. a nucleotide sequence encoding a polypeptide, wherein the encoded polypeptide has an activity of the polypeptide set forth in any of SEQ ID NO: 2 or SEQ ID NO: 5, the nucleotide sequence; (b) sequence The nucleotide sequence set forth in either SEQ ID NO: 1 or SEQ ID NO: 4, D in either ATCC Accession No. PTA-1479 or PTA-1480
The nucleotide sequence of the NA insert, or the nucleotide sequence of (a), the nucleotide sequence encoding the allelic or splice variants; encoding the polypeptide fragment of (c) at least about 25 amino acid residues, SEQ ID NO: 1 or any nucleotide sequence of SEQ ID NO: 4, ATCC accession No. PTA-1479 or DNA insert in either PTA-1480, a region of the nucleotide sequence of (a) or (b), wherein the polypeptide A fragment is a region that has or is antigenic in activity of the encoded polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; (d) SEQ ID NO: 1, which comprises a fragment of at least about 16 nucleotides. Alternatively, any one of SEQ ID NO: 4 Plastid sequence, ATCC Accession No. PTA-14
DNA insert in either 79 or PTA-1480, or (a)-
(C) a region of any nucleotide sequence ; (e) a nucleotide sequence that hybridizes to the complement of any of (a)-(d) under moderately or highly stringent conditions; and (f ) A nucleotide sequence complementary to any of (a) to (d), and a nucleotide sequence selected from the group consisting of:

The invention further provides an isolated nucleic acid molecule, which is shown below: (a) set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 having at least one conservative amino acid substitution. a nucleotide sequence encoding a polypeptide, wherein the encoded polypeptide has an activity of the polypeptide set forth in any of SEQ ID NO: 2 or SEQ ID NO: 5, the nucleotide sequence; (b) at least one a nucleotide sequence encoding a polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with a one amino acid insertion, wherein the encoded polypeptide is either SEQ ID NO: 2 or SEQ ID NO: 5 A nucleotide sequence having the activity of the polypeptide shown in (c) having at least one amino acid deletion A nucleotide sequence encoding a polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 that poly wherein the encoded polypeptide is shown in any of SEQ ID NO: 2 or SEQ ID NO: 5 A nucleotide sequence having the activity of a peptide; (d) a nucleotide sequence encoding the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5 having a C-terminal truncation and / or an N-terminal truncation, wherein: This encoded polypeptide has a nucleotide sequence having the activity of the polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 5; (e) amino acid substitution, amino acid insertion, amino acid deletion, C-terminal truncation, and N SEQ ID NO: 2 or SEQ ID NO: 5 having at least one modification selected from the group consisting of terminal truncation A nucleotide sequence encoding a polypeptide set forth in either Re, wherein the encoded polypeptide has an activity of the polypeptide set forth in any of SEQ ID NO: 2 or SEQ ID NO: 5, the nucleotide sequence; (F) A nucleotide sequence of any of (a)-(e) that comprises a fragment of at least about 16 nucleotides; (g) Any of (a)-(f) under moderately or highly stringent conditions. A nucleotide sequence which hybridizes to the complement of the nucleotide sequence; and (h) a nucleotide sequence complementary to any of (a) to (e), and a nucleotide sequence selected from the group consisting of:

The present invention provides an isolated polypeptide, which polypeptide comprises: (a) the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; and (b) ATCC Deposit Number. An amino acid sequence encoded by the DNA insert in either PTA-1479 or PTA-1480, the amino acid sequence being selected from the group consisting of:

The present invention also provides an isolated polypeptide, which polypeptide is set forth in either of the following: (a) as set forth in either SEQ ID NO: 3 or SEQ ID NO: 6, optionally at the amino terminus. An amino acid sequence further comprising methionine; (b) an amino acid sequence for the ortholog of either SEQ ID NO: 2 or SEQ ID NO: 5; (c) at least about 70% identical to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5. a certain amino acid sequence, wherein the polypeptide has an activity of the polypeptide set forth in any of SEQ ID NO: 2 or SEQ ID NO: 5, the amino acid sequence; (d) sequence comprising at least about 25 amino acid residues a fragment of the amino acid sequence shown in any of the No. 2 or SEQ ID NO: 5, wherein the Furagume <br/> cement, SEQ ID No. 2 or a fragment having the activity of the polypeptide set shown in SEQ ID NO: 5 or being antigenic; and (e) the amino acid sequence shown in either SEQ ID NO: 2 or SEQ ID NO: 5, A
DN in either TCC Deposit Number PTA-1479 or PTA-1480
An amino acid sequence encoded by the A insert, or an amino acid sequence of an allelic variant or splice variant of the amino acid sequence described in any of (a) to (c), Isolated polypeptide.

The present invention further provides an isolated polypeptide, which polypeptide is set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, which has: (a) at least one conservative amino acid substitution. an amino acid sequence, wherein the polypeptide has an activity of the polypeptide set forth in any of SEQ ID NO: 2 or SEQ ID NO: 5, the amino acid sequence; (b) SEQ ID NO: 2 having at least one amino acid insertion or an amino acid sequence shown in any of SEQ ID NO: 5, wherein the polypeptide has an activity of the polypeptide set forth in any of SEQ ID NO: 2 or SEQ ID NO: 5, the amino acid sequence; (c) an amino acid sequence shown in any of SEQ ID NO: 2 or SEQ ID NO: 5 having at least one amino acid deletion, wherein this The polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, the amino acid sequence; (d) a C-terminal truncated and / or N SEQ ID NO: having terminal truncation 2 or SEQ ID NO: 5 an amino acid sequence as set forth in either, wherein the polypeptide has an activity of the polypeptide set forth in any of SEQ ID NO: 2 or SEQ ID NO: 5, the amino acid sequence; and (e) amino acid substitutions, amino acid insertion, amino acid deletion, C-terminal, truncated, and an amino acid sequence shown in any of SEQ ID NO: 2 or SEQ ID NO: 5 having at least one modification selected from the group consisting of N-terminal truncated, wherein the The polypeptide consists of an amino acid sequence having the activity of the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5. Comprising an amino acid sequence selected from the group.

The present invention still further provides an isolated polypeptide, which polypeptide comprises: leucine or methionine at position 2; methionine at position 5; lysine at position 6; alanine at position 7; position 8 Isoleucine; phenylalanine at position 14; leucine at position 15;
Threonine at position 23; Leucine at position 25; Valine at position 27; Glutamic acid at position 30; Tyrosine at position 32; Methionine at position 34; Glutamine at position 36; Lysine at position 39; Alanine at position 41; Alanine at position 41; Threonine at position 45; 55th Valine; 59th Valine; 60th Asparagine; 66th Proline; 68th Asparagine; 72th Serine or Threonine; 74th Valine; 75th Arginine; 94th Arginine; 99th Arginine Asparagine; Serine at position 100; Lysine at position 105; 10
6th Valine; 113th Tyrosine; 116th Serine; 118th Serine; 1
Arginine at position 20; Leucine at position 123; Alanine at position 125; Alanine at position 129; Alanine at position 139; Threonine at position 142; Serine at position 144; 14
7th asparagine; 148th valine; 149th serine; 159th alanine; 160th alanine; 161st alanine; 164th valine; 166th valine; 173rd valine; 175th arginine; Aspartic acid at position 177; Alanine at position 190; Phenylalanine at position 191; Arginine at position 192;
Leucine at position 194; Asparagine at position 196; Leucine at position 205; Alanine at position 210; Alanine at position 212; Serine at position 213; Alanine at position 216; 2
Serine at position 17; Alanine at position 218; Isoleucine at position 219; Alanine at position 222; Leucine at position 225; Phenylalanine at position 226; Leucine at position 230; Glutamine or arginine at position 233; Glutamine at position 241; Glutamine at position 242; Leucine; isoleucine at position 244; glutamine or asparagine at position 245; glutamine at position 249; leucine or valine at position 251; alanine at position 256; asparagine at position 257; serine at position 259; alanine at position 260; 26
Glutamine at position 1; phenylalanine at position 265; valine at position 268; leucine at position 269; leucine at position 272; valine at position 275; valine at position 278; 28
Glutamic acid at position 4; Glutamic acid at position 288; Alanine or isoleucine at position 292; Serine at position 300; Isoleucine at position 306; Valine at position 313; 3
Serine at position 14; Leucine at position 319; Glutamine at position 323; Threonine at position 324; Alanine at position 326; Alanine at position 327; Serine at position 329; 331
Serine at position 334; Leucine at position 334; Asparagine at position 337; Valine at position 339; Leucine at position 340; Serine at position 342; Phenylalanine at position 344; 34
Glutamic acid at position 9; isoleucine or valine at position 354; methionine at position 356; valine at position 366; methionine or valine at position 368; isoleucine at position 371; leucine at position 375; leucine at position 376; glutamine at position 377;
Phenylalanine at position 381; Asparagine at position 383; Isoleucine at position 384; Leucine at position 393; Arginine at position 395; Valine at position 398; Alanine or valine at position 399; Tyrosine at position 403; Asparagine at position 406; 40
An isolation comprising the amino acid sequence set forth in SEQ ID NO: 5 having at least one conservative amino acid substitution selected from the group consisting of isoleucine at position 9; alanine or valine at position 415; isoleucine at position 417; and leucine at position 421. A polypeptide which has the activity of the polypeptide shown in SEQ ID NO: 5.

[0020]   Fusion polypeptides comprising the CHL2 amino acid sequence are also provided.

The present invention also includes an expression vector comprising an isolated nucleic acid molecule as set forth herein, a recombinant host cell comprising a recombinant nucleic acid molecule as set forth herein, and CHL2. A method of producing a polypeptide is provided, the method comprising culturing the host cell, optionally isolating the polypeptide so produced.

Also included in the invention are transgenic non-human animals containing a nucleic acid molecule encoding a CHL2 polypeptide. The CHL2 nucleic acid molecule is introduced into an animal in a manner that allows expression of the CHL2 polypeptide and increased levels of CHL2 polypeptide, which may include increased circulating levels. Alternatively, CHL2
The nucleic acid molecule is introduced into the animal in a manner that inhibits expression of the endogenous CHL2 polypeptide (ie, creates a transgenic animal carrying a CHL2 polypeptide gene knockout). The transgenic non-human animal is preferably a mammal, more preferably a rodent (eg rat or mouse).

[0023]   Derivatives of CHL2 polypeptides of the invention are also provided.

Further provided are selective binding agents (eg, antibodies and peptides) capable of specifically binding the CHL2 polypeptides of the present invention. Such antibodies and peptides may be agonistic or antagonistic.

Also included by the invention is a pharmaceutical composition comprising a nucleotide, polypeptide, or selective binding agent of the invention and one or more pharmaceutically acceptable formulation agents. The pharmaceutical composition is used to provide a therapeutically effective amount of a nucleotide or polypeptide of the invention. The invention also provides the polypeptide, nucleic acid molecule,
And methods of using selective binding agents.

The CHL2 polypeptides and CHL2 nucleic acid molecules of the present invention can be used in diseases and disorders (
(Including those listed herein) can be used to treat, prevent, ameliorate, and / or detect.

The present invention also provides for identifying a test molecule that binds to a CHL2 polypeptide,
A method of assaying a test molecule is provided. The method involves contacting a CHL2 polypeptide with a test molecule and determining the extent of binding of the test molecule to the polypeptide. The method further comprises the use of such a test molecule for CHL2
Determining whether the polypeptide is an agonist or antagonist. The invention further provides a method of testing the effect of a molecule on CHL2 polypeptide expression or CHL2 polypeptide activity.

Also included by the invention are methods of modulating CHL2 polypeptide expression and methods of modulating (ie, increasing or decreasing) CHL2 polypeptide levels. One method involves administering a nucleic acid molecule encoding a CHL2 polypeptide to an animal. In another method, nucleic acid molecules containing elements that modulate or regulate the expression of CHL2 polypeptides can be administered. Examples of these methods include gene therapy, cell therapy, and antisense therapy, as described further herein.

In another aspect of the invention, the CHL2 polypeptide has its receptor (“C
HL2 polypeptide receptor "). Various "expression cloning" in the form of, have been extensively used to cloning receptors for protein ligands. For example, Simonsen and Lo
dish, 1994, Trends Pharmacol. Sci. 15:43
7-41 and Tartaglia et al., 1995, Cell 83: 1263.
See -71. Isolation of CHL2 polypeptide receptors is useful for identifying or developing novel agonists and antagonists of CHL2 polypeptide signaling pathway. Such agonists and antagonists include soluble CHL2 polypeptide receptors, anti-CHL2 polypeptide receptor selective binding agents (eg, antibodies and their derivatives), small molecules, and antisense oligonucleotides, any of which Or may be used for the treatment of one or more diseases or disorders, including those disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. . All references listed in this application are expressly incorporated herein by reference.

Definitions The term “CHL2 gene” or “CHL2 nucleic acid molecule” or “CHL2 polynucleotide” refers to a nucleotide sequence shown in either SEQ ID NO: 1 or SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 5. Nucleotide sequence encoding any of the polypeptides, ATCC Deposit No. PTA-1479 or PTA
A nucleotide sequence of a DNA insert in any of -1480, and a nucleic acid molecule that comprises or consists of a nucleic acid molecule as defined herein.

The term “CHL2 polypeptide allelic variant” refers to any of several possible naturally occurring alternative forms of a gene that occupy a defined locus on the chromosome of an organism or population of organisms. One of the.

The term “CHL2 polypeptide splice variant” is generated by the selective processing of intron sequences in the RNA transcript of the CHL2 polypeptide amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, A nucleic acid molecule (usually RNA).

The term “isolated nucleic acid molecule” refers to (1) at least a protein, lipid, carbohydrate, or other substance with which total nucleic acid is naturally found when isolated from a source cell. A nucleic acid molecule of the present invention isolated from about 50 percent, (2)
A nucleic acid molecule of the invention that is not linked to all or part of a polynucleotide to which the "isolated nucleic acid molecule" is naturally linked; (3) The invention operably linked to a polynucleotide that is not naturally linked. Or a nucleic acid molecule of the present invention that does not naturally occur as part of a larger polynucleotide sequence than (4). Preferably, the isolated nucleic acid molecule of the present invention is any other contaminating nucleic acid molecule, or other contaminant found in its natural environment, either for its use in producing a polypeptide, or for its therapeutic use. , Interfering with diagnostic, prophylactic, or research use).

The term “nucleic acid sequence” or “nucleic acid molecule” refers to a DNA or RNA sequence. The term includes molecules formed from any of the known base analogs of DNA and RNA, including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinyl-. Cytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1 -Methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5 -Methoxyamino-methyl-2-thiouracil, β-D-mannosylkyuosin (
β-D-mannosylqueosine), 5′-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxoxo Xybutoxine, pseudouracil,
Queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, queucin, 2 -Thiocytosine, and 2,6-diaminopurine.

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

The term “expression vector” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing (if an intron is present).

The term “operably linked” is used herein to refer to the arrangement of flanking sequences, where the flanking sequences so described perform their normal function. Configured or constructed as. Thus, a flanking sequence operably linked to a coding sequence may be capable of replicating, transcribing and / or translating the coding sequence. For example, a coding sequence is operably linked to a promoter if it is capable of directing the transcription of the coding sequence. The flanking sequences need not be contiguous with the coding sequence, as long as it functions correctly. Thus, for example, an intervening, non-translated, but transcribed sequence may be present between the promoter and coding sequences, and the promoter sequence still "operably linked" to the coding sequence. Can be considered to be

The term “host cell” has been or can be transformed with a nucleic acid sequence,
It is then used to refer to cells capable of expressing the selected gene of interest. The term includes the progeny of the parent cell, whether the progeny is identical in morphology or genetic structure to the original parent, so long as the selected gene is present.

The term “CHL2 polypeptide” refers to a polypeptide comprising the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5, and related polypeptides. Related polypeptides include CHL2 polypeptide fragments, CHL2 polypeptide orthologs, CHL2 polypeptide variants, and CHL2 polypeptide derivatives, which are of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. It possesses at least one activity. CHL2 polypeptides can be mature polypeptides as defined herein, and may or may not have an amino terminal methionine residue, depending on the method by which they are prepared.

The term “CHL2 polypeptide fragment” refers to a truncation of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 at the amino terminus (with or without a leader sequence) and / or Refers to a polypeptide that includes a truncation at the carboxyl terminus. The term "CHL2 polypeptide fragment" also refers to CH
L2 polypeptide orthologs, CHL2 polypeptide derivatives, or amino-terminal and / or carboxyl-terminal truncations of CHL2 polypeptide variants, or amino acids of CHL2 polypeptide allelic variants or polypeptides encoded by CHL2 polypeptide splice variants Refers to terminal and / or carboxyl terminal truncation. The CHL2 polypeptide fragment is a selective RNA
It can result from splicing, or in vivo protease activity. Membrane-bound forms of CHL2 polypeptides are also contemplated by the present invention. In preferred embodiments, the truncation and / or deletion comprises about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids. The polypeptide fragment thus produced is about 25 contiguous amino acids, or about 50 amino acids, or about 7 amino acids.
5 amino acids, or about 100 amino acids, or about 150 amino acids, or about 20
Contains 0 amino acids, or more than about 200 amino acids. Such CHL2 polypeptide fragments may optionally include an amino terminal methionine residue. It is understood that such fragments can be used, for example, to raise antibodies to the CHL2 polypeptide.

The term “CHL2 polypeptide ortholog” refers to a polypeptide from another species that corresponds to the CHL2 polypeptide amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. For example, the mouse and human CHL2 polypeptides are
They are considered to be orthologs of each other.

The term “CHL2 polypeptide variant” refers to one or more compared to a CHL2 polypeptide amino acid sequence (with or without a leader sequence) set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. CHL2 polypeptide comprising an amino acid sequence having an amino acid sequence substitution, deletion (eg, internal deletion and / or CHL2 polypeptide fragment), and / or addition (eg, internal addition and / or CHL2 fusion polypeptide). Say. Variants can be naturally occurring (eg, CHL2 polypeptide allelic variants, CHL2 polypeptide orthologs, and CHL2 polypeptide splice variants) or can be artificially constructed. Such CHL2 polypeptide variants can be prepared from the corresponding nucleic acid molecule having a DNA sequence that varies from the DNA sequence shown in either SEQ ID NO: 1 or SEQ ID NO: 4. In a preferred embodiment, this variant is 1 to 3.
, Or 1-5, or 1-10, or 1-15, or 1-20, or 1
-25, or 1-50, or 1-75, or 1-100, or more than 100 amino acid substitutions, insertions, additions, and / or deletions, wherein the substitutions are conservative, Or it may be non-conservative, or any combination thereof.

The term “CHL2 polypeptide derivative” refers to a polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, a CHL2 polypeptide fragment, a CHL2 polypeptide ortholog, or a CHL2 polypeptide as defined herein. Refers to peptide variants, which have been chemically modified. The term "CHL2 polypeptide derivative" also refers to a polypeptide encoded by a CHL2 polypeptide allelic variant or CHL2 polypeptide splice variant as defined herein, which is chemically modified. .

The term “mature CHL2 polypeptide” refers to a CHL2 polypeptide lacking the leader sequence. Mature CHL2 polypeptide may also include other modifications (e.g., more of the amino-terminus (with or without a leader sequence) and / or carboxyl-terminal <br/> end proteolytic processing of larger precursor Cleavage of small polypeptides, N-linked and / or O-linked glycosylation, etc.). An exemplary mature CHL2 polypeptide is represented by the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 6.

The term “CHL2 fusion polypeptide” refers to SEQ ID NO: 2, as defined herein.
Alternatively, the polypeptide shown in any of SEQ ID NO: 5, CHL2 polypeptide fragment, CHL2 polypeptide ortholog, CHL2 polypeptide variant,
Alternatively, it refers to a fusion of one or more amino acids (eg, a heterologous protein or peptide) at the amino or carboxy terminus of a CHL2 derivative. The term "CHL2
A "fusion polypeptide" also includes one or more amino acids at the amino or carboxyl termini of a polypeptide encoded by a CHL2 polypeptide allelic variant or CHL2 polypeptide splice variant as defined herein. Refers to fusion.

The term “biologically active CHL2 polypeptide” refers to a CHL2 polypeptide having at least one activity characteristic of a polypeptide comprising the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5. Further, the CHL2 polypeptide is
It may be active as an immunogen; that is, the CHL2 polypeptide comprises at least one epitope against which an antibody may be raised.

The term “isolated polypeptide” refers to (1) at least about 50% of a polypeptide, lipid, carbohydrate, or other substance found together in nature when isolated from a source cell. A polypeptide of the invention isolated from (2) an "isolated polypeptide" is not linked to all or part of the polypeptide to which it is naturally linked (
A polypeptide of the invention (by covalent or non-covalent interaction), (
3) a polypeptide of the invention that is operably linked (by covalent or non-covalent interactions) to a polypeptide that is not naturally linked, or (4) a polypeptide of the invention that does not occur in nature. Say. Preferably, the isolated polypeptide is any other contaminating polypeptide or other contaminant found in its natural environment, which has therapeutic, diagnostic, prophylactic, or research uses. (Preventing use) is substantially not included.

The term “identity” refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences, as is known in the art. In the art, "identity" also refers to the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between two or more nucleotide or two or more amino acid sequences. Means "Identity" is the match between the smaller of two or more sequences and the gap alignment (if any) focused by a particular mathematical model or computer program (ie, "algorithm"). Evaluate the same percentage of.

The term “similarity” relates to a concept, but in contrast to “identity”, “similarity” refers to a measure of relatedness that includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids and the rest are all non-conservative substitutions, then the percent identity and percent similarity are both 50%. In the same example, if there are five more positions that are conservative substitutions, the percent identity remains 50%, but the percent similarity is 7.
It is 5% (15/20). Thus, if there are conservative substitutions, the percent similarity between the two polypeptides will be higher than the percent identity between these two polypeptides.

The terms “naturally occurring” or “native” are found in nature when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, etc. Material that has not been manipulated by. Similarly, "non-naturally occurring" or "non-native" as used herein refers to
Refers to materials that are not found in nature or have been structurally modified or synthesized by humans.

The terms “effective amount” and “therapeutically effective amount” each refer to CHL as indicated herein.
2 refers to the amount of CHL2 polypeptide or CHL2 nucleic acid molecule used to support an observable level of one or more biological activities of a polypeptide.

The term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refers to a CHL2 polypeptide, CHL2 nucleic acid molecule, or pharmaceutical composition as a pharmaceutical composition. Refers to one or more formulation materials suitable for achieving or enhancing delivery of a CHL2 selective binding agent.

The term “antigen” is a molecule or a molecule that can be bound by a selective binding agent (eg, an antibody) and is further used in an animal to produce an antibody capable of binding an epitope of that antigen. Part. An antigen can have one or more epitopes.

The term “selective binding agent” refers to a molecule (with specificity for a CHL2 polypeptide (
Singular or plural). As used herein, the terms "specific" and "specificity" refer to the ability of a selective binding agent to bind a human CHL2 polypeptide and not a human non-CHL2 polypeptide. However, the selective binding agent may also be an ortholog of the polypeptide as described in either SEQ ID NO: 2 or SEQ ID NO: 5 (ie an interspecies variation thereof (eg mouse CHL2 polypeptide and rat CHL2 polypeptide)). It is understood that

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

The term “transfection” is used to refer to the uptake of heterologous or exogenous DNA by a cell, and the cell is “transfected” if the exogenous DNA has been introduced inside the cell membrane. . Many transfection techniques are well known in the art and disclosed herein. For example, Grah
am et al., 1973, Virology 52: 456; Sambrook et al., M.
olecular Cloning, A Laboratory Manual
(Cold Spring Harbor Laboratories, 19
89); Davis et al., Basic Methods in Molecular.
r Biology (Elsevier, 1986); and Chu et al., 198.
1, Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

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

Relatedness of Nucleic Acid Molecules and / or Polypeptides Related nucleic acid molecules include allelic or splice variants of the nucleic acid molecule of either SEQ ID NO: 1 or SEQ ID NO: 4, and above. It is understood to include sequences that are complementary to any of the nucleotide sequences of. A related nucleic acid molecule also contains or consists essentially of substitutions, modifications, additions and / or deletions of one or more amino acid residues compared to the polypeptide in either SEQ ID NO: 2 or SEQ ID NO: 5. A nucleotide sequence encoding a polypeptide of Such related CHL2 polypeptides include, for example, the addition and / or deletion of one or more N-linked or O-linked glycosylation sites, or the addition and / or deletion of one or more cysteine residues. obtain.

Related nucleic acid molecules also include CHL2 of either SEQ ID NO: 2 or SEQ ID NO: 5.
At least about 25 consecutive amino acids of the polypeptide, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150
Fragments of the CHL2 nucleic acid molecule encoding a polypeptide of 1 amino acid, or about 200 amino acids, or more than 200 amino acid residues.

In addition, related CHL2 nucleic acid molecules may also be CHL2 nucleic acid molecules of either SEQ ID NO: 1 or SEQ ID NO: 4 , or polypeptides under moderately or highly stringent conditions as defined herein. (the polypeptide SEQ ID NO: 2 or is any amino acid comprising sequence shown in one of SEQ ID NO: 5) as defined in the molecule encoding or nucleic acid fragment as defined herein, or herein, molecules that contain a completely complementary sequence to the hybridizes Zusuru nucleotide sequence of a nucleic acid fragment encoding the polypeptide. Hybridization probes can be prepared using the CHL2 sequences provided herein for screening a cDNA library , a genomic DNA library or a synthetic DNA library for related sequences. Regions of the CHL2 polypeptide DNA and / or amino acid sequence that show significant identity to a known sequence are readily determined using the sequence alignment algorithms described herein, and Regions can be used to design probes for screening.

The term “highly stringent conditions” refers to DNs where the sequences are highly complementary.
Refers to conditions designed to allow hybridization of the A strand and exclude hybridization of significantly mismatched DNA. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents (eg, formamide). An example of "highly stringent conditions" for hybridization and washing is 0.015 at 65-68 ° C.
M Sodium Chloride, 0.0015M Sodium Citrate or 0.015M Sodium Chloride at 42 ° C
CHL2oride), 0.0015M sodium citrate and 50% formamide. Sambrook, Fritsch and Maniatis, M
olecular Cloning: A Laboratory Manual
(2nd edition, Cold Spring Harbor Laboratory, 1
989); Anderson et al., Nucleic Acid Hybrids.
ation: A Practical Approach Chapter 4 (IRLP
See Less Limited).

More stringent conditions (eg higher temperature, lower ionic strength,
Higher formamide or other denaturants) can also be used, but the rate of hybridization is affected. Other agents may be included in the hybridization and wash buffers for the purpose of reducing non-specific and / or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1
% Sodium dodecyl sulfate, NaDodSO ₄ , (SDS), Ficoll (fi
Coll), Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is largely independent of pH.
Anderson et al., Nucleic Acid Hybridization
: A Practical Approach Chapter 4 (IRL Press
Limited).

Factors affecting the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by those of skill in the art to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation: T _m (° C.) = 81.5 + 16.6 (log [Na +]) + 0.41 (% G + C)
) -600 / N-0.72 (% formamide) where N is the length of the duplex formed and [Na +] is the molar concentration of sodium ion in the hybridization or wash solution. And% G +
C is the percentage of (guanine + cytosine) bases in the hybrid. For imperfectly matched hybrids, the melting temperature drops by about 1 ° C. for every 1% mismatch.

The term “moderately stringent conditions” refers to conditions under which a DNA duplex having a higher degree of base pair mismatch than can occur under “highly stringent conditions” can be formed. An example of a typical “moderately stringent condition” is 5
0.015M sodium chloride (sodium CHL2orid at 0-65 ° C)
e), 0.0015M sodium citrate or 0.015M at 37-50 ° C
Sodium CHL2oride, 0.0015M sodium citrate and 20% formamide. By way of illustration, "moderately stringent conditions" of 0.015 M sodium ion at 50 ° C allow a discrepancy of about 21%.

It will be appreciated by those skilled in the art that there is no absolute distinction between “highly stringent conditions” and “moderately stringent conditions”. For example, 0
． For 015M sodium ion (without formamide), a long, perfectly matched D
The melting temperature of NA is about 71 ° C. With a wash at 65 ° C. (at the same ionic strength), this allows a mismatch of about 6%. To capture the more closely related sequences, one of ordinary skill in the art can simply lower the temperature or increase the ionic strength.

A good estimate of the melting temperature in 1M NaCl ^* for oligonucleotide probes up to about 20 nt is given by: Tm = 2 ° C. per AT base pair + 4 ° C. per GC base pair ^* The sodium ion concentration in 6 × sodium citrate salt (SSC) is 1M. Suggs et al., Developmental Biology Usin.
g Purified Genes 683 (Brown and Fox, eds., 19)
81).

High stringency wash conditions for oligonucleotides are typically 6 ×.
SSC, 0 ° C to 5 than Tm of the oligonucleotide in 0.1% SDS
It is at a temperature lower by ℃.

In another embodiment, a related nucleic acid molecule comprises or consists of a nucleotide sequence that is at least about 70 percent identical to the nucleotide sequence set forth in either SEQ ID NO: 1 or SEQ ID NO: 4, Alternatively, it comprises or consists essentially of a nucleotide sequence that encodes a polypeptide that is at least about 70 percent identical to the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. In a preferred embodiment, the nucleotide sequence is about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95 percent to the nucleotide sequence set forth in either SEQ ID NO: 1 or SEQ ID NO: 4. 96, 97, 98, or 99 percent identical, or the nucleotide sequence is about 75 percent, or about 80 percent, or about 85 percent to the polypeptide sequence shown in either SEQ ID NO: 2 or SEQ ID NO: 5. Percent, or about 90 percent, or about 95
, 96, 97, 98, or 99 percent identical polypeptides. Related nucleic acid molecules encode a polypeptide that retains at least one activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.

Differences in the nucleic acid sequences can result in conservative and / or non-conservative modifications of the amino acid sequence as compared to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5.

Conservative modifications to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5 (
And corresponding modifications to the encoding nucleotides) result in a polypeptide having functional and chemical characteristics similar to those of the CHL2 polypeptide. In contrast, substantial modification in the functional and / or chemical characteristics of the CHL2 polypeptide may be achieved by selecting a substitution in the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5, which is Its effect on the maintenance of the following is significantly different: (a) the structure of the molecular scaffold in the substitution region, such as the sheet or helical conformation, (b) the charge of the molecule at the target site or Hydrophobicity, or (c) bulkiness of side chains.

For example, a “conservative amino acid substitution” can include a substitution of a native amino acid residue with a non-native residue that has little or no effect on the polarity or charge of the amino acid residue at that position. In addition, any native residue in the polypeptide can also be replaced by alanine, as previously described for "alanine scanning mutagenesis."

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

Naturally occurring residues can be divided into multiple classes based on common side chain properties: 1) Hydrophobicity: Norleucine, Met, Ala, Val, Leu, Ile; 2) Neutral hydrophilicity. 3) Acidity: Asp, Glu; 4) Basicity: Asn, Gln, His, Lys, Arg; 5) Residues affecting chain orientation: Gly, Pro; and 6). Aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into a region of the human CHL2 polypeptide that is homologous to the non-human CHL2 polypeptide, or in a heterologous region of this molecule.

In making such changes, the hydropathic index of amino acids (hydropat)
hic index) may be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. The hydropathy index is
The following are: isoleucine (+4.5); valine (+4.2); leucine (+3)
． 8); Phenylalanine (+2.8); Cysteine / cystine (+2.5);
Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1) .3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3. 5); lysine (-3.9); and arginine (-4.5)
.

[0077] in conferring biological function the interaction in the protein, the importance of the hydropathic amino acid index is generally understood in the art (Kyte et al., 1
982, J. Mol. Biol. 157: 105-31). It is known that certain amino acids can be replaced with other amino acids having a similar hydropathic index or score, and still maintain a similar biological activity. In making changes based on the hydropathic index, substitution of amino acids whose hydropathic index is within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. .

Substitution of similar amino acids can be made effectively on the basis of hydrophilicity (especially if the biologically functionally equivalent protein or peptide produced thereby is immunologically as in this case). (When intended for use in embodiments)
It is also understood in the art. The greatest local average hydrophilicity of a protein correlates to its immunogenicity and antigenicity, ie, the biological properties of the protein, when governed by the hydrophilicity of its flanking amino acids.

The following hydrophilicity values were assigned to these amino acid residues: arginine (
+3.0); Lysine (+3.0); Aspartic acid (+ 3.0 ± 1); Glutamic acid (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2) ); Glycine (0); Threonine (-0.4); Proline (-
0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (
-1.0); methionine (-1.3); valine (-1.5); leucine (-1.
8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (
-2.5); and tryptophan (-3.4). When making changes based on similar hydrophilicity values, substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are preferred. , Even more particularly preferred. From the primary amino acid sequence, epitopes can also be identified on the basis of hydrophilicity. These regions are also referred to as "epitope core regions".

Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions may be used to identify key residues in a CHL2 polypeptide or may increase or decrease the affinity of a CHL2 polypeptide described herein. Exemplary amino acid substitutions are listed in Table I.

[0081]

[Table 1] One of ordinary skill in the art can determine suitable variants of the polypeptides set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 using well known techniques. To identify suitable regions of the molecule that can be altered without destroying biological activity, one of skill in the art can target regions that are not considered important for activity. For example, if a similar polypeptide with similar activity is known, from the same species or from another species, one of skill in the art will recognize the amino acid sequence of a CHL2 polypeptide as such similar polypeptide. You can compare. Such comparisons can be used to identify residues and portions of molecules that are conserved between similar polypeptides. It is understood that changes in regions of the CHL2 molecule that are not conserved for such similar polypeptides do not appear to adversely affect the biological activity and / or structure of the CHL2 polypeptide. It One of ordinary skill in the art also knows that chemically similar amino acids can be substituted for naturally occurring residues that maintain activity (replacement of conservative amino acid residues), even in relatively conserved regions. ). Thus, even regions that may be important for biological activity or structure may be subject to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure. .

In addition, one of skill in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such comparisons, one can predict the importance of amino acid residues in the CHL2 polypeptide that correspond to amino acid residues that are important for activity or structure in similar polypeptides. One of skill in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues in the CHL2 polypeptide.

One of skill in the art can also analyze the three-dimensional structure and amino acid sequence for the three-dimensional structure in similar polypeptides. In view of such information, those skilled in the art
Alignments of amino acid residues in CHL2 polypeptides can be predicted with respect to their three-dimensional structure. One of skill in the art can choose not to make abrupt changes to amino acid residues predicted to be present on the surface of the protein. Because such residues may be involved in important interactions with other molecules. In addition, one of skill in the art can generate test variants that contain a single amino acid substitution at each amino acid residue. These variants can be screened using activity assays known to those of skill in the art. Such variants can be used to gather information about suitable variants. For example, if a change to a particular amino acid residue is found to result in disruption, undesirably diminishing, or undesired activity, then variants with such changes are avoided. In other words, on the basis of information gathered from such routine experimentation, one of skill in the art would appreciate that amino acids for which further substitutions should be avoided either alone or in combination with other mutations. , Can be easily determined.

Numerous scientific publications have been devoted to the prediction of secondary structure. Mault, 199
6, Curr. Opin. Biotechnol. 7: 422-27; Chou.
Et al., 1974, Biochemistry 13: 222-45; Chou et al.
1974, Biochemistry 113: 211-2; Chou et al., 1
978, Adv. Enzymol. Relat. Areas Mol. Biol
． 47: 45-48; Chou et al., 1978, Ann. Rev. Biochem
． 47: 251-276; and Chou et al., 1979, Biophys. J.
26: 367-84. Moreover, computer programs are currently available to assist with predicting secondary structure. One method of estimating secondary structure is based on homology modeling. For example, two polypeptides or proteins with greater than 30% sequence identity or greater than 40% similarity often have similar structural topologies. Protein structure database (PDB)
The recent growth of A. has provided enhanced predictability of secondary structure, including the number of potential folds in the structure of a polypeptide or protein. Holm et al., 199
9, Nucleic Acids Res. 27: 244-47.
It has been suggested that there is a limited number of folds in a given polypeptide or protein, and that once a significant number of structures have been analyzed, structure estimation becomes dramatically more accurate (Brenner. Et al., 1997, Curr. O.
pin. Struct. Biol. 7: 369-76).

A further method of estimating secondary structure is by “threading”.
) ”(Jones, 1997, Curr. Opin. Struct. Biol.
7: 377-87; Sippl et al., 1996, Structure 4: 15-.
19), "Profile Analysis" (Bowie et al., 1991, Science, 2).
53: 164-70; Gribskov et al., 1990, Methods Enz.
ymol. 183: 146-59; Gribskov et al., 1987, Proc.
Nat. Acad. Sci. U. S. A. 84: 4355-58), and "Evolutionary Linkages" (see Holm et al., Supra, and Brenner et al., Supra).

Preferred CHL2 polypeptide variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Is mentioned. In one embodiment, the CHL2 polypeptide variant comprises a greater or lesser number of N-linked glycosylation sites than the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. The N-linked glycosylation site is the sequence Asn-X-Ser or Asn-X-.
The amino acid residue characterized by Thr, where X is indicated, can be any amino acid residue other than proline. Substitution of amino acid residues to create this sequence provides a potential new site for the addition of N-linked carbohydrate chains. Alternatively, substitutions that eliminate this sequence remove the existing N-linked carbohydrate chain. 1
Rearrangements of N-linked carbohydrate chains also eliminate one or more N-linked glycosylation sites (typically the naturally occurring glycosylation sites) and create one or more new N-linked sites. Provided. Further preferred CHL2 variants include:
Cysteine, in which one or more cysteine residues are deleted as compared to the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or substituted with another amino acid (eg, serine). Examples include modified forms. The cysteine variant is C
It is useful when the HL2 polypeptide must be refolded into a biologically active conformation, eg, after isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have a cysteine residue in even number to minimize interactions resulting from cysteine unpaired.

[0087] In other embodiments, related nucleic acid molecules have at least one has an amino acid insertion, and the activity of a polypeptide according to any one of the polypeptide is SEQ ID NO: 2 or SEQ ID NO: 5, SEQ comprises a nucleotide sequence encoding a polypeptide according to any one of the numbers 2 or SEQ ID NO: 5, or consisting of the sequence, or at least one amino acid deletion, wherein the polypeptide is SEQ ID NO: 2 Alternatively, it comprises or consists of a nucleotide sequence that encodes the polypeptide according to either SEQ ID NO: 2 or SEQ ID NO: 5, which has the activity of the polypeptide according to SEQ ID NO: 5. A related nucleic acid molecule is also one in which the polypeptide has a carboxyl-terminal and / or amino-terminal truncation, and further wherein the polypeptide has an activity of the polypeptide according to either SEQ ID NO: 2 or SEQ ID NO: 5. Comprising or consisting of a nucleotide sequence that encodes the polypeptide of either SEQ ID NO: 2 or SEQ ID NO: 5. A related nucleic acid molecule also includes at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, carboxyl terminal truncations, and amino terminal truncations, wherein the polypeptide is SEQ ID NO: 2 or It comprises or consists of a nucleotide sequence coding for the polypeptide according to either SEQ ID NO: 2 or SEQ ID NO: 5, which has the activity of the polypeptide according to SEQ ID NO: 5.

Further, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, or any of the other CHL2 polypeptides, may be fused to a homologous polypeptide to form a homodimer, or a heterologous polypeptide. To form a heterodimer. Heterologous peptides and polypeptides include, but are not limited to, epitopes that allow detection and / or isolation of CHL2 fusion polypeptides; transmembrane receptor proteins or portions thereof (eg, extracellular domain). Or a transmembrane domain and an intracellular domain); a ligand or portion thereof that binds to a transmembrane receptor protein; an enzyme or portion thereof that is catalytically active; a polypeptide or peptide that promotes oligomerization (eg, leucine zipper domain) ); A polypeptide or peptide that increases stability (eg, an immunoglobulin constant region); and a polypeptide comprising the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, or different from other CHL2 polypeptides Therapeutic activity Polypeptide having.

[0089] fusion polypeptide comprising an amino acid sequence as set forth in any of SEQ ID NO: 2 or SEQ ID NO: 5 or other CHL2 polypeptide may be such at either the amino or carboxyl terminus. Fusion may be direct fusion using no linker or adapter <br/> chromatography molecule may be through a linker or adapter molecule. The linker or adapter molecule can be one or more amino acid residues, typically about 20 to about 50 amino acid residues. Linker or adapter molecules may also be designed with cleavage sites for DNA restriction endonucleases or proteases to allow separation of fused moieties. It is understood that, once constructed, the fusion polypeptide can be derivatized according to the methods described herein.

In a further embodiment of the invention, a polypeptide comprising the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5, or another CHL2 polypeptide is fused to one or more domains of the Fc region of human IgG. To do. Antibodies include two functionally independent moieties: the variable domain known as "Fab" that binds antigens and is involved in effector functions such as complement activation and attack by phagocytic cells. A constant domain known as "Fc". Fc has a long serum half-life, while Fab has a short life span. Capon et al., 1989, Nature.
337: 525-31. When assembled with a therapeutic protein, the Fc domain may provide a longer half-life, or may perform functions such as Fc receptor binding, protein A binding, complement fixation, and perhaps placental transfer. Can be incorporated. Ibid. Table II summarizes the use of certain Fc fusions known in the art.

[0091]

[Table 2] In one example, the human IgG hinge region, CH2 region, and CH3 region are:
The CHL2 polypeptide can be fused at either the amino or carboxyl terminus using methods known to those of skill in the art. In another example, human IgG
The hinge, CH2, and CH3 regions of E. coli can be fused at either the amino or carboxyl termini of the CHL2 polypeptide fragment (eg, the putative extracellular portion of the CHL2 polypeptide).

The resulting CHL2 fusion polypeptide can be purified by use of a Protein A affinity column. Peptides and proteins fused to the Fc region were found to exhibit a substantially longer half-life in vivo than their unfused counterparts. Also, fusion to the Fc region allows for dimerization / multimerization of the fusion polypeptide. The Fc region can be a naturally occurring Fc region or can be modified to improve certain qualities such as therapeutic quality, circulation time, or reduced aggregation.

Identity and similarity of related nucleic acid molecules and polypeptides can be readily calculated by known methods. One such method is Computation
al Molecular Biology (AM Lesk ed., Oxford)
d University Press 1988); Biocomputin
g: Informations and Genome Projects (D.
W. Smith, Academic Press 1993); Comput
er Analysis of Sequence Data (Part 1,
A. M. Griffin and H.C. G. Griffin, Humana Pr
ess 1994); von Heinle, Sequence Anal
ysis in Molecular Biology (Academic P
ress 1987); Sequence Analysis Primer (
M. Gribskov and J.M. Devereux, M .; Stockton
Press 1991); and Carillo et al., 1988, SIAM J.
． Applied Math. , 48: 1073, but are not limited thereto.

Preferred methods to determine identity and / or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are described in publicly available computer programs. A preferred computer program method for determining identity and similarity between two sequences is the GCG program package (GAP (Dever
eux et al., 1984, Nucleic Acids Res. 12: 387; G
genetics Computer Group, University of
Wisconsin, Madison, WI), BLASTP, BLASTN
, And FASTA (Altschul et al., 1990, J. Mol. Biol.
215: 403-10)), but is not limited thereto. BL
The ASTX program is the National Center for Biote
chnology Information (NCBI) and other sources (A
ltschul et al., BLAST Manual (NCB NLM NIH, Be.
thesda, MD); Altschul et al., 1990, supra). The well-known Smith Waterman algorithm can also be used to determine identity.

A particular alignment scheme for aligning two amino acid sequences can result in a match of only the short regions of these two sequences, and this small alignment region is 2
Very high sequence identity can be achieved even when there is no significant association between the two full length sequences. Thus, in a preferred embodiment, the selected alignment method (GAP program) results in an alignment over at least 50 consecutive amino acids of the claimed polypeptide.

For example, the computer algorithm GAP (Genetics Comput) is used.
er Group, University of Wisconsin, Mad
ison, WI), two polypeptides whose percent sequence identities are to be determined are aligned for optimal match of their respective amino acids (" matched span" determined by the algorithm). To be done. Gap opening penalty (this is calculated as 3 times the mean diagonal: "mean diagonal" means the comparison matrix (comp) used.
arison matrix) diagonal mean; “diagonal” is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and gap extension penalties (gap extensio).
n penalty) (this is typically a cap opening penalty of 0.
1 ×), as well as comparison matrices such as PAM 250 or BLOSUM 62 are used in combination with this algorithm. A standard comparison matrix is also used by this algorithm (Dayhoff et al., 5 Atlas).
of Protein Sequence and Structure (Appendix 3 1978) (PAM250 comparison matrix); Henikoff et al., 1992, P.
roc. Natl. Acad. Sci USA 89: 10915-19 (BL
OSUM 62 comparison matrix)).

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, 1970.
J. Mol. Biol. 48: 443-53; Comparison matrix: BLOSUM 62 (Henikof).
f et al., supra); Gap Penalty: 12 Gap Length Penalty: 4 Threshold of Similarity: 0 This GAP program is useful with the above parameters. The above parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisons include the following: Algorithm: Needleman and Wunsch, supra; Comparison matrix: matches = + 10, misma.
tch = 0 Gap Penalty: 50 Gap Length Penalty: 3 This GAP program is also useful with the above parameters. The above parameters are the default parameters for nucleic acid molecule comparison.

Program Manual, Wisconsin Package, Ve
rsion 9, September, 1997 including those described in other exemplary algorithms, gap opening penalties, gap Extended Deployment penalty, comparison matrix, and similarity threshold (threshold The o
f similarity ) can be used. The particular choices to be made will be apparent to those skilled in the art and the particular comparisons to be made (eg DNA vs. DNA,
Protein-to-protein, protein-to-DNA); and, further, whether the comparison is between a given pair of sequences, in which case GAP or BestFit is generally preferred, and one sequence and a large database sequence. between (in this case, FASTA or BLASTA are preferred) it depends on whether.

Nucleic Acid Molecules Nucleic acid molecules encoding polypeptides comprising the amino acid sequence of CHL2 polypeptides can be expressed in various ways (chemical synthesis, cDNA or genomic library screening, expression library screening, and / or cDNA PCR
Amplification, but is not limited thereto).

Recombinant DNA methods used herein generally refer to Sambrook.
Et al., Molecular Cloning: A Laboratory Man.
ual (Cold Spring Harbor Laboratory Pr
ess, 1989) and / or Current Protocols in
Molecular Biology (Edited by Ausubel et al., Green P)
ublishers Inc. And Wiley and Sons 1994
) Is the method described in. The present invention provides nucleic acid molecules as described herein, and methods for obtaining such molecules.

When the gene encoding the amino acid sequence of CHL2 polypeptide is identified from one species, all or part of this gene may be used as a probe to identify orthologs or related genes from the same species. Can be used. This probe or primer can be used to screen cDNA from various tissue sources that are suspected of expressing the CHL2 polypeptide. In addition, part or all of the nucleic acid molecule having a sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4 is used to screen a genomic library to identify the gene encoding the amino acid sequence of the CHL2 polypeptide. Can be identified and isolated. Typically, moderate or high stringency conditions are used for the screen to minimize the number of false positives obtained from this screen.

Nucleic acid molecules encoding the amino acid sequence of CHL2 polypeptides can also be identified by expression cloning using detection of positive clones based on the characteristics of the expressed protein. Typically, nucleic acid libraries are screened by binding an antibody or other binding partner (eg, receptor or ligand) to the cloned protein expressed and displayed on the host cell surface. The antibody or binding partner is modified with a detectable label to identify cells that express the desired clone.

These polynucleotides can be produced and the encoded polypeptides expressed according to recombinant expression techniques performed according to the instructions set forth below.
For example, one can readily generate large amounts of the desired nucleotide sequence by inserting the nucleic acid sequence encoding the amino acid sequence of CHL2 polypeptide into a suitable vector. These sequences can then be used to generate detection probes or amplification primers. Alternatively, the polynucleotide encoding the amino acid sequence of CHL2 polypeptide can be inserted into an expression vector. By introducing the expression vector into a suitable host, the encoded CHL2 polypeptide can be produced in large quantities.

Another method for obtaining the appropriate nucleic acid sequence is the polymerase chain reaction (PCR). In this method, the cDNA is converted into poly (
A) + RNA or total RNA. Two primers, typically complementary to two distinct regions of the cDNA encoding the amino acid sequence of the CHL2 polypeptide, were then added to this cDNA along with a polymerase such as Taq polymerase, The polymerase then amplifies the region of the cDNA between these two primers.

Another means of preparing nucleic acid molecules encoding the amino acid sequence of CHL2 polypeptide is described by Engels et al., 1989, Angew. Chem. Intl. Chapter. 28
: 716-34, chemical synthesis using methods well known to those skilled in the art. These methods include phosphotriester, phosphoramidite, and H-phosphonate methods for nucleic acid synthesis, among others. The preferred method for such chemical synthesis is polymer-supported synthesis using standard phosphoramidite chemistry. Typically, the DNA encoding the amino acid sequence of CHL2 polypeptide is several hundred nucleotides long. Nucleic acids longer than about 100 nucleotides can be synthesized as several fragments using these methods. These fragments can then be ligated together to form the full length nucleotide sequence of the CHL2 gene. Usually, the DNA fragment encoding the amino terminus of this polypeptide has ATG, which
Encodes a methionine residue. This methionine, depending on whether the polypeptide produced in the host cell is designed to be secreted from that cell, CH
It may or may not be present in the mature form of the L2 polypeptide. Other methods known to those of skill in the art may be used as well.

In certain embodiments, the nucleic acid variants contain codons that have been altered for optimal expression of the CHL2 polypeptide in a given host cell. The particular codon change depends on the CHL2 polypeptide and host cell selected for expression. Such "codon optimization" can be performed by a variety of methods, such as by selecting preferred codons for use in the highly expressed gene in a given host cell. For codon preference of highly expressed bacterial genes
Eco_high. Computer algorithms that incorporate codon frequency tables, such as "Cod", can be used, and University of Wisconsi
n Package Version 9.0 (Genetics Compu
ter Group, Madison, WI). Other useful codon frequency tables include "Celegans_high.cod", "Cele
gans_low. cod "," Drosophila_high.cod ",
"Human_high.cod", "Maize_high.cod", and "Yeast_high.cod."

In some cases, it may be desirable to prepare a nucleic acid molecule that encodes a CHL2 polypeptide variant. Nucleic acid molecules encoding the variants can be generated using site-directed mutagenesis, in which the primer has the desired point mutation, PCR amplification, or other suitable method (see Sambrook for a description of mutagenesis techniques).
Et al., Supra, and Ausubel et al., Supra). Chemical synthesis using the methods described by Engels et al., Supra, can also be used to prepare such variants. Other methods known to those of skill in the art may be used as well.

Vectors and Host Cells Nucleic acid molecules encoding the amino acid sequence of CHL2 polypeptide are inserted into an appropriate expression vector using standard ligation techniques. Vectors are typically chosen to be functional in the particular host cell used (ie, the vector is compatible with the host cell machinery, so that gene amplification and / or gene expression may occur). Sex). A nucleic acid molecule encoding the amino acid sequence of a CHL2 polypeptide can be amplified / expressed in a prokaryotic host cell, a yeast host cell, an insect (baculovirus system) host cell and / or a eukaryotic host cell. The choice of host cell depends in part on whether the CHL2 polypeptide is post-translationally modified (eg, glycosylated and / or phosphorylated). If so, a yeast host cell,
Insect host cells or mammalian host cells are preferred. For a review of expression vectors, see Meth. Enz. , Volume 185 (D.V. Goeddel, edited, Acad
See emric Press 1990).

[0110] Typically, expression vectors used in any host cell will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences (collectively referred to as "flanking sequences"), in certain embodiments, typically include one or more of the following nucleotide sequences: promoter,
One or more enhancer sequences, origins of replication, transcription termination sequences, complete intron sequences including donor and acceptor splice sites, sequences encoding leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, expressed A polylinker region for inserting a nucleic acid encoding a polypeptide, and a selectable marker element. Each of these sequences is discussed below.

Optionally, the vector may include a "tag" coding sequence (ie, an oligonucleotide molecule located at the 5'or 3'ends of the CHL2 polypeptide coding sequence); the oligonucleotide sequence may be polyHis. (For example, hexa
His), or another “tag” (eg, FLAG, HA (hemagglutinin (he
maglutinin) influenza virus)) or myc for which commercially available antibodies are present. This tag is typically fused to the polypeptide upon expression of the polypeptide and can serve as a means for affinity purification of CHL2 polypeptide from host cells. Affinity purification can be achieved, for example, by column chromatography using an antibody against the tag as an affinity matrix. If desired, the tag is then removed from the purified CHL2 polypeptide by various means, such as by using a particular peptidase for cleavage.

The flanking sequences can be homologous (ie, from the same species and / or strain as the host cell), heterologous (ie, from a species other than the host cell species or strain), or a hybrid (ie, Can be a combination of flanking sequences from more than one source) or can be synthetic, or the flanking sequences can be native sequences that function normally to regulate CHL2 polypeptide expression. .
Thus, the source of flanking sequences can be any prokaryotic or eukaryotic, any vertebrate or invertebrate, or any plant, provided the flanking sequences are functional in the host cell machinery. And can be activated by the host cell machinery.

The flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein, other than the CHL2 gene flanking sequences, have been previously identified by mapping and / or restriction endonuclease digestion, and thus using appropriate restriction endonucleases. , May be isolated from a suitable tissue source. In some cases, the full length nucleotide sequence of the flanking sequences may be known. Here, flanking sequences can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

If all or only part of the flanking sequences are known, PCR is used and / or a genomic library is constructed using appropriate oligonucleotides and / or flanking sequence fragments from the same or another species. It can be obtained by screening. Where the flanking sequence is not known, DN flanking sequences fragment of DNA containing, for example, a large fragment which may include a coding sequence or a different gene
Can be isolated from A. Isolation includes restriction endonuclease digestion to generate the appropriate DNA fragment, followed by isolation using agarose gel purification, Qiage.
n <(R)> column chromatography (Chatsworth, CA), or other methods known to those of skill in the art. Selection of the appropriate enzyme to achieve this end will be readily apparent to the skilled person.

[0115] origin of replication is typically a part of those prokaryotic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. Amplification of the vector to a particular copy number may be important for optimal expression of the CHL2 polypeptide in some cases. If the vector of choice does not contain an origin of replication site, it can be chemically synthesized based on known sequences and ligated into the vector. For example, plasmid pBR322 (New England Biolabs, Bev).
origin of replication from erly, MA) is suitable for most Gram-negative bacteria, and is different in origin (eg, SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or HPV or BPV). Papillomavirus) is useful for cloning vectors in mammalian cells. Generally, the origin of replication component is a mammalian expression vector (eg, the SV40 origin is
It is often not needed for) because it contains the early promoter.

A transcription termination sequence will typically be located 3 ′ to the termini of the polypeptide coding region and serves to terminate transcription. Usually, the transcription termination sequence in prokaryotic cells is a GC rich fragment followed by a poly-T sequence. Is this sequence can be easily cloned from a library or be city sales as part of a vector, which is readily synthesized using methods for nucleic acid synthesis such as hereinabove obtain.

Selectable marker genetic elements encode proteins necessary for the survival and growth of host cells grown in a selective culture medium. An exemplary selectable marker gene confers (a) resistance to a prokaryotic host cell to antibiotics or other toxins (eg, ampicillin, tetracycline, or kanamycin); (b) cell auxotrophy. Complement the deficiency; or (c) encode a protein that supplies important nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, ampicillin resistance gene, and tetracycline resistance gene. The neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells.

Other selection genes can be used to amplify the expressed gene. Amplification is the process by which genes, which are largely required for the production of proteins important for growth, are tandemly repeated within the chromosome of continuous production of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DH
FR) and thymidine kinase. Mammalian cell transformants are placed under selection pressure, where only the transformants are uniquely adapted to survive by virtue of the selection gene present in the vector. Selection pressure is the culturing of transformed cells under conditions in which the concentration of the selection factor in the medium changes continuously, thereby leading to the amplification of both the selection gene and the DNA encoding the CHL2 polypeptide. Imposed by. As a result, increased amounts of CHL2 polypeptide are synthesized from the amplified DNA.

The ribosome binding site is normally required for translation initiation of mRNA, and Sh
ine-Dalgarno sequence (prokaryote) or Kozak sequence (eukaryote)
Characterized by This element is typically located 3'to the promoter of the expressed CHL2 polypeptide and 5'to the coding sequence. Sh
The ine-Dalgarno sequence is variable, but is typically polypurine (ie, has a high AG content). Many Shine-Dalgar
No sequences have been identified, each of which can be readily synthesized using the methods described herein and used in prokaryotic vectors.

A leader sequence, or signal sequence, can be used to direct the CHL2 polypeptide from the host cell. Typically, the nucleotide sequence encoding the signal sequence is located in the coding region of the CHL2 nucleic acid molecule or is directly CHL2.
It is located 5'to the 2 polypeptide coding region. Many signal sequences have been identified, and any signal sequence that is functional in the host cell of choice may be used in combination with the CHL2 nucleic acid molecule. Therefore, the signal sequence is C
It may be homologous (naturally occurring) or heterologous to the HL2 nucleic acid molecule. Additionally, the signal sequence can be chemically synthesized using the methods described herein. To a large extent, CHL2 from the host cell is present due to the presence of the signal peptide.
Secretion of the polypeptide results in removal of the signal peptide from the secreted CHL2 polypeptide. The signal sequence can be a component of the vector, or it can be part of the CHL2 nucleic acid molecule inserted into the vector.

Use of either a nucleotide sequence encoding a native CHL2 polypeptide signal sequence linked to a CHL2 polypeptide coding region or a nucleotide sequence encoding a heterologous signal sequence linked to a CHL2 polypeptide coding region is used in the invention. Within the range of. The heterologous signal sequence selected should be one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native CHL2 polypeptide signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, or thermostable enterotoxin II leaders. To be done. For yeast secretion, the native CHL2 polypeptide signal sequence may be replaced by the yeast invertase, alpha factor, or acid phosphatase leader. For mammalian cell expression, the native signal sequence is satisfactory, but other mammalian signal sequences may be suitable.

In some cases, where glycosylation is desired in eukaryotic host cell expression systems, various signal peptides (p
resource) can be manipulated. For example, the peptidase cleavage site of a particular signal peptide may be altered or pro-sequenced.
) Can be added, which can also affect glycosylation. The final protein product may have one or more additional amino acids at position 1 (relative to the first amino acid of the mature protein) associated with expression, which may not be completely removed. For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site attached to the amino terminus. Alternatively, some enzyme cleavage sites may result in a slightly truncated form of the desired CHL2 polypeptide if the enzyme is cleaved at such regions within the mature polypeptide.

In many cases, transcription of the nucleic acid molecule is increased by the presence of one or more introns within the vector; which results in the polypeptide being produced in a eukaryotic host cell, particularly a mammalian host cell. This is especially true when The intron used may be naturally present in the CHL2 gene, especially if the gene used is the full-length genomic sequence or a fragment thereof. If the intron is not naturally present in the gene (for most cDNAs), the intron may be obtained from another source. The position of the intron with respect to flanking sequences and the CHL2 gene is generally important as the intron must be transcribed effectively. Therefore,
When the CHL2 cDNA molecule is transcribed, the preferred position of the intron is 3'to the transcription start site and 5'to the poly-A transcription termination sequence. Preferably,
Introns are placed on one or the other side of the cDNA (ie, 5'or 3 ') so as not to interfere with the coding sequence. The invention can be practiced with any intron from any source, including viruses, prokaryotes and eukaryotes (plants or animals), provided that the intron is present in the host cell into which it is inserted. It is compatible. Synthetic introns are also included herein. If desired, more than one intron can be used in the vector.

Expression and cloning vectors of the invention will typically contain a promoter that is recognized by the host organism and is operably linked to the molecule encoding the CHL2 polypeptide. A promoter is a non-transcribed sequence located upstream (ie, 5 ') to the start codon of a structural gene (generally about 100 to 1000 bp) that controls the transcription of the structural gene. Promoters are usually grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some changes in culture conditions (eg, the presence or absence of nutrients, or changes in temperature). On the other hand, constitutive promoters initiate continuous gene product production; that is, little or no regulation of the gene for gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to the DNA encoding the CHL2 polypeptide by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector. The native CHL2 promoter sequence is used to amplify and / or amplify a CHL2 nucleic acid molecule.
Or it can be used to direct expression. However, a heterologous promoter is preferred if it allows for higher transcription and higher production of expressed proteins as compared to the native promoter, and is compatible with the host cell line chosen for use.

Suitable promoters for use with prokaryotic hosts include the β-lactamase and lactose promoter systems; alkaline phosphatase; tryptophan (trp).
) Promoter system; and hybrid promoters (eg tac promoter). Other known bacterial promoters are also suitable. These sequences are publicly available so that one of skill in the art will ligate them to the desired DNA sequence using linkers or adapters required to supply any useful restriction sites. You can

Suitable promoters for use with yeast hosts are also well known in the art.
Yeast enhancers are advantageously used for use with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, polyoma virus, fowlpox virus, adenovirus (eg, Adenovirus 2),
Bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferred simian virus 40 (SV40
) Such as a promoter obtained from the genome of a virus. Other suitable mammalian promoters include heterologous mammalian promoters (eg, heat shock promoter and actin promoter).

Additional promoters that may be of interest in controlling CHL2 gene expression include, but are not limited to: SV40 early promoter region (Bern).
oist and Chamber, 1981, Nature 290: 304.
-10); CMV promoter; promoter contained in the 3'long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787).
-97); herpes thymidine kinase promoter (Wagner et al., 1981).
, Proc. Natl. Acad. Sci. U. S. A. 78: 1444-45
); Regulatory sequences of the metallothionein gene (Brinster et al., 1982, Na
296: 39-42); a prokaryotic expression vector such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. N.
atl. Acad. Sci. U. S. A. , 75: 3727-31); or t
ac promoter (DeBoer et al., 1983, Proc. Natl. Acad.
Sci. U. S. A. , 80: 21-25). Also of interest are the following animal transcriptional regulatory regions that exhibit tissue specificity and have been utilized in transgenic animals: Elastase I gene regulatory region active in pancreatic acinar cells (Swift).
Et al., 1984, Cell 38: 639-46; Ornitz et al., 1986, C.
old Spring Harbor Symp. Quant. Biol. Fifty
: 399-409 (1986); MacDonald, 1987, Hepato.
7: 425-515); an insulin gene regulatory region active in pancreatic β cells (Hanahan, 1985, Nature 315: 115-22).
); Immunoglobulin gene regulatory region active in lymphoid cells (Grossc)
hedl et al., 1984, Cell 38: 647-58; Adames et al. , 19
85, Nature 318: 533-38; Alexander et al., 1987.
, Mol. Cell. Biol. , 7: 1436-44); mouse mammary adenocarcinoma virus regulatory region (Leder) active in testis, breast, lymphocytes, and mast cells.
Et al., 1986, Cell 45: 485-95); the albumin gene regulatory region active in liver (Pinkert et al., 1987, Genes and Dev).
el. 1: 268-76); the α-fetoprotein gene regulatory region active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol., 5:
1639-48; Hammer et al., 1987, Science 235: 53-.
58); α1-antitrypsin gene regulatory region active in liver (Kelsey)
Et al., 1987, Genes and Devel. 1: 161-71); β-globin gene regulatory region active in myeloid cells (Mogram et al., 1985.
Nature 315: 338-40; Kollias et al., 1986, Cell.
46: 89-94); a myelin-based protein gene regulatory region active in oligodendrocytes of the brain (Readhead et al., 1987, Cell 48:
703-12); myosin light chain-2 gene regulatory region (Sa) active in skeletal muscle.
ni, 1985, Nature 314: 283-86); and the gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 19).
86, Science 234: 1372-78).

Enhancer sequences can be inserted into the vector to increase transcription of the DNA encoding the CHL2 polypeptides of the present invention by higher eukaryotes . Enhancers act on promoters to increase transcription, usually about 10-300b.
p of is death to act d Remento of the length of the DNA. Enhancers are relatively independent in orientation and position. It has been found 5 'and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (eg, globin, elastase, albumin, α-fetoprotein and insulin). However, typically viral-derived enhancers are used. SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer and adenovirus enhancers are exemplary increase Tsuyoe Remento for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5 'or 3' to CHL2 nucleic acid molecule, it is typically located 5 'from the promoter.

The expression vector of the present invention may be constructed from a starting vector such as a commercially available vector. Such a vector may or may not contain all desired flanking sequences. If one or more of the flanking sequences described herein are not already in the vector, then they can be obtained individually and ligated into the vector. The methods used to obtain each of the flanking sequences are well known to those of skill in the art.

Preferred vectors for carrying out the present invention include bacterial host cells, insect host cells,
And a vector compatible with mammalian host cells. Such vectors include, among others, pCRII, pCR3, and pcDNA3.1 (Invitrog
en, San Diego, CA), pBSII (Stratagene, La)
Jolla, CA), pET15 (Novagen, Madison, WI)
, PGEX (Pharmacia Biotech, Piscataway, N
J), pEGFP-N2 (Clontech, Palo Alto, CA), p
ETL (BlueBacII, Invitrogen), pDSR- α (PCT
Publication number WO 90/14363) and pFastBacDual (Gib
co-BRL, Grand Island, NY).

[0131]   Further suitable vectors include, but are not limited to, cosmids, plasmids, or
Or modified viruses, these vector systems can be used with selected host cells.
It is understood that and must be compatible. Such vectors include:
Without limitation, a Bluescript® plasmid derivative (high copy
Number ColEl-based phagemid, Stratagene Cloning
  Systems, La Jolla CA), Taq amplification PCR product
PCR cloning plasmids designed to ^TM   TA Cloning (registered trademark) Kit, PCR2.1 (registered trademark)
Rasmid derivatives, Invitrogen, Carlsbad, CA), and
Mammalian vector such as baculovirus expression system, yeast vector or virus
Svector (pBacPAK plasmid derivative, Clontech, Palo
Alto, CA).

After the vector has been constructed and the nucleic acid molecule encoding the CHL2 polypeptide has been inserted into the vector at an appropriate site, the complete vector can be inserted into a host cell suitable for amplification and / or polypeptide expression. . CHL2 transformation into a selected host cell expression vectors for polypeptides, transfection, infection, calcium chloride, electroporation, microinjection, Ripofeku cane down, DEAE-dextran method, or other known as technology, Can be achieved by well-known methods including various methods. The method chosen will be in part a function of the type of host cell used. These methods and other suitable methods are well known to those skilled in the art, for example, Sambrook et al., Described supra.

The host cell can be a prokaryotic host cell (eg, E. coli) or a eukaryotic host cell (eg, yeast cell, insect cell, or vertebrate cell). The host cells, when cultured under suitable conditions, synthesize the CHL2 polypeptide, which can subsequently be harvested from the culture medium ( if the host cell secretes the polypeptide into the medium ) or directly. Collected from host cells that produce the polypeptide (if the polypeptide is not secreted). Selection of the appropriate host cell will depend on various factors such as desired expression levels, polypeptide modifications desired or necessary for activity (eg, glycosylation or phosphorylation), and ease of folding into biologically active molecules. Depends on factors.

Many suitable host cells are known in the art, many of which are American.
an Type Culture Collection (ATCC), Man
available from Assass, VA. Examples include, but are not limited to, Chinese hamster ovary cells (CHO), CHO DHFR (−) cells (Urlaub).
Et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 97: 4
216-20), human embryonic kidney (HEK) 293 or 293T cells, or mammalian cells such as 3T3 cells. Methods for selection and transformation, culture, amplification, screening, product production, and purification of suitable mammalian host cells are known in the art. Other suitable mammalian cell lines are monkey COS
-1 and COS-7 cell lines, and CV-1 cell line. Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines derived from in vitro cultures of primary tissues, as well as primary explants are also suitable. Candidate cells are
The selection gene may be genotypically deficient or may include a preferentially acting selection gene. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, Swiss-derived 3
Examples include T3 strain, Balb-c or NIH mouse, BHK or Hak hamster cell line. Each of these cell lines is known and available to those of skill in the art of protein expression.

Similarly, bacterial cells are useful as suitable host cells for the present invention. For example, E
． coli (eg, HB101, DH5α, DH10, and MC1061)
Various strains of S. alba are well known as host cells in the field of biotechnology. B. su
btilis, Pseudomonas spp. , Other Bacillus
pp. , Streptomyces spp. Various strains such as can also be used in the method.

Many strains of yeast cells known to those of skill in the art are also available as host cells for expression of the polypeptides of the invention. Examples of preferable yeast cells include Sa
ccharomyces cerevisiae and Pichia pasto
ris is mentioned.

Furthermore, if desired, insect cell lines can be utilized in the methods of the invention.
Such systems are described, for example, in Kitts et al., 1993, Biotechnique.
s, 14: 810-17; Lucklow, 1993, Curr. Opin. B
iotechnol. 4: 564-72; and Lucklow et al., 1993,
J. Virol. , 67: 4566-79. Preferred insect cells are
Sf-9 and Hi5 (Invitrogen).

Transgenic animals can also be used to express glycosylated CHL2 polypeptides. For example, transgenic milk-producing animals (eg, cows or goats) can be used to obtain the glucosylated polypeptides of the invention in animal milk. Plants may also be used to produce CHL2 polypeptides, but in general, the glycosylation that occurs in plants is different from that produced in mammalian cells and is not suitable for human therapy. Can occur.

Polypeptide Production Host cells containing the CHL2 polypeptide expression vector can be cultured using standard media well known to those of skill in the art. This medium usually contains all the nutrients necessary for cell growth and survival. E. Suitable media for culturing E. coli cells include, for example, Luria Broth (LB) and / or Terrific
Broth (TB) is mentioned. Suitable media for culturing eukaryotic cells include the Roswell Park Memorial Institute.
medium 1640 (RPMI 1640), Minimal Ese
ntial Medium (MEM) and / or Dulbecco's M
modified Eagle Medium (DMEM), all of which can be supplemented with serum and / or growth factors required for the particular cell line being cultured. Appropriate media for insect cells are yeast rate (y
Eatate), lactalbumin hydrolysates, and / or Grace's medium supplemented with fetal bovine serum.

Typically, antibiotics or other compounds useful for the selective growth of transfected or transformed cells are added to the medium as a supplement.
The compound used is detectable by the selectable marker element present on the plasmid with which the host cell is transformed. For example, if the selectable marker element is kanamycin resistance, the compound added to the culture medium is kanamycin. Other compounds for selective growth include ampicillin, tetracycline, and neomycin.

The amount of CHL2 polypeptide produced by a host cell can be evaluated using standard methods known in the art. Such methods include, but are not limited to, Western blot analysis, SDS-polyacrylamide gel electrophoresis,
Activity assays such as non-denaturing gel electrophoresis, high performance liquid chromatography (HPLC) separations, immunoprecipitation, and / or DNA binding gel migration assays are included.

If the CHL2 polypeptide is designed to be secreted from the host cell, the majority of the polypeptide may be found in cell culture medium. However, if the CHL2 polypeptide is not secreted from the host cell, it is present in the cytoplasm and / or nucleus (for eukaryotic host cells) or in the cytosol (for Gram-negative bacterial host cells).

For CHL2 polypeptides located in the host cell cytoplasm and / or nucleus (for eukaryotic host cells) or in the cytosol (for bacterial host cells), intracellular material (inclusion bodies for Gram-negative bacteria). Can be extracted from the host cells using any standard technique known to those of skill in the art. For example, host cells can be lysed to release their periplasm / cytoplasmic content by French press, homogenization, and / or sonication followed by centrifugation.

When the CHL2 polypeptide forms inclusion bodies within the cytosol, the inclusion bodies can often be associated with inner and / or outer cell membranes and are therefore primarily found in pellet material after centrifugation. The pellet material can then be treated at pH extremes or at an alkaline pH in the presence of a reducing agent such as dithiothreitol or at an acidic pH in the presence of triscarboxyethylphosphine.
Can be treated with a chaotropic agent, such as a detergent, guanidine, a guanidine derivative, urea, or a urea derivative, to release, cleave, and dissolve the inclusion bodies. The lysed CHL2 polypeptide can then be analyzed using gel electrophoresis, immunoprecipitation and the like. When it is desired to isolate a CHL2 polypeptide, isolation is described herein and in Marston et al., 1990, Meth. En
z. , 182: 264-75, can be achieved using standard methods.

In some cases, the CHL2 polypeptide may not be biologically active upon isolation. "Refolding," that is, a variety of methods for converting a polypeptide into its tertiary structure and creating disulfide linkages, can be used to duplicate biological activity. Such methods include the step of exposing the solubilized polypeptide to a pH (usually above 7) and the presence of a particular concentration of chaotropic agent. The choice of chaotropic agent is very similar to the options used for inclusion body lysis, but usually chaotropic agents are used at low concentrations and are not necessarily the same as the chaotropic agents used for lysis. Often, the refolding / oxidation solution also contains a reducing agent, or a specific ratio of reducing agent and its oxidized form, to generate a specific redox potential, resulting in disulfide shuffling that results in the formation of cysteine bridges in the protein. To enable. Some commonly used redox couples include cysteine / cystamine, glutathione (GSH) / dithiobisGS
H, copper (II) chloride, dithiothreitol (DTT) / dithian DTT, and 2,2-mercaptoethanol (bME) / dithio-b (ME).
In many cases, cosolvents may be used or may be necessary to increase the efficiency of refolding, and a more common reagent used for this purpose is glycerol, of various molecular weights. Examples thereof include polyethylene glycol and arginine.

When inclusion bodies are not formed to a significant extent in the expression of CHL2 polypeptide, the polypeptide is found primarily in the supernatant after centrifugation of cell homogenates. The polypeptide can be further isolated from the supernatant using methods as described herein.

Purification of CHL2 polypeptides from solution can be accomplished using a variety of techniques. The polypeptide is hexahistidine (CHL2 polypeptide / hexaHi
s) or other small peptides such as FLAG (Eastma).
n Kodak Co. , New Haven, CT) or myc (Invi
trogen, Carlsbad, Calif.)) was synthesized in one step by passing the solution through an affinity column having a high affinity for the tag when the column matrix was synthesized to include either at its carboxy or amino terminus. .

For example, polyhistidine binds to nickel with great affinity and specificity. Therefore, nickel affinity columns (eg Qiage
n (R) nickel column) can be used for the purification of CHL2 polypeptide / polyHis. For example, Current Protocols i
n Molecular Biology § 10.11.8 (Ausube
l et al., eds. , Green Publishers Inc. and Wi
See ley and Sons 1993).

In addition, CHL2 polypeptides can be purified by the use of monoclonal antibodies that can specifically recognize and bind the CHL2 polypeptide.

Other suitable means for purification include, but are not limited to, affinity chromatography, immunoaffinity chromatography, ion exchange chromatography, molecular sieve chromatography, HPLC, electrophoresis (native gel electrophoresis). , Followed by gel elution and preparative isoelectric focusing (“Isoprime” machine / technology, Hoefer Scientific).
c, San Francisco, CA). In some cases, two or more purification techniques may be combined to achieve increased purity.

CHL2 polypeptides have also been described by Merrifield et al., 1963, J. Am. Am
． Chem. Soc. 85: 2149; Houghten et al., 1985, Pro.
c Natl Acad. Sci. USA 82: 5132; and Stewa.
rt and Young, Solid Phase Peptide Synth
chemistry (Pierce Chemical Co. 1984), using techniques known in the art, such as chemical synthesis methods (eg, solid phase peptide synthesis). Such polypeptides can be synthesized with or without a methionine at the amino terminus. Chemically synthesized CHL2 polypeptides can be oxidized using the methods described in these references to form disulfide bridges. A chemically synthesized CHL2 polypeptide is expected to have comparable biological activity to the corresponding CHL2 polypeptide that is recombinantly produced or purified from natural sources, and thus recombinant. Alternatively it can be used interchangeably with the native CHL2 polypeptide.

Another means of obtaining CHL2 polypeptides is by purification from biological samples such as tissue and / or fluids of the source in which the CHL2 polypeptides are naturally found. Such purification can be performed using methods for protein purification as described herein. During purification, the presence of CHL2 polypeptides can be monitored, for example, using antibodies prepared against recombinantly produced CHL2 polypeptides or peptide fragments thereof.

Many additional methods for producing nucleic acids and polypeptides are known in the art, and this method can be used to produce polypeptides with specificity for CHL2 polypeptides. For example, Roberts et al., 19
97, Proc. Natl. Acad. Sci. U. S. A. 94: 12297
See -303. This describes the production of fusion proteins between mRNA and its encoded peptide. Roberts, 1999, Curr. Op
in. Chem. See also Biol 3: 268-73. In addition, US Pat. No. 5,824,469 describes methods for obtaining oligonucleotides capable of performing particular biological functions. This procedure involves generating a heterogeneous pool of oligonucleotides, each having a 5'randomized sequence, a central preselected sequence, and a 3'randomized sequence. The resulting heterologous pool is introduced into a population of cells that do not exhibit the desired biological activity. Subpopulations of cells are then screened for those exhibiting the desired biological function. From that subpopulation, oligonucleotides that can perform the desired biological function are isolated.

US Pat. Nos. 5,763,192; 5,814,476; 5,72.
3,323; and 5,817,483 describe processes for producing peptides or polypeptides. This is accomplished by producing stochastic genes or fragments thereof and then introducing those genes into host cells that produce one or more proteins encoded by the stochastic genes. The host cell is then screened to identify those clones that produce the peptide or polypeptide with the desired activity.

Another method for producing a peptide or polypeptide is Athersys
, Inc. PCT / US98 / 20094 (WO99 / 15
650) ("Random Activation of Gene Expr
(known as "session for Gene Discovery" (known as RAGE-GD)), which involves activation of endogenous gene expression or gene overexpression by in situ recombination methods. For example, expression of an endogenous gene is activated or increased by incorporating regulatory sequences into target cells that may activate expression of the gene by heterologous or aberrant recombination. This target DNA is first subjected to radiation and then inserted into the gene promoter. This promoter is finally placed in front of the gene and initiates transcription of the gene. This results from the expression of the desired peptide or polypeptide.

These methods can be used to generate a comprehensive CHL2 polypeptide expression library, which can be followed by a variety of assays (eg, biochemical, cellular and whole organism assays (eg. It is understood that it can be used for high throughput phenotypic screening, eg in plants, mice etc.)).

(Synthesis) It will be appreciated by those of skill in the art that the nucleic acid and polypeptide molecules described herein can be produced by recombinant and other means.

Selective Binding Agent The term “selective binding agent” refers to a molecule that has specificity for one or more CHL2 polypeptides. Suitable selective binding agents include, but are not limited to, antibodies and their derivatives, polypeptides, and small molecules. Suitable selective binding agents can be prepared using methods known in the art. Exemplary CHL2 polypeptide selective binding agents of the present invention are capable of binding a particular portion of a CHL2 polypeptide, thereby inhibiting the binding of the polypeptide to the CHL2 polypeptide receptor.

Selective binding agents such as antibodies and antibody fragments that bind CHL2 polypeptides are within the scope of the invention. This antibody is polyclonal, including monospecific polyclonal; monoclonal (MAb); recombinant; chimeric; humanized (
For example, CDR grafting); human; single chain; and / or bispecific; and fragments; variants; or derivatives thereof. Antibody fragments include those portions of antibodies that bind to an epitope on the CHL2 polypeptide. Examples of such fragments include Fab and F (ab ′) 2 fragments produced by enzymatic cleavage of full length antibodies. Other binding fragments include fragments produced by recombinant DNA techniques (eg, expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions).

Polyclonal antibodies specific for CHL2 polypeptides are generally produced in animals (eg, rabbits or mice) by multiple subcutaneous or intraperitoneal injections of CHL2 polypeptide and adjuvant. It may be useful to couple the CHL2 polypeptide to a carrier protein, which protein
It is immunogenic in the species immunized, such as keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregating agents such as alum are used to enhance the immune response. Following immunization, the animals are bled and the serum is assayed for anti-CHL2 antibody titer.

Monoclonal antibodies specific for CHL2 polypeptides are produced using any method provided to produce antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies are the hybridoma method of Kohler et al., 1975, Nature 256: 495-97, and the human B cell hybridoma method (Kozbor, 1984, J. Immunol.
133: 3001; Brodeur et al., Monoclonal Antibod.
y Production Techniques and Applicat
ions 51-63 (Marcel Dekker, Inc., 1987)).
Is mentioned. Hybridoma cell lines that produce monoclonal antibodies that react with CHL2 polypeptides are also provided by the invention.

The monoclonal antibodies of the invention can be modified for use as therapeutic agents. One embodiment is a “chimeric” antibody, the heavy chain (H) and / or light chain (
Part of L) is derived from a particular species or is identical or homologous to the corresponding sequence in an antibody of a particular antibody class or subclass, while the rest of the chain Is derived from another species or is identical or homologous to the corresponding sequence in an antibody belonging to another antibody class or subclass. Also included are fragments of these antibodies so long as the fragments of the antibody exhibit the desired biological activity. U.S. Pat. No. 4,816,567; Morrison
Et al., 1985, Proc. Natl. Acad. Sci. 81: 6851-55
checking ...

In another embodiment, the monoclonal antibodies of the invention are "humanized" antibodies. Methods for humanizing non-human antibodies are well known in the art. See U.S. Patents 5,585,089 and 5,693,762. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Humanization has been described, for example, in the art (Jones et al., 1986, Nature 3
21: 522-25; Riechmann et al., 1998, Nature 332.
: 323-27; Verhoeyen et al., 1988, Science 239:
1534-36), using the method described in 1534-36).
By substituting at least a portion of a corresponding region of the human antibody.

Human antibodies that bind CHL2 polypeptides are also encompassed by the invention. Using a transgenic animal (eg, a mouse) that is capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin products, such an antibody is used to generate a CHL2 polypeptide antigen (ie, at least 6). Individual contiguous amino acids), and optionally conjugated to a carrier. For example, Jakobovits et al., 1993, Proc. Natl
． Acad Sci. 90: 2551-55; Jakobovits et al., 199.
3, Nature 362: 255-58; Bruggermann et al., 199.
3, Year in Immuno. See 7:33. In one method, such transgenic animals disable the endogenous loci encoding heavy and light chain immunoglobulins herein and insert human heavy and light chain proteins into their genome. Is produced by The partially modified animal, which has less than the full complement of variants, is then cross-breaded to obtain an animal with all of the desired immune system variants. When the immunogen is administered, these transgenic animals have an antibody with a human (eg, non-mouse) amino acid sequence, which amino acid sequence contains modified regions that are immunospecific for these antigens. To produce. For example, PCT application number PC
See T / US96 / 05928 and PCT / US93 / 06926.
Further methods are described in US Pat. No. 5,545,807, PCT application number PCT / US.
91/245 and PCT / GB89 / 01207, and European Patent No. 546.
073B1 and 546073A1. Human antibodies can also be produced by expression of recombinant DNA in host cells or expression in hybridoma cells as described herein.

In an alternative embodiment, human antibodies can also be produced from phage display libraries (Hoogenboom et al., 1991, J. Mol. Biol.
． 227: 381; Marks et al., 1991, J. Am. Mol. Biol. 222:
581). These handle mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by binding to selected antigens. One such technique is PCT application number PCT
/ US98 / 17364, which describes the isolation of high affinity and functional antagonist antibodies for MPL- and msk-receptors using such an approach.

Chimeric, CDR-grafted, and humanized antibodies are typically produced by recombinant methods. Nucleic acid encoding the antibody is introduced into a host cell and expressed using the materials and procedures described herein. In a preferred embodiment, the antibody is treated in mammalian host cells (eg CHO cells). Monoclonal (eg, human) antibodies are as described herein.
It can be produced by expression of recombinant DNA in host cells or expression in hybridoma cells.

The anti-CHL2 antibodies of the invention can be used in any known assay method (eg, competitive binding assay, direct and indirect sandwich assay, and immunoprecipitation assay) for detection and quantification of CHL2 polypeptides. Can be used (S
ola, Monoclonal Antibodies: A Manual o
f Technologies 147-158 (CRC Press, Inc.,
1987)). This antibody binds the CHL2 polypeptide with an affinity that is appropriate for the assay method used.

[0168]   For diagnostic applications, in certain embodiments, the anti-CHL2 antibody is detectable
Can be labeled with different moieties. This detectable moiety is either direct or indirect
, And can be any moiety capable of producing a detectable signal. For example, detectable
This part is a radioactive isotope (for example,^ThreeH,¹⁴C,³²P,³⁵S,¹²⁵I, ⁹⁹ Tc,¹¹¹In, or⁶⁷Ga); fluorescent compound or chemiluminescent compound
(Fluorescein isothiocyanate, rhodamine, or luciferin),
Or enzymes (alkaline phosphatase, β-galactosidase, or hose
Radish peroxidase) (Bayer et al., 1990, Meth
． Enz. 184: 138-63).

Competitive binding assays rely on the ability of labeled standards (eg, CHL2 polypeptides, or immunologically active portions thereof) to test samples for binding to limited amounts of anti-CHL2 antibody. Compete with the sample (CHL2 peptide). The amount of CHL2 polypeptide in the test sample is inversely proportional to the amount of standard that binds the antibody. To facilitate determining the amount of standard that binds, the antibody is typically immunized before or after competition, and the standards and analytes that bind the antibody are separated from those that remain unbound. It can be conveniently separated.

The sandwich assay typically involves the use of two antibodies, each capable of binding to a different immune portion or epitope of the protein to be determined and / or quantified. In a sandwich assay, the test sample analyte is typically bound by a primary antibody that is immunized on a solid support, and then a secondary antibody is bound to the analyte to form a soluble three-part complex. Form. For example, US Patent No. 4
, 376, 110. The secondary antibody itself may be labeled with a detectable moiety (direct sandwich assay) or anti-labeled with a detectable moiety.
It can be measured using immunoglobulin antibodies (indirect sandwich assay). For example, one type of sandwich assay is the enzyme linked immunosorbent assay (ELISA), where the detectable moiety is an enzyme.

Selective binding agents, including anti-CHL2 antibodies, are also useful for in vivo imaging. An antibody labeled with a detectable moiety can be administered to an animal, preferably the bloodstream, and the presence and location of the labeled antibody in the host assayed. The antibody may be labeled with any moiety that is detectable in the animal, either by nuclear magnetic resonance, radiology, or other detection means known in the art.

Selective binding agents of the invention, including antibodies, can be used as therapeutic agents. These therapeutic agents are generally agonists or antagonists, which either enhance or decrease the biological activity of at least one CHL2 polypeptide, respectively. In one embodiment, an antagonist antibody of the invention specifically binds a CHL2 polypeptide and specifically binds a CHL2 polypeptide that is capable of inhibiting or eliminating the functional activity of a CHL2 polypeptide in vivo or in vitro. Antibody or binding fragment thereof. In a preferred embodiment, the selective binding agent (eg, antagonist antibody) is at least about 50%, and preferably at least about 80% CH.
Inhibits the functional activity of the L2 polypeptide. In another embodiment, the selective binding assay comprises a CHL2 polypeptide binding partner (ligand or receptor).
Is an anti-CHL2 polypeptide antibody capable of interacting with and thereby inhibiting or eliminating CHL2 polypeptide activity in vitro or in vivo. Selective binding agents, including agonist and antagonist antibody-CHL2 polypeptide antibodies, are identified by screening assays well known in the art.

The present invention also relates to kits containing CHL2 selective binding agents (eg, antibodies) and other reagents useful for detecting CHL2 polypeptide levels in biological samples. Such reagents may also include detectable labels that block serum, positive and negative control samples, and detection reagents.

Microarray It is understood that DNA microarray technology can be utilized in accordance with the present invention. DNA microarrays are small, high-density arrays of nucleic acids arranged on a solid support (eg glass). Each cell or element in the array contains multiple copies of a single nucleic acid species, which acts as a label for hybridizing with complementary nucleic acid sequences (mRNA). In expression profiles using DNA microarray technology, mRNA is first extracted from a cell or tissue sample and then converted to enzymatically fluorescently labeled cDNA. This material is hybridized to the microarray and unbound cDNA is removed by washing. The expression of the individual genes shown on this array is then visualized by quantifying the amount of labeled cDNA that is specifically bound to each target the nucleic acid molecule. In this way, expression of thousands of genes can be quantified in a high-throughput, parallel fashion from a single sample of biological material.

This high-throughput expression profile has widespread application in the context of the CHL2 molecules of the invention, including but not limited to: CHL2 disease as a target for treatment. Identification and confirmation of related genes; related CH
Molecular toxicity of the L2 molecule and its inhibitors; population and production stratification of surrogate markers for clinical trials; and aid in identifying selected compounds in high-throughput screens to identify relevant CHL2 polypeptide small molecule drug discoveries. To strengthen.

Chemical Derivatives Chemically modified derivatives of CHL2 polypeptides can be prepared by one of skill in the art, as this disclosure is described herein. CHL2 polypeptide derivatives are modified in different ways, either in the type or position of the molecules naturally attached to the polypeptide. Derivatives may include molecules formed by the removal of one or more naturally attached chemical groups. SEQ ID NO: 2, SEQ ID NO: 5, or other CHL
Polypeptides containing the amino acid sequence of any of the two polypeptides can be modified by covalent attachment of one or more polymers. For example, the selected polymers are typically water soluble so that the bound proteins do not precipitate in an aqueous environment (eg, physiological environment). Mixtures of polymers are included within the scope of suitable polymers. Preferably, for therapeutic use in the preparation of the desired product, the polymer is
It is pharmaceutically acceptable.

Each of the polymers can have any molecular weight and can be branched or unbranched. Each of the polymers typically has an average molecular weight of between about 2 kDa and about 100 kDa (the term "about" is used in the preparation of water-soluble polymers, with some higher than the above molecular weight). It has less weight). The average molecular weight of each polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 12 kDa and about 40 kDa, and most preferably about 20 kDa.
It is between Da and about 35 Da.

Suitable water-soluble polymers or mixtures thereof include, but are not limited to: N-linked or O-linked carbohydrates, sugars, phosphates, polyethylene glycol (PEG), which is mono-. (Including C ₁ -C ₁₀ ), alkoxy-, or aryloxy-polyethylene glycol, including forms of PEG used to derive proteins), monomethoxy-polyethylene glycol, dextran (eg, low molecular weight (eg, , Dextran) of about 6 kD), cellulose, or other carbohydrate-based polymers, poly- (N-vinylpyrrolidine) polyethylene glycols, propylene glycol homopolymers, polypropyleneoxy / ethylene oxide copolymers, polyoxyethylated polyols (eg Glycerol), and polyvinyl alcohol. Also included in the invention are bifunctional cross-linking molecules that can be used to prepare covalently linked CHL2 polypeptide multimers.

In general, chemical induction can be carried out under any suitable conditions used to react proteins with activated polymer molecules. Methods for preparing chemical derivatives of polypeptides generally include the following steps: (a) SEQ ID NO: 2.
, SEQ ID NO: 5, or a polypeptide comprising the amino acid sequence of any of the other CHL2 polypeptides under conditions that bind to one or more polymer molecules (eg, a reactive ester of a polymer molecule or A step of reacting an aldehyde derivative) with a polypeptide, and (b) a step of obtaining a reaction product. Optimal reaction conditions are determined based on known parameters and desired results. For example, the greater the ratio of polymer molecules to protein, the greater the percentage of polymer molecules bound. In one embodiment, the CHL2 polypeptide derivative is
It has an amino-terminated single polymer molecular moiety. For example, US Pat.
See No. 784.

Pegylation of a polypeptide can be specifically performed using any pegylation reaction known in the art. Such reactions include, for example,
It is described in the following references: Francis et al., 1992, Focus.
on Growth Factors 3: 4-10; European Patent 015431.
6 and 0401384; and U.S. Pat. No. 4,179,337. For example, pegylation can be carried out via an acylation or alkylation reaction with a reactive polyethylene glycol molecule (or similar reactive water-soluble polymer), as described herein. For the acylation reaction, the polymer selected has a single reactive ester group. For reductive alkylation, the polymer selected has a single reactive aldehyde group. For example, the reactive aldehyde is
Polyethylene glycol propionaldehyde, which is water soluble, or mono C ₁ -C ₁₀ alkoxy or its aryloxy derivatives (see US Pat. No. 5,252,714).

In another embodiment, the CHL2 polypeptide can be chemically linked to biotin. The biotin / CHL2 polypeptide molecule can then be bound to avidin, resulting in a tetravalent avidin / biotin / CHL2 polypeptide molecule. CHL2 polypeptides may also be covalently coupled to dinitrophenol (DNP) or Application Benefits nitrophenol (TNP), and the resulting conjugate precipitated with anti -DNP or anti-TNP-IgM, of 10-valent ten Form a conjugate of monomers.

Generally, conditions that can be alleviated or modulated by administration of a CHL2 polypeptide derivative of the invention include those described herein for CHL2 polypeptides. However, the CHL2 polypeptide derivatives disclosed herein have further activity, enhanced or decreased biological activity, when compared to underivatized molecules,
Or it may have other properties, such as increased or decreased half-life.

Genetically Engineered Non-Human Animals Non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other farm animals are further included within the scope of the invention, Where natural CH
The gene encoding the L2 polypeptide is disrupted (ie, "knocked out") and the expression level of the CHL2 polypeptide is significantly reduced or abolished. Such animals can be prepared using the techniques and methods as described in US Pat. No. 5,557,032.

[0184] The present invention is a mouse, rat, or other rodent; rabbits, further comprising goats, non-human animals such as sheep or other livestock, the native of CHL2 gene of the animal form or non Either of the homologous CHL2 genes is overexpressed by the animal, thereby creating a "transgenic" animal. Such transgenic animals can be prepared using well known methods such as those described in US Pat. No. 5,489,743 and PCT Publication No. WO94 / 28122.

The present invention further includes non-human animals, wherein the promoter of one or more CHL2 polypeptides of the present invention is activated (eg, by using homologous recombination methods). , Or not activated, alters the level of expression of one or more native CHL2 polypeptides.

These non-human animals can be used for drug candidate screening. In such a screen, the effect of the drug candidate on the animal can be measured.
For example, the drug candidate may reduce or increase expression of the CHL2 gene. In certain embodiments, the amount of CHL2 polypeptide produced can be measured after exposing the animal to the drug candidate. Moreover, in certain embodiments, the actual effect of the drug candidate on the animal may be detected. For example, due to overexpression of certain genes,
Causes or is associated with a disease or pathological condition. In such cases, the ability of the drug candidate to reduce gene expression or the ability to prevent or inhibit a pathological condition may be tested. In another example, the production of a particular metabolite, such as a fragment of a polypeptide, results in or is associated with a disease or pathological condition. In such cases, the ability of the drug candidate to reduce the production of such metabolites or the ability to prevent or inhibit a pathological condition may be tested.

Assays for Other Modulators of CHL2 Polypeptide Activity In some situations it may be desirable to identify molecules ( ie agonists or antagonists) that are modulators of CHL2 polypeptide activity. Natural or synthetic molecules that modulate a CHL2 polypeptide can be identified using one or more screening assays, as described herein. Such molecules can be administered by injection, or oral delivery, implantation devices, etc., in either an in vitro or ex vivo manner.

A “test molecule” refers to a molecule that evaluates for its ability to modulate (ie, increase or decrease) the activity of a CHL2 polypeptide. Most commonly, the test molecule interacts directly with the CHL2 polypeptide. However, the test molecule can also be used, for example, by affecting CHL2 gene expression or in CHL2.
2 polypeptide binding partner (e.g., receptor or ligand) by binding to, it is believed et al are capable of modulating indirectly the CHL2 polypeptide activity. In one embodiment, the test molecule has an affinity constant (at least about 10 ⁻⁶ M, preferably about 10 ⁻⁸ M, more preferably about 10 ⁻⁹ M, and even more preferably about 10 ⁻¹⁰ M). Affinity constant) CHL
It binds to 2 polypeptides.

Methods for identifying compounds that interact with a CHL2 polypeptide are encompassed by the present invention. In certain embodiments, the CHL2 polypeptide is incubated with the test molecule under conditions that allow the test molecule to interact with the CHL2 polypeptide and the extent of interaction measured. Test molecules can be screened in a substantially purified form or crude mixture.

In certain embodiments, the CHL2 polypeptide agonist or antagonist can be a protein, peptide, carbohydrate, lipid, or low molecular weight molecule, which interacts with the CHL2 polypeptide to modulate its activity. To do. CH
A molecule that regulates expression of an L2 polypeptide is complementary to a nucleic acid encoding a CHL2 polypeptide, or a nucleic acid molecule that is complementary to a nucleic acid sequence that directs or controls expression of the CHL2 polypeptide, and expression Nucleic acid molecules that act as antisense regulators of.

Once a test compound has been identified once it interacts with the CHL2 polypeptide, the molecule can be further evaluated for its ability to increase or decrease CHL2 polypeptide activity. Measuring the interaction of a test molecule with a CHL2 polypeptide can be performed in several forms, including cell-based binding assays, membrane binding assays, solution phase assays, and immunoassays. Generally, the test molecule is incubated with the CHL2 polypeptide for a specified period of time and C
HL2 polypeptide activity is determined by one or more assays to measure biological activity.

[0192] The interaction of test molecules with CHL2 polypeptide in an immunoassay, can be directly assayed using the polyclonal antibody or monoclonal antibody. Alternatively, CHL containing an epitope tag as described herein.
Modified forms of the two polypeptides can be used in solution and immunoassays.

In cases where a CHL2 polypeptide exhibits biological activity via interaction with a binding partner (eg, receptor or ligand), various in vitro assays will detect the corresponding binding partner ( eg, selective Can be used to measure the binding of CHL2 polypeptides to binding agents, receptors, or ligands). These assays can be used to screen test molecules to increase or decrease the rate and / or rate of binding of CHL2 polypeptides to binding partners. In one assay, CHL2 polypeptide is immobilized in the wells of a microtiter plate. Radiolabeled CHL2 polypeptide binding partner (eg, iodinated CHL2 polypeptide binding partner) and test compound are then added to this well one at a time (
Or in any order) or simultaneously. After incubation, the wells are washed and the radioactivity is counted using a scintillation counter to determine the extent to which the binding partner binds the CHL2 polypeptide. Typically, molecules are tested over a range of concentrations, and a series of control wells lacking one or more of the components of the test assay can be used for the accuracy of evaluation of the results.
An alternative to this method is to reverse the "position" of the protein (ie immobilize the CHL2 polypeptide binding partner to the microtiter plate well, incubate the test molecule and radiolabeled CHL2 polypeptide,
And determining the extent of CHL2 polypeptide binding). For example,
Current Protocols in Molecular Biolo
gy, Chapter 18 (Edited by Ausubel et al., Green Publishers I)
nc. And Wiley and Sons 1995).

[0194] As an alternative to radiolabeling, CHL2 polypeptide or its binding partner may be conjugated to biotin and the presence of biotinylated protein, then enzyme (e.g., horseradish peroxidase (HRP) or alkaline Phosphatases (AP)), which are detected colorimetrically, can be detected using streptavidin or can be detected by fluorescent tagging of streptavidin. Antibodies to CHL2 polypeptides or CHL2 polypeptide binding partners, which are bound to biotin, may also be used for detection purposes following incubation of the complex with enzyme-linked streptavidin linked to AP or HRP. Can be used for.

CHL2 polypeptides or CHL2 polypeptide binding partners can be immobilized by attachment to agarose beads, acrylic beads, or other types of such inert solid phase substrates. The substrate-protein complex can be placed in a solution containing the complementary protein and the test compound. After incubation, the beads can be pelleted by centrifugation and the amount of binding between the CHL2 polypeptide and its binding partner can be assessed using the methods described herein. Alternatively, the substrate-protein complex can be immobilized within the column with the test molecule and complementary protein passing through the column. The formation of complexes between the CHL2 polypeptide and its binding partner can then be assessed using any of the techniques described herein (eg, radiolabel or antibody binding).

Another in vitro assay useful for identifying test molecules that increase or decrease the formation of a complex between a CHL2 polypeptide binding protein and a CHL2 polypeptide binding partner is a surface plasmon resonance detector system (eg, , BI
Acore assay system (Pharmacia, Piscataway, N
J)). The BIAcore system is utilized as specified by the manufacturer. This assay is essentially a CHL2 polypeptide or CHL2
Covalent attachment of any of the polypeptide binding partners to the dextran coated sensor chip, which is present on the detector. The test compound and other complementary protein are then either simultaneously or sequentially
It can be injected into the chamber containing the sensor chip. The amount of complementary protein that binds can be assessed based on the change in mass of a molecule physically associated with the dextran-coated side of the sensor chip, which change in mass is measured by a detector system.

In some cases, it may be desirable to evaluate two or more test compounds together for their ability to increase or decrease the formation of a complex between a CHL2 polypeptide and a CHL2 polypeptide binding partner. Can be done. In these cases, the assays described herein can be readily modified by the addition of such additional test compounds, either concomitantly with or subsequent to the first test compound. The rest of the steps in this assay are described herein.

In vitro assays, such as those described herein, are used to screen large numbers of compounds for their effect on the formation of complexes between CHL2 polypeptides and CHL2 polypeptide binding partners. It can be used to advantage. These assays can be automated to screen compounds produced in phage display, synthetic peptides, and chemically synthesized libraries.

A compound that increases or decreases the formation of a complex between a CHL2 polypeptide and a CHL2 polypeptide binding partner may also be a CHL2 polypeptide or CH
Cells and cell lines expressing any of the L2 polypeptide binding partners F can be used to screen in cell culture. The cells and cell lines can be obtained from any mammal, but are preferably derived from humans or other primate, canine or rodent sources. The binding of the CHL2 polypeptide to cells expressing the CHL2 polypeptide binding partner at its surface is assessed in the presence or absence of the test compound and the degree of binding is determined, for example, to the CHL2 polypeptide binding partner. It can be determined by flow cytometry using a biotinylated antibody. Cell culture assays can be advantageously used to further evaluate compounds that score positive in the protein binding assays described herein.

Cell cultures can also be used to screen the effects of drug candidates. For example, the drug candidate may reduce or increase expression of the CHL2 gene. In certain embodiments, the amount of CHL2 polypeptide or CHL2 polypeptide fragment produced can be measured after exposure of the cell culture to the drug candidate.
In certain embodiments, the substantial effect of the drug candidate on cell culture can be detected. For example, overexpression of a particular gene can have a particular effect on cell culture. In such cases, the drug candidate's ability to increase or decrease gene expression, or its ability to prevent or inhibit a particular effect on cell culture, can be tested. In other examples, the production of particular metabolites (eg, fragments of polypeptides, etc.) can result in or be associated with a disease or pathological condition.
In such cases, the ability of drug candidates to reduce the production of such metabolites in cell culture can be tested.

Internalization Proteins The tat protein sequence (from HIV) can be used to internalize proteins into cells. For example, Falwell et al., 1994, Proc. N
atl. Acad. Sci. U. S. A. 91: 664-68. For example, the 11-amino acid sequence of the HIV tat protein (Y-G-R-K-K-
R-R-Q-R-R-R; SEQ ID NO: 10) ("protein transduction domain",
Or TAT PDT) has been described as mediating delivery across the cytoplasmic and nuclear membranes of cells. Schwarze et al., 1999, Scien.
285: 1569-72; and Nagahara et al., 1998, Nat.
． Med. 4: 1449-52. In these procedures, FITC
Construct (FITC labeled G-G-G-G-Y-G-R-K-K-R-R-Q-R-
R-R; SEQ ID NO: 11) (which, following intraperitoneal administration penetrating tissue) is prepared and the binding to cells such constructs, fluorescence activated cell sorting (f luo
rescue activated cell sorting; FACS
) Detected by analysis. Cells treated with the tat-β-gal fusion protein show β-gal activity. Following injection, expression of such constructs can be detected in many tissues (liver, kidney, lung, heart and brain tissues). It is believed that such a construct may undergo some modification to enter the cell and may itself require refolding following import into the cell.

Thus, it is understood that tat protein sequences can be used to internalize a desired polypeptide into a cell. For example, using the tat protein sequence, a CHL2 antagonist (eg, anti- CHL2 selective binding agent, small molecule,
Soluble receptors, or antisense oligonucleotides) can be administered intracellularly to inhibit the activity of the CHL2 molecule. As used herein,
The term "CHL2 molecule" refers to both CHL2 nucleic acid molecules and CHL2 polypeptides, as defined herein. If desired, the CHL2 protein itself can also be administered intracellularly using these procedures. Straus, 1
See also 999, Science 285: 1466-67.

Identification of Cell Sources Using CHL2 Polypeptides In accordance with certain embodiments of the invention, it is useful to be able to determine the source of particular cell types associated with CHL2 polypeptides. obtain. For example, it may be useful to determine the origin of a disease or pathological condition as an aid in selecting an appropriate treatment. In certain embodiments, the nucleic acid encoding the CHL2 polypeptide is used as a probe, by screening the nucleic acids of the cells with such a probe can identify cells described herein. In other embodiments, anti-CHL2 polypeptide antibodies are used to detect CHL2 in cells.
One can test for the presence of the polypeptide, and thus determine whether such cells are of the type described herein.

CHL2 Polypeptide Compositions and Administration Therapeutic compositions are within the scope of this invention. Such a CHL2 polypeptide pharmaceutical composition comprises a therapeutically effective amount of a CHL2 polypeptide or CHL2 nucleic acid molecule with a pharmaceutically or physiologically acceptable prescription agent selected to be compatible with the mode of administration. It may be included in the mixture. The pharmaceutical composition comprises a therapeutically effective amount of one or more CHL.
The two polypeptide selective binding agents may be included in admixture with a pharmaceutically or physiologically acceptable formulation agent selected to be compatible with the mode of administration.

Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed.

A pharmaceutical composition may, for example, modify pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissociation or release, adsorption, or penetration of the composition. ,
It may include formulation materials for maintenance or storage. Suitable formulation materials include:
These include, but are not limited to: amino acids (eg, glycine, glutamine, asparagine, arginine, or lysine), antibacterial agents, antioxidants (eg, ascorbic acid, sodium sulfite, or sodium bisulfite (so.
), buffer (eg borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acid).
, Bulking agents (eg mannitol or glycine), chelating agents (ethylenediaminetetraacetic acid (EDTA)), complexing agents (eg caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin). , Fillers, monosaccharides, disaccharides, and other carbohydrates (eg glucose, mannose or dextrin), proteins (eg serum albumin, gelatin, or immunoglobulins), colorants, flavoring and diluents, emulsifiers, hydrophilic Polymers (eg polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (eg sodium), preservatives (eg benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propyiene. Luparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (eg, glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (eg, mannitol or sorbitol)
, Suspending agents, surface-active agents or wetting agents (eg pluronic
); PEG; sorbitan ester; polysorbate (for example, polysor
bate 20 or polysorbate 80); triton; tromethamine; lecithin; cholesterol or tyloxapol).
, Stability enhancers (eg sucrose or sorbitol), tonicity enhancers (eg alkali metal halides (preferably sodium chloride or potassium chloride)
, Or mannitol, sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Remington's Pharmaceu
Tal Sciences (18th Edition, AR Gennaro, Ed., Ma
ck Publishing Company 1990 ) .

Optimal pharmaceutical compositions will be determined by those of skill in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. For example, Remington
See's Pharmaceutical Sciences, supra.
Such compositions may affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the CHL2 molecule.

The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection is water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. There is. Neutral buffered saline or saline mixed with serum albumin is a further exemplary vehicle. Other exemplary pharmaceutical compositions have a Tr of about pH 7.0-8.5.
is buffer or acetate buffer at about pH 4.0-5.5, which further comprises
It may include sorbitol or a suitable substitute. In one embodiment of the invention, the CHL2 polypeptide composition provides a selected composition having the desired degree of purity in any prescription drug (Remington's Pharmaceutical).
It can be prepared for storage in the form of a lyophilized cake or an aqueous solution by mixing with Sciences, supra). Further, the CHL2 polypeptide product can be formulated as a lyophilizate using appropriate excipients such as sucrose.

The CHL2 polypeptide pharmaceutical composition may be selected for parenteral delivery. Alternatively, these compositions may be selected for inhalation or delivery via the gastrointestinal tract (eg, orally). Preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation components are present in concentrations that are acceptable at the site of administration. For example, buffers are used to maintain the composition at physiological pH or slightly lower pH (typically within the pH range of about 5 to about 8).

When intended for parenteral administration, therapeutic compositions for use in the present invention are pyrogen-free, parenterally acceptable containing a desired CHL2 molecule in a pharmaceutically acceptable vehicle. It can be in the form of an aqueous solution. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the CHL2 molecule is formulated as a sterile, isotonic solution, and stored properly. Yet another preparation is the desired molecule and drug (eg, injectable microspheres, bio-erodible drugs, polymeric compounds (polylactic or polyglycolic acid), beads or liposomes).
With a formulation that provides a controlled or sustained release of the product that can then be delivered via depot injection. Hyaluronic acid can also be used,
And this can have the effect of promoting a sustained duration in the circulation.
Other suitable means for introduction of the desired molecule include implantable drug delivery devices.

[0212] In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, the CHL2 polypeptide can be formulated as a dry powder for inhalation. CH
Inhalation solutions of L2 polypeptides or nucleic acid molecules can also be formulated with a nebulizer for aerosol delivery. In yet another embodiment, the solution may be nebulized.
Pulmonary administration is further described in PCT Publication No. WO94 / 20069, which describes pulmonary delivery of chemically modified proteins.

It is also contemplated that certain formulations may be administered orally. In one embodiment of the invention, the CHL2 polypeptide administered in this manner is formulated with or without carriers conventionally used in the formulation of solid dosage forms (eg tablets or capsules). obtain. For example, capsules may be designed to release the active portion of the formulation at a point within the gastrointestinal tract when bioavailability is maximized and presystemic degradation is minimized. Further drugs are CHL
It may be included to facilitate absorption of the two polypeptides. Diluents, seasonings, low melting waxes, vegetable oils, lubricants, suspensions, tablet disintegrating agents, and binders are also
Can be used.

Another pharmaceutical composition may include an effective amount of CHL2 polypeptide in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents (calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate); or binders (eg starch, gelatin or acacia). ); Or a lubricant (eg magnesium stearate, stearic acid or talc)
.

Additional CHL2 polypeptide pharmaceutical compositions will be apparent to those of skill in the art and include formulations that include CHL2 polypeptides in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible microspheres or porous beads and depot injections, are also provided.
It is known to those skilled in the art. See, eg, PCT / US93 / 00829, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions.

Further examples of sustained release formulations include semipermeable polymer matrices in the form of shaped articles, eg films, or microcapsules. Sustained-release matrices include polyesters, hydrogels, polylactides (US Pat. No. 3,773,919 and EP 058481), copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., 1983, Bi.
Polymers 22: 547-56), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater.
Res. 15: 167-277 and Langer, 1982, Chem. Te
ch. 12: 98-105), ethylene vinyl acetate (Langer et al., Supra) or poly-D (-)-3-hydroxybutyric acid (European Patent No. 133988).
. Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. For example, Eppstein et al., 19
85, Proc. Natl. Acad. Sci. USA 82: 3688-92.
European Patent Nos. 0366676, 088046, and 1439;
See No. 49.

[0217] Pharmaceutical compositions of CHL2 to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through a sterile filtration membrane. Where the composition is lyophilized, sterilization using this method can be performed either before, or subsequent to lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in solution. In addition, parenteral compositions are generally placed in a container having a sterile access port such as an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form that requires reconstitution prior to administration (lyophilized form).

In a particular embodiment, the present invention relates to a kit for producing a single dose dosage unit. The kit may each include both a first container having a dry protein and a second container having an aqueous formulation. Also included within the scope of the invention are kits including single and multi-chambered pre-filled syringes (eg, liquid syringes and lyosyringes).

The effective amount of the pharmaceutical composition of CHL2 used therapeutically depends, for example, on the therapeutic content and purpose. Those of ordinary skill in the art will appreciate that the appropriate dosage level for treatment will be
Depends in part on the molecule delivered, the indication that the CHL2 molecule is used, the route of administration, and the size (body weight, body surface, or organ size) and condition (age and general health) of the patient. And understand that it changes. Thus, the clinician may titer the dosage and modify the route of administration in order to obtain the optimal therapeutic effect. A typical dosage will range from about 0.1 μg / kg to depending on the factors mentioned above.
It can range up to about 100 mg / kg or more. In other embodiments, the dosage is from 0.1 μg / kg to about 100 mg / kg; or 1 μg / kg to about 10
It can range up to 0 mg / kg; or up to 5 μg / kg up to about 100 mg / kg.

The frequency of dosing will depend on the pharmacokinetic parameters of the CHL2 molecule in the formulation used. Typically, the clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition, and therefore the composition, as a single dose over time,
It can be administered as two or more doses, which may or may not contain the same amount of the desired molecule, or as a continuous infusion through an implantation device or catheter. Further refinement of the appropriate dosage is well within the scope of the problems routinely made and carried out by those skilled in the art. The proper dosage is
It can be confirmed through the use of appropriate dose-response data.

The route of administration of the pharmaceutical composition is consistent with known methods as follows: orally; intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular. , Via an intraarterial, intraportal, or intralesional route of injection; by a sustained release system; or by an implantable device. If desired, these compositions can be administered by bolus injection, or continuously by infusion, or by implantation device.

Alternatively or additionally, the composition may be administered locally via implantation of a membrane, sponge, or other suitable material onto which the desired molecule is absorbed or encapsulated. If an implant device is used, the device can be implanted in any suitable tissue or organ and delivery of the desired molecule can be via diffusion, timed release bolus or continuous administration.

In some cases, it may be desirable to use pharmaceutical compositions of CHL2 polypeptides in an ex vivo manner. In such instances, cells, tissues, or organs removed from the patient are exposed to the CHL2 polypeptide after the cells, tissues, or organs have been subsequently transplanted back into the patient.

In other cases, the CHL2 polypeptide is genetically engineered to transplant specific cells expressing and secreting the CHL2 polypeptide using the methods described herein. Can be delivered. Such cells can be animal or human cells, and can be autologous, xenogeneic, or exogenous cells. If desired, the cells can be immortalized. To reduce the chance of an immunological response, cells can be encapsulated to avoid infiltration of surrounding tissue. The encapsulating material is typically a biocompatible, semi-permeable polymeric enclosure or membrane that allows the release of the protein product, but which is otherwise free of the patient's immune system or surrounding tissues. Prevents destruction of cells by harmful factors.

As discussed herein, treating an isolated cell population (eg, stem cells, lymphocytes, red blood cells, chondrocytes, neurons, etc.) with one or more CHL2 polypeptides comprises: May be desirable. This can be accomplished by exposing the isolated cells directly to the polypeptide when it is in a permeable form to the cell membrane.

Further embodiments of the invention include cells and methods (eg, homologous recombination and / or homologous recombination and / or) for both in vitro production of therapeutic polypeptides and production and delivery of therapeutic polypeptides by gene therapy or cell therapy. Or other recombinant production methods). Homologous recombination and other methods of recombination can be used to modify cells that normally contain a CHL2 gene that is silent for transcription (ie, an underexpressed gene), thereby producing a therapeutically effective amount of CHL2. Cells can be produced which express the polypeptide.

Homologous recombination was originally a technique developed to target genes to induce or correct mutations in transcriptionally active genes. Kucherl
apati, 1989, Prog. in Nucl. Acid Res. & M
ol. Biol. 36: 301. Because basic techniques introduce specific mutations into specific regions of the mammalian genome (Thomas et al., 1986, Cell 44: 4.
19-28; Thomas and Capecchi, 1987, Cell 51.
: 503-12; Doetschman et al., 1988, Proc. Natl. A
cad. Sci. U. S. A. 85: 8583-87), or to correct specific mutations in the defective gene (Doetschman et al., 1987, Natu.
Re 330: 576-78). Exemplary homologous recombination techniques are described in US Pat. Nos. 5,272,071; European Patents 9193051 and 505500; PCT / US90 / 07642, and PCT Publication No. WO 91/09955). .

By homologous recombination, the DNA sequence to be inserted into the genome is
By attaching to A, it can be directed to a specific region of the gene of interest. This target DNA is a nucleotide sequence that is complementary (homologous) to a region of genomic DNA. A small piece of target DNA complementary to a particular region of the genome is a DNA
During the replication process, it is contacted with the parent strand. This is a general property of hybridized DNA inserted into cells and, therefore, recombines with other fragments of endogenous DNA via shared homologous regions. If this complementary strand is attached to an oligonucleotide containing a mutation or a different sequence or additional nucleotides, it will also be incorporated into the newly synthesized strand as a result of this recombination.
As a result of the proofreading function, new sequences of DNA can serve as templates. Therefore, the transferred DNA is integrated into the genome.

DNA capable of interacting with CHL2 polypeptide or controlling its expression
Regions (eg, flanking sequences) attach to these fragments of the target DNA. For example, a promoter / enhancer element, suppressor, or exogenous transcriptional regulatory element is sufficient to affect the transcription of this DNA in the vicinity of the DNA encoding the desired CHL2 polypeptide, in the genome of the intended host cell. With a proper orientation,
Is inserted. The control elements control the portion of DNA present in the host cell genome. Thus, expression of the desired CHL2 polypeptide is not due to transfection of the DNA encoding the CHL2 gene itself, but rather CHL2.
This can be accomplished by the use of target DNA (including regions of homology with the endogenous gene of interest) linked to a DNA regulatory segment that provides the endogenous gene sequence with a recognizable signal for transcription of the two genes.

In an exemplary method, expression of a desired target gene (ie, a desired endogenous cellular gene) in a cell is achieved through at least regulatory sequences via homologous recombination into the cell genome at a preselected site, It is modified by the introduction of DNA containing exons and splice donor sites. These components are introduced into the chromosomal (genomic) DNA in such a way that they actually result in the production of new transcription units (where the regulatory sequences, exons, and splice donor sites present in the DNA construct are Operably linked to an endogenous gene). The introduction of these components into chromosomal DNA results in altered expression of the desired endogenous gene.

As described herein, altered gene expression can result in activation (or expression) of a gene that is normally silent (unexpressed) in cells that have been obtained, as well as It involves increased expression of genes that are not expressed at physiologically significant levels in intact cells. This embodiment further includes the step of altering the pattern of regulation or induction so that it differs from the pattern of regulation or induction that occurs in the as-obtained cells and in the as-obtained cells. Decrease (including eliminate) expression of the expressed gene.

One way in which phase-identifying recombination can be used to increase or cause the production of CHL2 polypeptide from the endogenous CHL2 gene of a cell is to first use homologous recombination. , A recombination sequence derived from a site-specific recombination system (eg,
Cre / loxP, FLP / FRT) (Sauer, 1994, Curr. Op.
in. Biotechnol. , 5: 521-2; Sauer, 1993, Me.
methods Enzymol. , 225: 890-900) to the endogenous C of cells.
Locating upstream of the HL2 polypeptide coding region (ie 5 '). A plasmid containing a recombination site homologous to a region located immediately upstream of the genomic CHL2 polypeptide coding region is introduced into the modified cell line along with the appropriate recombinase enzyme. This recombinase allows the plasmid to integrate through the recombination site of the plasmid into the recombination site located immediately upstream of the genomic CHL2 polypeptide coding region of the cell line (Bauboni).
s and Sauer, 1993, Nucleic Acids Res. 21:
2025-29; O'Garman et al., 1991, Science 251: 1.
351-55). Any flanking sequences known to increase transcription (eg, enhancers / promoters, introns, translational enhancers), when properly placed in this plasmid, will not be transferred to the endogenous CHL2 gene of the cell. Alternatively, they are combined in such a way as to create a new or modified transcription unit that results in increased CHL2 polypeptide production.

A further method using a cell line in which the site-specific recombination sequences are located immediately upstream of the cell's endogenous genomic CHL2 polypeptide coding region is a second recombination at another location in the genome of the cell line. The use of homologous recombination to introduce the site. The appropriate recombinant enzyme is then introduced into the two recombination site cell line to reduce recombination (
Deletion, conversion, and metastasis) (Sauer, 1994, Curr. Opin
． Biotechnol. , 5: 521-27; Sauer, 1993, Met.
hods Enxymol. , 225: 890-900), which creates new or modified transcription units that result in novel or increased CHL2 polypeptide production from the endogenous CHL2 gene of the cell.

A further approach to increasing or causing expression of a CHL2 polypeptide from the endogenous CHL2 gene of the cell is a modality that results in the production of new or increased CHL2 polypeptide from the endogenous CHL2 gene of the cell. And / or increasing the expression of the gene (s) (eg transcription factors) and / or decreasing the expression of the gene (s) (eg transcription repressors). This method uses non-naturally occurring polypeptides (
(Eg, a polypeptide containing a site-specific DNA binding domain fused to a transcription factor domain), a new or increased CHL from the endogenous CHL2 gene of the cell.
Introducing into the cell such that production of the 2 polypeptide occurs.

The present invention further relates to DNA constructs useful in the method of altering the expression of a target gene. In certain embodiments, a representative DNA construct comprises: (a) one or more target sequences, (b) regulatory sequences, (c) exons, and (d).
Unpaired splice donor site. The target sequence in the DNA construct is element (a)-
It directs the integration of (d) into the target gene in the cell so that these elements (b)-(d) are operably linked to the sequence of the endogenous target gene. In another embodiment, the DNA construct comprises: (a) one or more target sequences, (
b) regulatory sequences, (c) exons, (d) splice donor sites, (e) introns, and (f) splice acceptor sites. Here, the target sequence directs the integration of elements (a)-(f), so that these elements (b)-(
f) is operably linked to the endogenous gene. The target sequence is homologous to a preselected site in the cell chromosomal DNA where homologous recombination occurs. In this construct, the exon is generally 3'to the control sequences and the splice donor site is 3'to this exon.

If the sequence of a particular gene (eg, the nucleic acid sequence of a CHL2 polypeptide described herein) is known, the DN complementary to the selected region of the gene
The portion of A may be synthetic or otherwise obtained, such as by appropriate restriction of native DNA at a particular recognition site bound to the region of interest. This part, when inserted into the cell, serves as the target sequence and hybridizes to the homologous region within its genome. If this hybridization occurs during DNA replication, a portion of this DNA, and any additional sequences attached to it, will act as an Okazaki fragment and be incorporated into the newly synthesized daughter strand of the DNA. Thus, the invention includes a nucleotide encoding a CHL2 polypeptide, which nucleotide can be used as a target sequence.

CHL2 polypeptide cell therapy (eg, transplantation of cells that produce CHL2 polypeptide) is also contemplated. This embodiment provides a biologically active form of CHL.
2 transplanting cells capable of synthesizing and secreting the 2 polypeptide. Such CHL2 polypeptide-producing cells, or it may be a cell, a natural Purodeyu Sa of CHL2 polypeptide, or a recombinant cell, capable of producing the CHL2 polypeptide, encoding the desired polypeptide Gene or CH
It may be a recombinant cell which has been expanded by transforming with a gene which increases the expression of the L2 polypeptide. Such modifications can be accomplished by appropriate vectors to deliver the gene and promote its expression and secretion. To minimize the strong immunological response in patients receiving CHL2 polypeptides,
When occurring with administration of a heterologous polypeptide, the natural cells producing the CHL2 polypeptide are of human origin and preferably produce the human CHL2 polypeptide. Similarly, recombinant cells that produce the CHL2 polypeptide can bind to human C
It is preferably transformed with an expression vector containing the gene encoding the HL2 polypeptide.

Transplanted cells can be encapsulated to avoid infiltration of surrounding tissue. Human or non-human animal cells are biocompatible semipermeable polymer inclusions or membranes (which are C
It allows the release of HL2 polypeptides but prevents the destruction of cells by other deleterious factors from the patient's immune system or surrounding tissues). Alternatively, the patient's own cells transformed to produce the CHL2 polypeptide ex vivo can be directly transplanted into the patient without such encapsulation.

Techniques for encapsulating live cells are known in the art, and preparation of encapsulated cells and their implantation into patients can be accomplished routinely. For example, Baetge et al. (PCT Publication WO 95/05452 and PCT / U
S94 / 09299) describes a membrane capsule containing cells that have been genetically engineered for effective delivery of biologically active molecules. The capsule is biocompatible and is easily removable. The capsule is transfected with a recombinant DNA molecule comprising a DNA sequence encoding a biologically active molecule operably linked to a promoter that is not down-regulated in vivo when transplanted into a mammalian host. Encapsulated cells. The device provides delivery of living cell-derived molecules to specific sites within the recipient. Furthermore, US Pat.
, 892,538; 5,011,472; and 5,106,62.
See No. 7. A system for encapsulating live cells is described in PCT Publication WO 91/10425 (Aebischer et al.). PCT release 9
1 / 10-40470 (Aebischer et al.); Winn et al. 1991, Expe.
r. Neurol. 113: 322-29; Aebischer et al., 1991.
Expert. Neurol. 111: 269-75; and Tresco et al., 1
See also 992, ASAIO 38: 17-23.

In vivo and in vitro gene therapy delivery of CHL2 polypeptides is also envisioned. One example of gene therapy technology is the CHL2 gene (genomic D) encoding a CHL2 polypeptide that can be operably linked to a constitutive or inducible promoter.
NA, cDNA and / or synthetic DNA) to form a "gene therapy DNA construct". This promoter is based on the endogenous CHL2
Homozygous or heterozygous for a gene, provided that it is active in the cell or tissue type into which the construct is inserted. Other components of gene therapy DNA constructs include DN, which is optionally designed for site-specific integration.
Identifies A molecules (eg, endogenous sequences useful for homologous recombination), tissue-specific promoters, enhancers or silencers, DNA molecules capable of providing selective dominance over parental cells, transformed cells Useful as a label for
Molecules, negative selection systems, cell-specific binding agents (eg for cell targeting)
Is mentioned. Cell-specific internalization, transcription factors that increase expression from vectors,
And factors that enable the production of the vector.

[0242] Gene therapy DNA construct can then (either ex vivo or in vivo) that can be introduced into cells using viral vectors or non-viral vector.
One method for introducing the gene therapy DNA construct is by the use of viral vectors as described herein. Certain vectors, such as retroviral vectors, deliver the DNA construct to the chromosomal DNA of the cell, and the gene can be integrated into the chromosomal DNA. Other vectors function as episomes and the gene therapy DNA construct remains cytosolic.

[0243] In yet another embodiment, the regulatory element is CHL2 in the target cell.
May be included for controlled expression of genes. Such elements are stimulated in response to appropriate effectors. In this way, the therapeutic polypeptide can be expressed when desired. One conventional control means is a chimeric protein containing a small molecule binding domain and a domain that can initiate biological processes (eg,
The use of low molecular weight dimerizers or rapalogs that dimerize DNA binding proteins or transcriptionally active proteins) (PCT Publication WO 96/4).
1865, WO 97/31898 and WO 97/31899).
Protein dimerization can be used to initiate transcription of a transgene.

An alternative regulation technique uses the method of storing the protein expressed from the gene of interest inside the cell as aggregates or clusters. The gene of interest, results in a retention of the aggregated protein in the endoplasmic reticulum, is expressed as a fusion protein comprising a conditional aggregation domain. The stored protein is stable and inactive inside the cell. However, the protein can be released by administering a drug that removes the conditional aggregation domain, such as a small molecule ligand, thereby destroying the aggregate or cluster, which results in the protein being secreted from the cell. Can be done. Aridor et al., 2000, Science 287: 816-1.
7 and Rivera et al., 2000, Science 287: 826-30.

Other suitable control means or gene switches include, but are not limited to, the systems described herein. Mifepristone (RU4
86) is used as a progesterone antagonist. Binding to the progesterone antagonist of the modified progesterone receptor ligand-binding domain, two transcription factors (which in turn leads to the nucleus, binds to DNA) by forming a dimer of, you activate transcription. This ligand binding domain is modified to eliminate the ability of the receptor to bind the natural ligand.
The modified steroid hormone receptor system is further described in US Pat.
791, and PCT publications WO 96/40911 and WO 97/1033.
7.

Yet another regulatory system binds to the ecdysone receptor (cytoplasmic receptor),
And ecdysone (Drosophila steroid hormone) which activates this is used. This receptor then translocates to the nucleus and binds to specific DNA response elements (promoters from ecdysone-responsive genes). The ecdysone receptor contains a transactivation domain, a DNA binding domain, and a ligand binding domain and initiates transcription. The ecdysone system is further described in US Pat. No. 5,514.
, 578, and PCT publications WO 97/38117, WO 96/37609, and WO 93/03162.

Another control means uses positive tetracycline controllable transactivators. This system consists of a mutated tet repressor protein DNA-binding domain linked to a polypeptide that activates transcription (a mutated tetR-4 amino acid change that resulted in a reverse tetracycline-regulated transactivator protein, ie, Binds to the tet operator in the presence of tetracycline). Such systems are described in US Pat. Nos. 5,464,758, 5,650,298, and 5,654,168.

Additional expression control systems and nucleic acid constructs can be found in US Pat. Nos. 5,741,679 and 5,834,186 (Innovir Laboratories).
Inc. ).

In vivo gene therapy involves replacing the gene encoding the CHL2 polypeptide with CHL2.
It may be achieved by local injection of the two nucleic acid molecules or by introduction into the cells by a suitable viral or non-viral delivery vector. Hefti, 19
94, Neurobiology 25: 1418-35. For example, a nucleic acid molecule encoding a CHL2 polypeptide can be included in an adeno-associated virus (AAV) vector for delivery to target cells (eg, Johnson, PCT Publication W).
See O95 / 34670; PCT application PCT / US95 / 07178.
Recombinant AAV genomes are typically C operably linked to a functional promoter.
It comprises a DNA sequence encoding the HL2 polypeptide and an AAV inverted terminal repeat flanked by polyadenylation sequences.

Alternative suitable viral vectors include retroviruses, adenoviruses,
Herpes simplex virus, lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papillomavirus vectors. US Pat. No. 5,672,344 describes an in vivo virus-mediated gene transfer system containing a recombinant neurotrophic HSV-1 vector. US Pat. No. 5,399,346
The issue provides an example of a process for providing a therapeutic protein to a patient by delivery of human cells treated in vitro to insert a DNA segment encoding the therapeutic protein. Additional methods and materials for practicing gene therapy techniques are described in US Pat. No. 5,631,236 (including adenovirus vectors),
No. 5,672,510 (including retrovirus vector), No. 5,635.
, 399 (including retroviral vectors expressing cytokines).

Non-viral delivery methods include liposome-mediated delivery, naked DNA delivery (direct injection), receptor-mediated delivery (ligand-DNA complex), electroporation, calcium phosphate precipitation, and particulate bombardment (eg, gene gun). )
But is not limited to these. Gene therapy materials and methods also include inducible promoters, tissue-specific enhancer-promoters, DNA sequences designed for site-specific integration, DNA sequences capable of providing selective dominance over parental cells, transformed Labels to identify cells, negative selection and expression control systems (safety indicators), cell-specific binding factors (for cell targeting)
, Cell-specific internalization factors, and transcription factors that enhance expression by the vector and methods of vector production. Such additional methods and materials for practicing gene therapy techniques are described in US Pat. Nos. 4,970,154 (including electroporation techniques), 5,679,559 (liposome for gene delivery). Protein-containing systems), 5,676,954 (including liposomal carriers), 5,593,875 (describing methods for calcium phosphate transfection), and 4, 945,050, ibid. (Describing the process by which biologically active particles are sprayed onto cells at a certain rate, whereby the particles penetrate the surface of the cells and are incorporated inside the cells), and P
CT publication WO 96/40958 (including nuclear ligands).

It is also contemplated that CHL2 gene therapy or cell therapy may further include delivery of one or more additional polypeptides in the same or different cell (s). Such cells can be introduced separately into the patient, the cells can be contained in a single implantable device (eg, the encapsulating membrane described above), or the cells can be separated by a viral vector. It can be modified.

A means of increasing expression of an endogenous CHL2 polypeptide in a cell by gene therapy is to insert one or more enhancer elements in the CHL2 polypeptide promoter, where the enhancer element is the transcriptional activity of the CHL2 gene. Can work to increase. The enhancer elements used are
Selected on the basis of the tissue in which it is desired to activate the gene-enhancer elements are known to confer promoter activation in that the tissue is selected. For example, if the gene encoding the CHL2 polypeptide "turns on" in T cells, the lck promoter enhancer element may be used. Thus, the added functional portion of the transcription element can be inserted into the fragment of DNA containing the CHL2 polypeptide promoter using standard cloning techniques (and, if desired, the vector and / or 5'and / Or inserted in the 3'flanking sequence). This construct, known as the "homologous recombination construct", can then be introduced into the desired cells, either ex vivo or in vivo.

Gene therapy can also be used to increase CHL2 polypeptide expression by modifying the nucleotide sequence of the endogenous promoter. Such modifications are typically accomplished by homologous recombination methods. For example, DNA containing all or part of the promoter of the CHL2 gene selected for inactivation
The molecule can be engineered to remove and / or replace a fragment of a promoter that regulates transcription. For example, the TATA box and / or binding site of the promoter's transcriptional activator can be deleted using standard molecular biology techniques; such deletion can inhibit promoter activity, Can block transcription of the corresponding CHL2 gene. Deletion of the TATA box or transcriptional activator binding site in the promoter results in the CHL2 polypeptide promoter (regulated C
D containing all or related parts (from the same or related species as the HL2 gene)
Can be achieved by generating an NA construct, wherein one or more TATA box and / or nucleotides of the transcriptional activator binding site are mutated by substitutions, deletions, and / or insertions of one or more nucleotides. To be done. As a result, the TATA box and / or activator binding site decreases activity,
Or completely deactivated. This construct, which is also typically the native (endogenous) 5'and 3'DN flanked by the modified promoter segments
Containing at least about 500 bases corresponding to the A sequence), either directly or via a viral vector as described herein, into appropriate cells (either ex vivo or in vivo). Can be introduced. Typically, the integration of this construct into the genomic DNA of the cell is by homologous recombination, where the 5'and 3'DNA sequences of this promoter construct are integrated into the endogenous chromosomal DNA.
By hybridizing to, may serve to help integrate the modified promoter region.

Therapeutic Uses CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof are used to treat many diseases, disorders or conditions, including those listed herein. , May be diagnosed, recovered or prevented.

CHL2 polypeptide agonists and antagonists include molecules that modulate CHL2 polypeptide activity and either increase or decrease the activity of at least one of the mature forms of the CHL2 polypeptide. Agonists or antagonists can be cofactors (eg, proteins, peptides, carbohydrates, lipids or small molecules) that interact with the CHL2 polypeptide and thereby regulate its activity. Potential polypeptide agonists or antagonists include antibodies that react with either soluble or membrane bound forms of CHL2 polypeptides that include some or all of the extracellular domain of the CHL2 protein. Molecules that regulate CHL2 polypeptide expression typically include C, which may act as an antisense regulator of expression.
Included are nucleic acids encoding HL2 polypeptides.

In adults, gene products of the BMP family (especially BMP2 and BMP
One of the major roles of 4) is the regulation of bone weight. BMP1 has been shown to cleave and inactivate CHD, and has been isolated from bone along with BMP2 and BMP3 (Wozney et al., 1988, Science 242).
: 1528-34; Celeste et al., 1990, Proc. Nat. Acad
． Sci. USA 87: 9843-47), CHL2 polypeptides may play important regulatory roles in bone formation. Thus, CHL2 nucleic acid molecules, polypeptides and agonists and antagonists thereof, including but not limited to anti-CHL2 selective binding agents, may be useful in diagnosing or treating diseases and conditions affecting bone density. Examples of such diseases and conditions include, but are not limited to, osteofibrosis and osteoporosis. Other diseases and conditions that affect bone density are included within the scope of this invention.

Direct delivery of BMP4 or other BMP family members to the regenerating bone via the bloodstream appears to be a direct therapeutic concept for the treatment of bone fibrosis.
However, this can be difficult to achieve because BMP4 is known to travel only short distances in vivo (Jones et al., 1996, Curr. Biol. 6: 1468-75). In the case of CHD during embryonic development, BMP and CHLII
Formation of a complex with the polypeptide can result in further migration of BMPs and the formation of a concentration gradient of BMP4 (Jones and Smith, 1998, Dev.
Biol. 194: 12-17).

Based on the spatially tight regulation of CHL2 gene expression on the surface of articular cartilage, where the first signs of cartilage damage are detected during the pathogenesis of osteoarthritis.
Altered expression levels of 2 polypeptides may play a role in the pathogenesis of osteoarthritis or rheumatoid arthritis. Thus, CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof, prevent cartilage fibrosis during the early stages of osteoarthritis and destroy surface areas once destroyed in the late stages of osteoarthritis or rheumatoid arthritis. Can be useful in regenerating or producing an appropriate superficial area (ie, surface) in transplanted cartilage.

BMP polypeptides have been shown to function in organogenesis during late embryogenesis. Organogenesis in embryonic kidney, lung and gastrointestinal tract has been shown to be affected by BMP4 expression (Hogan 1996, Genes D.
ev. 10: 1580-94). The combination of BMP4 and CHL2 polypeptides may be useful for controlling the proliferation and differentiation of progenitor cells in these tissues, thereby allowing the regulation of tissue regeneration or wound healing in vivo. Thus, CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may be useful in promoting tissue regeneration or wound healing.

CHL2 nucleic acid molecules, polypeptides, agonists and antagonists thereof, can also be used in the development and development of hematopoietic stem cells. It may be possible to use the BMP / CHL2 polypeptide complex to regulate primordial hematopoietic stem cells and thereby regulate repopulating stem cells of adult bone marrow. Alternatively, it may be possible to control the development of stem cells derived from mesodermal stem cells. Therefore, the CH of the present invention
L2 polypeptides and nucleic acids can be useful with BMPs for ex vivo expansion of hematopoietic stem cells and gene therapy performed through such cells.

BMP4 is an essential factor for generating hematopoietic progenitor cells from mouse ES cells. However, the effective concentration of BMP4 is in a narrow range (0.5 ng / ml to 5 ng / ml).
ml), which is consistent with the idea that differences in the active concentration of BMPs correlate with differences in cell types arising from totipotent ectoderm. No system for reproducible in vitro generation of hematopoietic stem cells from ES cells has yet been disclosed.
However, this can be achieved by precise control of the concentration of BMP4. The CHL2 nucleic acid molecules and polypeptides of the present invention may be useful in optimizing culture conditions for in vitro production of hematopoietic stem cells from ES cells.

Progenitor hematopoietic stem cells have recently been demonstrated in mouse yolk sac (Yode.
r et al., 1997, Proc. Natl. Acad. Sci. USA 94:67.
76-80). This subclass of hematopoietic stem cells does not show bone marrow regrowth activity in adults. However, when exposed to the environment of the neonatal liver, these progenitor stem cells are converted into long-term myelorepopulating stem cells (ie, secondary stem cells). This can also be achieved by culturing primordial stem cells on a particular stromal cell line. The long-term survival of this secondary stem cell in culture and the long-term maintenance of progenitor stem cells that spontaneously differentiate into secondary stem cells have not yet been established. BMP and CH
The interaction with the L2 polypeptide may function in the regulation of secondary stem cells.
The recombinant CHL2 polypeptides and CHL2 antibodies of the invention may be useful tools for long-term maintenance of hematopoietic stem cells in vitro and generation of secondary stem cells from primordial stem cells in vitro. Alternatively, BMP4 or putative novel C
HL2 interacting molecules may be useful for controlling these processes.

Moreover, primitive hematopoietic stem cells have not yet been well characterized. Progenitor stem cells can be of lymphocytic cell type, but such cells can also be mesoderm precursors, which can give rise to hematopoietic cell types and other mesodermal progeny. In support of this notion, adult bone marrow uses endothelial progenitor cells, cells that regenerate liver (Peterse).
n, et al., 1999, Science 284: 1168-70), as well as common stem cells that have the ability to induce endothelial cells, myocytes and hematopoietic cells in vivo (Ferrari et al., 1998, Science
279: 1528-30). Furthermore, the osteoblast lineage, which consists of bone marrow stroma, is known to be derived from mesodermal stem cells present in the bone marrow. It is also possible that common mesodermal stem cells may be responsible for the generation of both stromal and hematopoietic cells.
It has been previously speculated.

[0265] CHL2 polypeptide expression was detected in skeletal muscle, indicating that CHL2
2 polypeptides may also play a role in skeletal muscle development and function. Thus, CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may be useful in diagnosing or treating diseases and conditions affecting skeletal muscle. Examples of such diseases and conditions include, but are not limited to, cachexia and muscular dystrophy. Other diseases and conditions related to skeletal muscle development and function are included within the scope of this invention.

The expression of CHL2 polypeptide was detected in the heart, indicating that CHL2
Polypeptides may play a role in cardiac development and function. Therefore, C
HL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof, may also be useful in diagnosing or treating diseases and conditions affecting the heart. Examples of such diseases and conditions include, but are not limited to, arrhythmias, angina, hypertension, myocardial infarction and congestive heart failure. Other heart-related diseases and conditions are included within the scope of the present invention.

CHL2 polypeptides may play a role in gastric development and function, as expression of CHL2 polypeptides was detected in the stomach. Therefore, CHL
The 2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof, may also be useful in diagnosing or treating diseases and conditions related to the stomach. Examples of such diseases and conditions include, but are not limited to, gastric cancer and gastric ulcer. Other diseases and conditions associated with the stomach are included within the scope of this invention.

CHL2 polypeptide expression was detected in the liver, indicating that CHL2
Polypeptides may play a role in liver development and function. Therefore, C
HL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof, may also be useful in diagnosing or treating diseases and conditions involving the liver. Examples of such diseases and conditions include, but are not limited to, hepatitis and liver cancer. Other diseases and conditions associated with the liver are included within the scope of this invention.

An agonist or antagonist of CHL2 polypeptide function may be combined (simultaneously or together) with one or more cytokines, growth factors, antibiotics, anti-inflammatory and / or chemotherapeutic agents as appropriate for the condition being treated. Can be used continuously).

Other diseases caused or mediated by undesired levels of CHL2 polypeptides are included within the scope of this invention. Undesired levels include excess levels of CHL2 polypeptide and subnormal levels of CHL2 polypeptide.

Uses of CHL2 Nucleic Acids and Polypeptides Nucleic acid molecules of the present invention, including nucleic acid molecules that do not themselves encode biologically active polypeptides, can be used on a chromosome to integrate the CHL2 gene and related genes. The location of the gene to be mapped can be mapped. Mapping can be performed using techniques known in the art (for example,
PCR amplification and in situ hybridization).

CHL2 nucleic acid molecules, including nucleic acid molecules which themselves do not encode biologically active polypeptides, are qualitatively or quantitatively for the presence of CHL2 nucleic acid molecules in mammalian tissue or body fluid samples. May be useful as a hybridization probe in a diagnostic assay to test either.

Other methods may also be used where it is desired to inhibit the activity of one or more CHL2 polypeptides. Such inhibition may be provided by a nucleic acid molecule that is complementary to and hybridizes to the expression control sequences (triple helix) or CHL2 mRNA. For example, an antisense DNA or RNA molecule having a sequence that is complementary to at least a portion of the CHL2 gene can be introduced into the cell. Antisense probes can be designed by available techniques using the sequences of the CHL2 gene disclosed herein. Typically, each such antisense molecule is complementary to the start site (5 'end) of each selected CHL2 gene. This antisense molecule then forms the corresponding CHL2m
Translation of this mRNA is blocked or reduced when hybridized to RNA. Antisense inhibitors provide information associated with the reduction or absence of CHL2 polypeptides in cells or organisms.

Alternatively, gene therapy may be used to generate dominant negative inhibitors of one or more CHL2 polypeptides. In this context, DNA encoding each selected variant of CHL2 polypeptide polypeptide is prepared and the viral or non-viral method as described herein is used to It can be introduced into cells. Each such variant is typically designed to compete with the endogenous polypeptide in its biological role.

Further, CHL2 polypeptides (whether biologically active or not)
Can be used as an immunogen, ie the peptide contains at least one epitope against which antibodies can be raised. A selective binding agent that binds a CHL2 polypeptide (as described herein) is provided for in vivo and in vitro diagnostic purposes (for detecting the presence of a CHL2 polypeptide in a body fluid or cell sample, Can be used in, but is not limited to, use in labeled form. These antibodies can also be used to prevent, treat or diagnose a number of diseases and disorders, including the diseases and disorders listed herein. These antibodies bind to the CHL2 polypeptide and
At least one activity characteristic of the 2 polypeptide may be reduced or blocked or may be bound to the polypeptide to increase at least one activity characteristic of the CHL2 polypeptide (by increasing the pharmacokinetics of the CHL2 polypeptide. Including that).

The CHL2 polypeptides of the present invention can be used to clone CHL2 polypeptide receptors using expression cloning strategies. Radiolabeled ( ¹²⁵ iodine) CHL2 polypeptides or affinity / activity-tagged CHL2 polypeptides (eg, Fc fusions or alkaline phosphatase fusions) identify cell types or cell lines or tissues that express CHL2 polypeptide receptors. Can be used in a binding assay for RNA isolated from such cells or tissues is converted to cDNA, cloned into a mammalian expression vector and transfected into mammalian cells (eg, COS or 293 cells) to produce an expression library. You can The radiolabeled or tagged CHL2 polypeptide can then be used as an affinity ligand to identify and isolate from this library a subset of cells that express the CHL2 polypeptide receptor on their surface. DNA can then be isolated from these cells and transfected into mammalian cells to create a secondary expression library. The percentage of cells expressing the CHL2 polypeptide receptor in this library is several times higher than in the original library. This enrichment process can be iteratively repeated until a single recombinant clone containing the CHL2 polypeptide receptor is isolated. The isolation of this CHL2 polypeptide receptor is useful for identifying or developing novel agonists and antagonists of the CHL2 polypeptide signaling pathway. Such agonists and antagonists include soluble CHL2 polypeptide receptors, anti-CHL2 polypeptide receptor antibodies, small molecules or antisense oligonucleotides, and these are one or more of the diseases or disorders described herein. The treatment,
It can be used for prevention or diagnosis.

The mouse and human CHL2 nucleic acids of the invention also have their corresponding chromosomal CHL.
It is a useful tool for isolating 2 polypeptide genes. For example, CHL2
Mouse chromosomal DNA containing sequences can be used to construct knockout mice, which allows testing of their role in vivo for CHL2 polypeptides. Human CHL2 genomic DNA can be used to identify genetic tissue degenerative diseases.

Deposits of cDNAs encoding mouse and human CHL2 polypeptides were also subcloned into pSPORT1 (Gibco BRL) and Ameri.
can Type Culture Collection, 10801 Un
diversity Boulevard, Manassas, VA 20110
Accession No. PTA-147 created by Mar. 14, 2000 by Mar. 14, 2000
9 and PTA-1480.

The following examples are intended for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Example 1 Cloning of Mouse CHL2 Polypeptide Genes In general, the materials and methods as described in Sambrook et al. (Supra) are used to clone and analyze the gene encoding the mouse CHL2 polypeptide. Used to

Two mouse placental cDNA libraries were prepared to isolate sequences encoding the mouse CHL2 polypeptide. Total RNA was extracted from mouse placenta and poly-A + RNA was selected using standard extraction and isolation techniques. The random-primed cDNA is then cloned into S
upperscript Plasmid System for cDNA S
was synthesized from poly-A + RNA using synthesis (Gibco-BRL, Rockville, MD). The resulting cDNA was digested with NotI and fractionated on a 0.8% agarose gel. 300-1000bp electrophoresed
CDNA was isolated and ligated into the signal trap vector kFGF7, and a cDNA larger than 1.5 kb was isolated and ligated into pSPORT1. The ligation reaction was transformed into E. coli using standard transformation techniques. E. coli, these transformants were selected on medium containing ampicillin, and these transformants were harvested to make two cDNA libraries.

Clones containing a signal peptide sequence were enriched from a kFGF-based library using the kFGF signal trapping technique (US patent application Ser. No. 09 / 026,959). Plasmid DNA from this cDNA library was prepared from 10 pools of 100,000 colonies each using standard techniques. This DNA was transformed into NIH by calcium phosphate transfection.
Transfected cells were introduced into 3T3 cells and then the transfected cells were grown for 14 days in selection medium supplemented with 0.5% fetal bovine serum. Only transformants containing the plasmid with the signal peptide sequence produced colonies. These colonies were recovered by trypsinization and the total R
NA was treated with TRIzon reagent (Gibc according to the manufacturer's recommended protocol).
o-BRL). Poly-A + RNA was added to mRNA Purif
ication Kit (Amersham Pharmacia Biote
ch., Piscataway, NJ) was used to isolate from this total RNA.

To generate a cDNA library enriched for molecules containing a signal peptide sequence, the first-strand cDNA was ligated with SuperScript ^™ preamplification (pream).
preparation system (Gibco-BRL) was first used to prepare the poly-A + RNA isolated above. This first strand cDNA reaction
1 μg of poly-A + RNA and 2 pmol of primer 16 in a total volume of 15 μL
05-21

[0284]

[Chemical 1] And the template-primer mixture was heated at 70 ° C. for 10 minutes. The template-primer mixture was transferred to 50 ° C. and 2.5 μL 10 × buffer, 2.5 μL 25 mM MgCl ₂ , 1.3 μL
Of 10 mM dNTPs and 2.5 μL of 0.1 M dithiothreitol were added to the premixture. Then, reverse transcriptase (25
0 U) was added and the reaction was incubated at 50 ° C. for 1 hour. The first strand cDNA reaction is stopped by heating at 70 ° C for 15 minutes, and 2
RNA was digested by treatment with U RNAse H for 20 minutes at 37 ° C.

Following first-strand cDNA synthesis, 2 μL of first-strand cDNA, primer 1239-08, at a final concentration of 0.5 μM each in a volume of 100 μL.

[0286]

[Chemical 2] And 1605-22

[0287]

[Chemical 3] Containing 200 μM dNTPs and 2.5 U of Pfu polymerase (PE
Double-stranded cDNA was generated by PCR amplification in reactions containing Biosystems, Norwalk, CT). The PCR reaction was performed at 95 ° C for 1 minute for 1 cycle; 95 ° C for 30 seconds, 66 ° C for 45 seconds, and 72 ° C for 2 minutes for 30 cycles; and 72 ° C for 10 minutes for 1 cycle. Amplification product,
Digested with NotI and SalI and ligated into kFGF7. This ligation reaction was transformed into E. coli using standard transformation techniques. E. coli to generate a signal enriched cDNA library. Plasmid DNA was prepared from 400 selected clones and analyzed by sequencing.

The sequence of one clone (designated ymkz5-00011-c10) is X
Cordin (ch) derived from enopus (GenBank registration number Q91713)
ordin) precursor and was found to share significant homology. This identified clone encodes the N-terminal 115 amino acids of mouse CHL2, 418b.
It was found to contain a p insertion. The full-length cDNA clone was designated as ymkz5-0.
The 0011-c10 clone was used as a probe to isolate from a cDNA library cloned into pSPORT1.

To screen the pSPORT1 library, 1 × 10 ⁶ clones were plated on 150 mm plates at approximately 5 × 10 ⁴ clones per plate, and then these clones were plated from the plates onto nitrocellulose membranes. Filters were prehybridized in ExpressHyb Hybridization Solution (Clontech) for 30 minutes at 68 ° C., then ³² P-dCT.
Hybridization was performed overnight at 68 ° C. in the same hybridization solution containing P-labeled probe. The hybridized filters were washed twice in 2 × SSC and 0.1% SDS for 10 minutes at room temperature and in 0.1 × SSC and 0.1% SDS at 65 ° C. for 30 minutes, 2 Washed twice. After washing, the filters were subjected to autoradiography at -80 ° C overnight in the presence of an intensified screen. Positive clones were screened again. According to the secondary screen, two positive clones were identified and the DNA from these two clones was isolated and sequenced. One clone, designated pSPORTmCHL2, contained an insert of approximately 1.8 kb, which encoded the complete mouse CHL2 polypeptide.

Sequence analysis of the full-length cDNA for mouse CHL2 polypeptide showed that this gene contains a 1278 bp open reading frame encoding a protein of 426 amino acids. 1A-1C show mouse CHL
2 illustrates the nucleotide sequence of two genes (SEQ ID NO: 1) and the predicted amino acid sequence of mouse CHL2 polypeptide (SEQ ID NO: 2). In FIG. 1A, von
The signal peptide sequence predicted by the Heijne algorithm is underlined.

Computer analysis of the mouse CHL2 amino acid sequence revealed that this polypeptide
It has been shown to have three procollagen repeats (CR motifs), such as the CHL (CHL1) and CHLd5 polypeptides (co-pending and co-owned US patent application Ser. No. 09 / 724,915) (in contrast). In addition, four repeats are observed in CHD) (Fig. 5). The amino acid sequence of CHL2 is
Shares 24.4% identity with mouse cordin (or 2 in GAP study)
Share 8% identity). FIG. 2 shows mouse CHL2 polypeptide (mouse CHL2; SEQ ID NO: 2) and mouse cordin (Af069501;
The alignment of the amino acid sequence of SEQ ID NO: 7) is exemplified. The amino acid sequence of mouse CHL2 polypeptide also shares 50% identity with CHL1 and B
It shares structural similarities with MP and SOG, and this similarity is particularly high in the CR domain. The BLAST survey of the Celera Human Genome Database is CHL2
It was shown that the gene shares maximum identity with the CHL1 gene. These results suggest that the CHL2 gene is a novel member of the CHD / SOG gene family.

Example 2 Cloning of Human CHL Polypeptide Genes Generally, the materials and methods as described in Sambrook et al. (Supra) are used to clone and analyze the gene encoding the human CHL2 polypeptide. Used to

To isolate the sequence encoding the human CHL2 polypeptide, human placenta cd
An NA library was prepared. Total RNs using standard extraction and isolation techniques
A was extracted from human placenta and poly-A + RNA was selected. Then, the oligo d (T) -primed (Origo d (T) -primed) cDNA was
Superscript Plasmid System for cDNA
Synthesized from poly-A + RNA using Synthesis (Gibco-BRL). The resulting cDNA was digested with NotI and fractionated on a 0.8% agarose gel. The electrophoresed cDNA larger than 1.5 kb was isolated and
Ligated into RT1. The ligation reaction was transformed into E. coli using standard transformation techniques. co
li, the transformants were selected on medium containing ampicillin, and these transformants were harvested to generate a human placental cDNA library.

A ³² P-dCTP labeled mouse CHL2 cDNA fragment was used to screen a human placental cDNA library. Plasmid DNA,
1 μg from each pool, isolated from 12 pools of 100,000 clones each
Plasmid DNA was digested with NotI and SalI, electrophoresed on a 0.8% agarose gel and then transferred to a nitrocellulose membrane. The filter is placed in an ExpressHyb hybridization solution (C
prehybridized in lontech) and then mouse CHL2 cDNA
Hybridization was performed overnight at 60 ° C. in the same hybridization solution containing the probe. The hybridized filter is 2 × SSC and 0.1% SD
Wash twice in S for 10 minutes at room temperature and 0.5 × SSC and 0.1% S
Washed twice in DS at 60 ° C for 30 minutes. After washing, the filters were subjected to autoradiography at -80 ° C overnight in the presence of an intensified screen. Positive cDNA fragments were identified in 2 out of 12 plasmid pools.

Individual clones containing sequences encoding the human CHL2 polypeptide were cloned into 150
3 × 10 ⁵ clones from each pool were added to the mm plate at about 5 × per plate.
Isolated from the two plasmid pools identified above by plating at 10 ⁴ . These clones were then mounted from the plates onto nitrocellulose filters and the filters analyzed as described above. Two positive clones were identified and each was subjected to sequencing analysis. One clone (pSP
(Named ORThCHL2) contained an approximately 1.5 kb insert encoding the complete human CHL2 polypeptide.

Sequence analysis of the full-length cDNA of human CHL2 polypeptide showed that this gene contains a 1287 bp open reading frame encoding a protein of 429 amino acids. 3A-3C illustrate the nucleotide sequence of the human CHL2 gene (SEQ ID NO: 4) and the deduced amino acid sequence of the human CHL2 polypeptide (SEQ ID NO: 5). The amino acid sequence of CHL2 is similar to that of human cordin 2.
It was Heading to share 6.7 percent identity. FIG. 4 depicts an amino acid sequence alignment of human VHL2 polypeptide (human CHL2; SEQ ID NO: 5) and human cordin (Af076612; SEQ ID NO: 8) .

Example 3 CHL2 mRNA Expression Multiple Human Tissue Northern Blots or Mouse Tissue Northern Blots (Clo)
nCH) is ³² P-dCTP-labeled human or mouse CH, respectively.
It was probed with the L2 cDNA fragment. Northern blot, Ex
Prehybridization was performed in press Hyb hybridization solution (Clontech) for 30 minutes at 68 ° C , then hybridized overnight at 68 ° C in the same solution to which labeled probe was added. After hybridization, the filters are washed twice in 2x SSC and 0.1% SDS for 10 minutes at room temperature with 0.1x.
Washed twice in SSC and 0.1% SDS for 30 minutes at 68 ° C. After washing, the blot was subjected to audradiography in the presence of an intensifying screen at 80 ° C for 72 hours.

Northern blot analysis of human tissue blots shows that in the prostate, testis, uterus (very abundant expression), colon, small intestine, heart, skeletal muscle and stomach , there is a predominant C of about 2 kb.
Represented HL2 transcript. Weak expression was detected in trachea, placenta and bone marrow, and very weak expression was detected in liver. Northern blot analysis of mouse tissue blots revealed a predominant CHL2 transcript of approximately 1.8 kb in liver and kidney. Very weak expression was detected in the heart. Weak expression of a transcript of approximately 2 kb was detected in testis and skeletal muscle. CHL2 expression was not analyzed in mouse stomach tissue. Very weak expression was detected in mouse embryonic tissue of day 7, 11, 15 and 17 embryos.

Northern blot analysis strengthens the relationship between CHD / SOG and CHL2 polypeptides. CHD / SOG is expressed at relatively high levels in E7 embryos and expressed in E11, E15 and E17 embryos at lower levels (Pap
pano et al., 1998, Genomics 52: 236-39). CHL2
mRNA was also detected in E7, E11, E15 and E17 embryos, but at lower levels than CHD / SOG transcripts. CHD / SO G is spleen,
It was expressed in liver and kidney (Pappano et al., 1998). CHL2 m
RNA was similarly detected in liver and kidney. The similarity in expression pattern, combined with the similarity in structure, suggests that CHL2 and CHD / SOG have similar biological activities, although these proteins may function at different developmental stages.

Expression of CHL2 mRNA was localized by in situ hybridization. Embryos and adult mouse tissues in situ hybridization to sections, as serial loading was performed (Wilcox, 1993, J Histochem.Cytochem.41: . 1725~33). Mouse CH
The L2 probe was first loaded with 1.2 kb NcoI-S from pSPORTmCHL2.
It was prepared by removing the acI fragment, pSPmCHL2 C OO
H was generated which was then linearized with EcoRI. Furthermore, the HindIII fragment was taken out to obtain pSPmCHL2NH2. Antisense RNA
It was synthesized using SP6 RNA polymerase. The RNA synthesized from these plasmids contained non-overlapping sequences.

[0301] by in situ hybridization analysis, CHL2 mRNA expression was initiated in the sternum at about 12. Day 5 embryos in mice, and have been demonstrated to survive in a limited area of adult articular cartilage. CHL2 mRNA expression was not detected in sections tested in embryos 10.5 days old. 13 in. 5 day embryos, a strong CHL2 mRNA expression over chondrocytes in the extremities of the bone (Fig. 6 and 7) and the sternum, and was detected in mesenchymal cells of the perichondrium adjacent to the joint during development. Strong but very limited CHL2 mRNA expression was also detected in developing articular cartilage of vertebrae and chondrocytes in superficial regions within the epiphyses of long bones (FIGS. 6-8). CHL2 m until embryos up to 18.5 days and until adulthood
Skeletal expression of RNA was limited to a single layer of chondrocytes surface regions in articular cartilage. The epiphyseal growth plate did not show CHL2 mRNA expression at any time point in mouse development.

[0302] Among the non-cartilaginous tissues, significant CHL2 mRNA expression, the ovaries of female mice, oviduct and uterus, and testis of male mice epididymis and possibly other sub glandular (e.g., seminal vesicles, Observed in the coagulating gland , and prostate). The strong signal observed in the uterine wall was somewhat similar to CHL1 mRNA expression detected in the uterus. Weak expression of CHL2 mRNA was also detected on the colon surface. However, the location of CHL2 expression in the colon is distinctly different from that of CHL1, where CHL2 mRNA is
Detected in fibroblasts / connective tissue cells that separate submucosa and muscularis (Nak
ayama et al., 2001). In contrast to the CHL1 gene, CHL2 mRNA expression was not detected in the stomach or small intestine. Therefore, even among soft tissues, CH
The L2 gene was specifically expressed in the reproductive organs.

[0303]: Using (Example 4 Mouse CHL2 Chromosomal Mapping of polypeptide gene) fluorescence in situ hybridization (FISH) analysis, to determine the chromosomal station resident mouse CHL2 gene (Shi et al., 1997, Gnenom
ics 45: 42-47). FISH probe, standard technology and mouse CH
A primer corresponding to the 5'untranslated region of the L2 gene (5'-TCCTCTCAT
CCTCACCTTAG-3 ′; SEQ ID NOS: 15 and 5′-GGAGAAAAGT
A mouse ES-129 / SvJ II BAC chromosomal DNA library (Ge) by PCR using GAGATAAGGACAC-3 ′; SEQ ID NO: 16).
It was prepared from BAC clone (F1067) isolated from Nome Systems). Mice CHL2 gene, using the chromosome 7 centromere-specific P1 clones as a simultaneous hybridization (co-hyb ridization) probe was localized to chromosome 7 (Shi et al., 1997). Total analyzed 80 metaphases cells and 77 10 cells of which showed specific labeling was used for simultaneous hybridization experiments. As a result, CHL
2 gene, heterochromatin for telomere of the chromosome 7 - from euchromatin boundary 6
Located in a position which is a distance of 6% (that corresponds to the band 7E2-E3). Therefore,
CHL2 gene is different from the CHL1 gene in terms CHL2 gene is autosomal.

Example 5: Production of CHL2 Polypeptides A. Expression of CHL2 Polypeptides in Bacteria PCR is used to amplify a template DNA sequence encoding a CHL2 polypeptide. Primers corresponding to the 5'and 3'ends of this sequence are used for this PCR. The amplified DNA product may be modified to contain restriction enzyme sites to allow insertion into an expression vector. The PCR product is gel purified and inserted into an expression vector using standard recombinant DNA methodologies. pAMG21 (AT containing a gene encoding lux promoter and kanamycin resistance
An exemplary vector such as CC No. 98113) is digested with BamHI and NdeI for directional cloning of the inserted DNA. The ligated mixture was electroporated by E. coli. E. coli host strain and transformants are selected for kanamycin resistance. Plasmid DNA from selected colonies is isolated and subjected to DNA sequencing to confirm the presence of insert.

Transformed host cells are incubated at 30 ° C. in 2 × YT medium containing 30 μg / mL kanamycin prior to transfection. Gene expression is induced by the addition of N- (3-oxohexanoyl) -dl-homoserine lactone to a final concentration of 30 ng / mL, followed by 6 hours of incubation at either 30 ° C or 37 ° C. Expression of CHL2 polypeptide, centrifugation, resuspension and dissolve the bacterial pellet culture, and is assessed by analysis of host cell proteins by SDS-polyacrylamide gel electrophoresis.

Inclusion bodies containing CHL2 polypeptides are purified as follows. Bacterial cells are pelleted by centrifugation and resuspended in water. The cell suspension is lysed by sonication and pelleted by centrifugation at 195,000 xg for 5-10 minutes. The supernatant is discarded and the pellet washed and transferred to the homogenizer. Add 5 mL of Perc to the pellet until uniformly suspended.
Homogenize in an Oll solution (75% liquid Percoll and 0.15M NaCl), then dilute and centrifuge at 21,600 xg for 30 minutes. Gradient fractions containing inclusion bodies are collected and pooled. The isolated inclusion bodies are analyzed by SDS-PAGE.

E. A single band on the SDS polyacrylamide gel, corresponding to the CHL2 polypeptide produced by E. coli, was excised from the gel and the N-terminal amino acid sequence was determined essentially by Matsudaira et al., 1987, J. Mol. Biol. Chem.
262: 10-35.

(B. Construction of CHL2 Polypeptide Mammalian Expression Vector) Mouse CHL2 was transiently expressed using the pSRαmCHL2 vector, which was prepared as follows. The open reading frame of CHL2 polypeptide was determined using standard techniques, as well as primers 2360-40 (5'-
GCTATCTAGAGCCACCCATGGTTCCCCGGGGTGAGGGAT
CATC-3 '; SEQ ID NO: 17) and 2360-41 (5'-GCTAGTC
It was first amplified by PCR using GACCATTAATGTCTTGGTCACTTTGTCTG-3 '; SEQ ID NO: 18). The amplified produced product Xba I
And Sal I and digested, then SRα-based expression plasmid (
Takebe et al., 1988, Mol. Cell. Biol. 8: 466-72)
And inserted into, to produce a pSRαmCHL2.

[0309] FLAG tagged mouse CHL2 polypeptide expression construct was prepared as follows. Full length mouse CHL2 DNA fragment (where the stop codon is Sal I
Site-substituted) as a template for the full-length mouse cDNA clone as well as the primers 5′-GCTAGCGGCCGCGCCACCATGGGTTC.
CCGGGGGTGAGGATCATC-3 ′; SEQ ID NO: 19) and 5′-GC
TAGTCGAACTAATGTCTTGGTCACTTTGTCTGGGC-3
'; SEQ ID NO: 20) was used to obtain by PCR. The amplified PCR product was digested with NotI and SalI, then pFLAG-CMV-5a.
It was inserted into an expression vector (Sigma) with the FLAG sequence linked in-frame with the CHL2 sequence at its carboxy terminus. Obtained mCHL2-FLA
The G expression plasmid, named PFLAGmCHL2.

Expression of mCHL2-FLAG polypeptide was expressed in 5% (w / v) skim milk powder (T.
Rader Joe's, Thousand Oaks CA) at 3% (w /
v) 1 according to manufacturer's recommendations, except used in place of bovine serum albumin
Detection was by direct Western blot analysis using 0 μg / ml of anti-FLAG mouse monoclonal antibody, M2 (Sigma). Rabbit polyclonal antibody against mouse CHL2 was isolated from Harlow and Lane, Using A
ntibodies: A Laboratory Manual (Cold S
was initiated with a synthetic peptide based on the carboxy terminal sequence CPEDEAEDDHSEVISTR (SEQ ID NO: 21) as described in pring Harbor Laboratory Press, 1988). The resulting antisera were used directly or subjected to affinity purification with the corresponding peptides.

To generate clones that can stably express mCHL2-FLAG, linear pFLAGmCHL2 was used to transfect 293 cells. Select stable transformants, and the expression levels of the corresponding FLAG tagged proteins in both serum-free conditioned medium and cell lysates were compared by Western blot analysis (Nakayama et al., 2001, Dev.Biol. Printing During)
. Conditioned medium was concentrated prior to Western blot and then the protein was visualized. Expression based on transient transformation was performed using 293T cells. The 10-fold concentrated conditioned medium was then analyzed as described above.

For medium-scale preparation, stable clones were cloned into CL 1000 flasks (Inte.
gra Biosciences, Ijamsville, MD), 2
Cultivated in 93 SFM II serum-free medium and conditioned medium was collected every 2-3 days. Expression levels of about 3-4 μg / ml were obtained. The FLAG tagged protein high salt conditions (Piccolo et al., 1997, Cell 91: 407~16) in using anti-FLAG M2 affinity gel (Sigma), from supernatants collected in 500ml by affinity chromatography single step Purified.
The protein concentration of each preparation was determined by Western blot analysis with M2 and compared to FLAG bacterial alkaline phosphatase standard (Sigma).

[0313]: antibodies against (Example 6 Anti CHL2 production of polypeptide antibody) CHL2 polypeptide, using C HL2 peptides generated by purified proteins or biological synthesis or chemical synthesis acquired by immunizing obtain. Suitable procedures for producing antibodies are described by Hudson and Bay.
, Practical Immunology (second edition, Blackwel l S cientific Pubilication) includes a procedure described in.

In one procedure for antibody production, animals (typically mice or rabbits) were injected with a CHL2 antigen (eg, CHL2 polypeptide) and determined by ELISA for hybridoma production. Animals with sufficient serum titer are selected. The spleen of the immunized animal is harvested and prepared as a single cell suspension from which splenocytes are harvested. Spleen cells were fused to mouse myeloma cells (eg Sp2 / 0-Ag14 cells) and DMEM (containing 200 U / mL penicillin, 200 μg / mL streptomycin sulfate, and 4 mM glutamine).
Incubate first, then in HAT selection medium (hypoxanthine, aminopterin and thymidine). After selection, tissue culture supernatant is removed from each fusion well and tested by ELISA for anti-CHL2 antibody production.

Another procedure may also be used to obtain anti-CHL2 antibodies. This procedure can be used, for example, in the immunization of transgenic mice carrying the human Ig locus for the production of human antibodies, and in the screening of synthetic antibody libraries, such as those produced by mutagenesis of antibody variable domains. is there.

Example 7: Biological activity of mouse CHL2 in Xenopus embryo Cordin forms gastrulation by inhibiting the activity of BMP4 Xe.
It is known to dorsalize nopus embryos. The effect of CHL2 on the development of Xenopus embryos was tested as follows. EcoRI of pSPORTmCHL2
The NotI fragment was first labeled with pCS2 + (Ruppp et al., 1994, Ge.
nes Dev. 8: 1311-23), linearized with Not I, and capped mRNA was synthesized with SP6 polymerase and quantified (Nishinakamura et al., 1999, Dev. Biol. 2).
16: 481-90). The pSPORTmCHL2 plasmid also directly linearized with No t I, and transcribed with T7 polymerase. Both constructs induced secondary axis formation. As a negative control, elongation factor 1 (EF1) RNA was synthesized.

[0317] Each RNA sample was injected into two ventral blastomeres of Xenopus embryos 4-cell stage. After Note morphism, the embryos were cultured for 48 hours at 10% Steinberg solution, following Ide were scored for ectopic axis (Nishinakamura et al, 1
999; FIG. 9). When blastomeres were injected with 1 pg of mouse CHL2 RNA, the axial replication rate ranged from 77-87%, whereas the uninjected control and EF1 RNA injected embryos had a 0% rate . Experiments with mouse CHL1 RNA were also performed as a positive control. In these experiments, 10-3
An axial replication rate of 83% was obtained when 0 pg of CHL1 RNA was used. Therefore, mouse CHL2 RNA has the activity of antagonizing the endogenous ventrifying factor (probably BMP4).

Example 8 Inhibition of BMP4 Action by Mouse CHL2 Polypeptide Formation of the cordin - CHL1 complex is known to inhibit BMP4 function. Similar axial replication activity observed for CHL2 polypeptides (Example 7)
Suggested that the CHL2 polypeptide also directly inhibited BMP4 action. CD34 ⁺ / CD31 ^hi lymphocyte hematopoietic progenitor cells, CD34 ⁺ / CD
31 ^lo erythroid-myeloid progenitor cells, and CD45 ⁺ myelomonocytic cells have been shown to be dependent on the presence of 0.15-2 ng / ml BMP4 during the differentiation of mouse embryonic stem cell (ES) cells. (Nakayama et al., 2000, Blood 9
5: 2275: 83).

The ability of CHL2 polypeptides to inhibit the action of BMP4 was tested directly as follows. Rosa26 ES cells were transferred to fibronectin coated plates, conditioned in serum-free medium for 2 days and then differentiated in serum-free methylcellulose medium in the presence of 0.9 ng / ml BMP4 (Nakayama et al., 200.
0). A BMP4 concentration of 0.9 ng / ml produced near maximal levels. Embryoid bodies (EB) was collected on day 7, were separated by co-La Genaze. The these cells, C
It was stained with monoclonal antibodies for hematopoietic progenitor cell markers such as D31 and CD34. Then, the stained sample was subjected to FACScan (Becton Dick
inson, San Jose, CA).

[0320] If the mCHL2-FLAG affinity purified and added pressure beyond the ^{100ng / ml, CD34 - / CD31} + cell population and CD34 ⁺ / CD31 ⁺ cell population, background levels of (i.e., is achieved without BMP4 Level (see FIG. 10)). These results, with FLAG tag m
It suggests that the CHL2 protein could inhibit the action of BMP4 in vitro.

[0321]: Both (Example 9 BMP2 action and BMP4 inhibition by murine CHL2 polypeptide of action) BMP2 and BMP4, which is known to induce alkaline phosphatase expression in C2C12 myoblasts. BMP2 inhibition by CHL2 polypeptide of action and BMP4 effect was demonstrated in C2C12 alkaline phosphatase assay.

The pre-myoblast cell line C2C12 (ATCC Accession No. CRL-1722) was treated with 5% C at 37 ° C. in DMEM containing 10% fetal bovine serum (FBS) and antibiotics.
Maintained in a humid atmosphere of O ₂ . Receptor sensitivity can be reduced in highly passaged cells, so cells were discarded after 10-15 passages. C2C12 cells were plated in 100 μl DMEM (containing 2% bovine serum and antibiotics) in 96 well microtiter plates at a density of 3 × 10 ⁴ cells / well. The outermost wells of the microtiter plate were filled with 200 μl DMEM only to avoid excessive drying. Cells were then, 5 at 37 ° C.
Incubated overnight in a humid atmosphere of% CO ₂ .

[0323]   After plating, C2C12 cells were loaded with 909 ng / ml (35 nM) BMP2 or
Mouse CHL in the presence of either 566 ng / ml (22 nM) BMP4
Serial dilution of 2-FLAG (1.47, 2.93, 5.9, 11.7, 23.4,
46.9, 93.8, 187.5, 375, 750, and 1500 ng / ml
) Was exposed to. Then the cells,5% CO at 37 ° C_Two3 days in a humid atmosphere
Incubated. After incubation, the medium is removed and the cells are washed with 0.1 M T Rinse with ris, pH 7.4 and 150 μl glycine buffer
Solution (0.1 M glycine, 1 mM MgCl_Two, PH 10.5) (0.1% IGE
PAL CA-630 (containing Sigma)) was added to the wells. Then this
The cells were frozen at -80 ° C, thawed and 50 μl of cell supernatant was added to the Bradford cells.
Removed for use in protein assay and 100 μl phosphate p
-Nitrophenyl disodium (Sigma; 4 mg / ml in glycine buffer)
Diluted),Added to the remaining cell supernatant. Incubate this mixture at 37 ° C for 30 minutes.
Incubate and add 50 μl of 0.5 N NaOHAddReaction stopped
. The plate was then read at 405-410 nm. CHL2-FLA
G is BMP2 in a dose-dependent mannerActionAnd BMPActionInhibiting both
Were found (Fig. 11).

[0324]   Example 10: Direct interaction between CHL2 polypeptide and BMP   Mouse CHL1 polypeptide and human BMP4, BMP5, BMP6 and TG
Direct interaction with Fβ2 has been previously demonstrated (Nakayama et al., 20.
01). Except that mCHL2-FLAG protein was used at 600 ng / ml,
Similar immunoprecipitation experiments were performed with mouse CHL2 polypeptide. mCHL
2-FLAG proteins are BMP2, BMP4, BMP5, BMP6, GDF
5 (BMP14) or activin AJoint withImmunoprecipitates(Co-immu noprecipitate) Was found (Fig. 12). However, high concentration
BMP5 may exhibit detectable levels of interaction with mCHL2-FLAG.
Was needed. CHL2 seems to have a weaker affinity for BMP5 than for other BMPs
Met. Similar to mCHL1, activin A (another TGFβ superfamily
Members) have CHL2 and CHL2 under any set conditions.ofShow signs of interaction
There wasn't. However, with mouse CHL1In contrast, TGFβ interaction observed at all
Was not done. BMP2 and BMP4 are BMP5, BMP6 and BMP7 What is Form separate subfamilies (Celeste et al., 1990, Proc
． Natl. Acad. Sci. U. S. A. 87: 9843-47). Therefore
, CHL1 and CHL2 polypeptides, all BMPs
Is a protein that binds to (pan-BMP binding protein)
possible.

Example 11: Expression of CHL2 Polypeptides in Transgenic Mice To assess the biological activity of CHL2 polypeptides, encode CHL2 polypeptide / Fc fusion protein under the control of liver-specific ApoE promoter. To prepare the construct. Delivery of this construct is expected to produce pathological changes that are informative regarding the function of the CHL2 polypeptide. Similarly, a construct containing the full-length CHL2 polypeptide under the control of the β-actin promoter is prepared.
The delivery of this construct is expected to cause expression of the ubiquitous.

To generate these constructs, PCR is used to amplify the template DNA sequence encoding the CHL2 polypeptide. Primers corresponding to the 5'and 3'ends of the desired sequence are used for this PCR. This primer then incorporates a restriction enzyme site that allows the insertion of the amplification product into the expression vector.
After amplification, the PCR product is gel purified, digested with the appropriate restriction enzymes and ligated into the expression vector using standard recombinant DNA techniques. For example, amplified CHL2
The polypeptide sequence is described in Graham et al., 1997, Nature Geneti.
cs, 17: 272-74 and Ray et al., 1991 Genes Dev. 5
: 2265-73, it can be cloned into an expression vector under the control of the human β-actin promoter.

After ligation, the reaction mixture was used to transform E. E. coli host strains are transformed by electroporation and transformants selected for drug resistance. Plasmid DNA from selected colonies is isolated and subjected to DNA sequencing to confirm the presence of the appropriate insert and the absence of mutations. The CHL2 polypeptide expression vector is purified through two rounds of CsCl density gradient centrifugation, cut with the appropriate restriction enzymes, and the linearized fragment containing the CHL2 polypeptide transgene is purified by gel electrophoresis. The purified fragment was
2 mg / mL in mM Tris (pH 7.4) and 0.2 mM EDTA
Resuspend at a concentration of.

[0328] The single cell embryos from BDF1 × BDF1 bred mice are injected as in serial mounting (PCT Publication No. WO97 / 23614). Embryos are cultured overnight in a CO ₂ incubator and 15-20 2-cell embryos are transferred into oviducts of pseudopregnant CD1 female mice. The progeny obtained from the implantation of microinjected embryos are screened by PCR amplification of the transgene incorporated into the genomic DNA sample as follows. The ear sections ear buffer 20mL (20mM Tris, pH8.0,10mM EDTA, 0.5% SDS, and 500 mg / mL proteinase K)
Digest at 55 ° C. overnight. The sample is then diluted with 200 mL TE and 2 mL ear sample is used in a PCR reaction with appropriate primers.

At 8 weeks of age, transgenic founder animals and control animals are sacrificed for necropsy and pathological analysis. Remove a portion of spleen and remove Total RN
Total cellular RNA is isolated from spleens using the A Extraction Kit (Qiagen) and transgene expression is determined by RT-PCR.
RNA recovered from the spleen was analyzed by SuperScript ^™ Pre as follows.
c using amplification system (Gibco-BRL)
Convert to DNA. Appropriate primers (located in the expression vector sequence and CH
The 3'side of the L2 polypeptide transgene) is used to prime cDNA synthesis from the transgene transcript. 10 mg of total spleen RNA from transgenic founder animals and controls was used for 7 minutes with 1 mM primer.
Incubate at 0 ° C and place on ice. Then, the reaction was supplemented with 10 mM T
ris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl ₂ , 1
0 mM of each dNTP, 0.1 mM DTT, and 200 U of SuperScr
Supplement with iptII reverse transcriptase. After 50 minutes incubation at 42 ° C,
The reaction is stopped by heating at 72 ° C for 15 minutes and digested with 2U RNase H for 20 minutes at 37 ° C. The sample is then amplified by PCR with primers specific for the CHL2 polypeptide.

Example 12: Biological activity of CHL2 polypeptides in transgenic mice Prior to euthanasia, transgenic animals are weighed, anesthetized with isoflurane and bled by cardiac puncture. The sample is subjected to hematology and serum chemistry analysis. Radiographs will be taken after the final exsanguination. The major internal organs are subjected to gravimetric analysis during gross dissection.

Following gross dissection, tissues (ie liver, spleen, pancreas, stomach, whole gastrointestinal tract, kidneys, reproductive organs, skin and mammary glands, bones, brain, heart, lungs, thymus, trachea, esophagus, thyroid gland,
Adrenal glands, bladder, lymph nodes and skeletal muscle) are removed and fixed in 10% buffered Zn-formalin for histological examination. After fixation, the tissue is processed in a paraffin block and 3 mm sections are obtained. All sections are stained with hematoxylin and eosin and then subjected to histological analysis.

[0332] subjecting both the spleen of transgenic mice and control mice, the lymph nodes and Peyer's patches, the immunohistochemical analysis with B cell-specific antibodies and T cell specific antibodies as follows. Formalin fixed paraffin embedded sections are deparaffinized and hydrated in deionized water. The sections were quenched with 3% hydrogen peroxide and then Protein Block (Lipshaw, Pittsburgh)
, PA) and rat monoclonal anti-mouse B220 and C
Incubate in D3 (Harlan, Indianapolis, IN). Antibody binding was performed using DAB (BioTek, chromogen) as a chromogen.
Santa Barbara, CA) using biotinylated rabbit anti-rat immunoglobulin and peroxidase-conjugated streptavidin (BioGen).
ex, San Ramon, CA). Sections are counterstained with hematoxylin.

After necropsy, MLN and sections of spleen and thymus from transgenic animals and control littermates are removed. Using the flat end of a syringe, 100m
m nylon cell strainer (Becton Dickinson, Fran)
klin Lakes, by gently grinding the tissues with respect to the bottom of NJ), to prepare a single cell suspension. Cells were washed twice and counted, then approximately 1 × 10 ⁶ cells from each tissue were added to 0.5 μg CD16 / 32 (in 20 μL volumes.
Incubate with FcγIII / II) Fc block for 10 minutes. The sample was then conjugated to FITC or PE, CD90.2 (Thy-1.2.
), CD45R (B220), CD11b (Mac-1), Gr-1, CD4 or CD8 against monoclonal antibodies (PharMingen, San Di).
ego, CA) with 0.5 μg antibody in 100 μL volume of PBS (lacking Ca ⁺ and Mg ⁺ ), 0.1% bovine serum albumin and 0.01% sodium azide for 30 minutes at 2-8 ° C. To dye. After antibody binding, cells are washed and then F
Analyze by flow cytometry on ACSScan (Becton Dickinson).

Although the present invention has been described in terms of preferred embodiments, it will be appreciated that variations and modifications will occur to those skilled in the art. Therefore, the scope of the appended claims, is intended to encompass all such equivalent modifications that fall within the scope so that book onset Ming claimed.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Contents of correction]

[Brief description of drawings]

1A-1C depict the nucleotide sequence of the mouse CHL2 gene (SEQ ID NO: 1) and the deduced amino acid sequence of the mouse CHL2 polypeptide (SEQ ID NO: 2). The predicted signal sequence is shown (underlined).

FIG. 2 shows the amino acid sequence alignment of mouse CHL2 polypeptide (mouse CHL2; SEQ ID NO: 2) and mouse cordin (Af069501; SEQ ID NO: 7).

3A-3C depict the nucleotide sequence of the human CHL2 gene (SEQ ID NO: 4) and the deduced amino acid sequence of the human CHL2 polypeptide (SEQ ID NO: 5). The predicted signal sequence is shown (underlined).

FIG. 4 shows human cordin (huCHD; SEQ ID NO: 8), human CHL1 polypeptide (huCHL; SEQ ID NO: 9), and human CHL2 polypeptide (huCH).
L2: shows the amino acid sequence alignment ( alignment) of SEQ ID NO: 5).

FIG. 5 shows a schematic diagram of mouse Chordin, CHL1 polypeptide, and CHL2 polypeptide. Pre-collagen repeat (CR; homologous C
R domain is indicated by a gray box), signal peptide (SP), putative BMP1
/ Tolloid cleavage site ( ^* ) and sites of amino acid sequence variation in CHL1 (dE and d5).

FIG. 6. Detection by in situ hybridization in the E17.5 mouse hip joint (panels A and B) and the rib-chondral joint (panel C; showing signals in articular chondrocytes on both sites of the joint). Mouse CHL2
The expression of mRNA is shown.

FIG. 7: Normal adult spinal joints (panels A, B, and C; in articular cartilage cells on the surface of articular cartilage on either side of the articular process or in the facet joints between adjacent spinal processes). Signal) and the intervertebral disc fibrosis anulus fi
3 shows expression of mouse CHL2 mRNA detected by in situ hybridization in fibrocartilage of brosus (panel D).

FIG. 8 shows mouse CHL2 mRNA expression detected by in situ hybridization in the sternum and placenta of E18.5 mice and in the uterus, colon, and small intestine of normal adult mice.

[Figure 9]   Figure 9 shows the secondary axis forming activity of mouse CHL2 polypeptide.

FIG. 10 ^is, CD34 ⁺ / CD31 + red blood cells - bone marrow (erythro-myel
BMP of progenitor cells (R7) and CD34 ⁻ / CD31 + cells (R3)
4 shows the effect of CHL2 polypeptide on 4-dependent production.

FIG. 11 shows the effect of CHL2 polypeptide on the induction of BMP2-dependent alkaline phosphatase in C2C12 myoblasts.

FIG. 12 shows the results of Western blot using mCHL2-FLAG protein. In panel A, mCHL2-FLAG was mixed with BMP5 or activin A, treated with anti-mCHL2 antiserum and precipitated with protein A agarose beads. In panel B, mCHL2-FLAG is
BMP2, BMP4, BMP5, BMP6, GDF5 (BMP14), or activin A were mixed, treated with anti-mCHL2 antiserum, and precipitated with protein A agarose beads.

[Procedure 3]

[Document name to be corrected] Drawing

[Name of item to be corrected] Fig. 12

[Correction method] Change

[Contents of correction]

[Fig. 12]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 7/00 A61P 9/06 4H045 9/06 9/10 9/10 9/12 9/12 19/02 19/02 19/08 19/08 19/10 19/10 21/00 21/00 29/00 101 29/00 101 35/00 35/00 43/00 111 43/00 111 C07K 14/47 C07K 14 / 47 16/18 16/18 16/46 16/46 19/00 19/00 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 C12P 21/02 C 5/10 21/08 C12P 21/02 G01N 33/15 Z 21/08 33/50 Z G01N 33/15 33/53 D 33/50 C12N 15/00 ZNAA 33/53 5/00 AB (81) Designated country EP (AT, BE , CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, C) , CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW) ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, S L, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Ming Ling, Cam United States California 93065, Surumi Valley, Hazelwood Way 605, Unit Dee (72) Inventor Naoki Nakayama United States California 91360, Thousand Oaks, Briar Bluff Circle 325 F Term (reference) 2G045 AA25 AA29 AA40 BA13 BB03 BB20 CB01 CB17 CB21 DA12 DA13 DA14 DA36 DA77 FB03 FB04 4B024 AA01 AA11 BA41 BA58 BA80 CA04 DA01 DA02 DA05 DA06 DA11 EA04 GA01 GA11 HA08 4B064 AG26 AG27 CA02 CA05 CA10 CA11 CA11 CA19 CC50 CC21 CC24 DA01 A01 A57A02A01 A01 A57 A02 CA44 CA46 4C084 AA17 NA14 ZA362 ZA402 ZA422 ZA512 ZA662 ZA752 ZA942 ZA962 ZA972 ZB152 ZC022 ZC542 4H045 AA10 AA11 AA20 AA30 BA09 BA41 BA42 BA56 CA40 DA75 DA76 EA20 EA50 FA72 FA73 FA74

Claims (56)

[Claims] 1. An isolated nucleic acid molecule comprising: (a) the nucleotide sequence set forth in either SEQ ID NO: 1 or SEQ ID NO: 4; (b) ATCC Accession No. PTA-1479 or PTA-1480. A nucleotide sequence of the DNA insert in any of them; (c) a nucleotide sequence encoding the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5; (d) the complement of any of (a) to (c) A nucleotide sequence that hybridizes under moderately or highly stringent conditions; and (e) a nucleotide sequence complementary to any of (a) to (c), a nucleotide sequence selected from the group consisting of: An isolated nucleic acid molecule comprising. 2. An isolated nucleic acid molecule comprising the following: (a) less than the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5.
A nucleotide sequence encoding a polypeptide which is at least about 70% identical,
, Wherein the encoded polypeptide is either SEQ ID NO: 2 or SEQ ID NO: 5.
A nucleotide sequence having the activity of the polypeptide shown therein; (b) the nucleotide sequence shown in either SEQ ID NO: 1 or SEQ ID NO: 4.
, D in either ATCC Deposit Number PTA-1479 or PTA-1480
The nucleotide sequence of the NA insert, or (a) an allelic variant or
A nucleotide sequence encoding a splice variant; (c) encodes a polypeptide fragment of at least about 25 amino acid residues
The nucleotide sequence of either SEQ ID NO: 1 or SEQ ID NO: 4,
DNA inserts in either Accession Number PTA-1479 or PTA-1480
Region of (a) or (b), wherein said polypeptide fragment
Is the encoded polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5.
A region having the activity of tide or being antigenic; (d) comprising a fragment of at least about 16 nucleotides, SEQ ID NO: 1
Or the nucleotide sequence of any of SEQ ID NO: 4, ATCC Deposit No. PTA-14
DNA insert in either 79 or PTA-1480, or (a)-
(C) any region; (e) under moderately or highly stringent conditions, (a)-(d)
An isolated nucleotide sequence that hybridizes to one of its complements; and (f) a nucleotide sequence that is complementary to any of (a)-(d). Nucleic acid molecule. 3. An isolated nucleic acid molecule comprising: (a) a nucleotide sequence encoding the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5 having at least one conservative amino acid substitution. Where the encoded polypeptide is a nucleotide sequence having the activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; (b) SEQ ID NO: 2 having at least one amino acid insertion. Alternatively, a nucleotide sequence encoding the polypeptide shown in any of SEQ ID NO: 5, wherein the encoded polypeptide has the activity of the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5. (C) Any of SEQ ID NO: 2 or SEQ ID NO: 5 having a nucleotide sequence having: A nucleotide sequence encoding the polypeptide shown in, wherein the encoded polypeptide has the activity of the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5; d) a nucleotide sequence encoding the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 having a C-terminal truncation and / or an N-terminal truncation, wherein the encoded polypeptide is SEQ ID NO: 2 or a nucleotide sequence having the activity of the polypeptide shown in SEQ ID NO: 5; (e) at least selected from the group consisting of amino acid substitution, amino acid insertion, amino acid deletion, C-terminal truncation, and N-terminal truncation Encodes the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with one modification A nucleotide sequence, wherein the encoded polypeptide has the activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; (f) a fragment of at least about 16 nucleotides. A nucleotide sequence of any of (a)-(e); (g) a nucleotide sequence that hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(f) And (h) a nucleotide sequence complementary to any of (a) to (e), an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: 4. A vector comprising the nucleic acid molecule according to any one of claims 1, 2 and 3. 5. A host cell containing the vector of claim 4. 6. The host cell according to claim 5, which is a eukaryotic cell. 7. The host cell according to claim 5, which is a prokaryotic cell. 8. A process for producing a CHL2 polypeptide comprising:
A process comprising culturing a host cell according to claim 5 under suitable conditions for expressing said polypeptide, and optionally isolating said polypeptide from said culture. 9. A polypeptide produced by the process of claim 8. 10. The process of claim 8, wherein the nucleic acid molecule is operably linked to DNA encoding a CHL2 polypeptide, a promoter other than the promoter DNA for the native CHL2 polypeptide. A process involving DNA. 11. The isolated nucleic acid molecule of claim 2, wherein:
Percent identity is GAP, BLASTN, FASTA, BLASTA, BL
An isolated nucleic acid molecule as determined using a computer program selected from the group consisting of ASTX, BestFit, and Smith-Waterman algorithm. 12. A process for determining whether a compound inhibits CHL2 polypeptide activity or CHL2 polypeptide production, according to any one of claims 5, 6 or 7. A process comprising exposing a cell to the compound and measuring CHL2 polypeptide activity or CHL2 polypeptide production in the cell. 13. An isolated polypeptide comprising: (a) the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; and (b) ATCC Accession No. PTA-1479 or PTA-1480. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence encoded by the DNA insert in any of: 14. An isolated polypeptide, wherein: (a) the amino acid sequence set forth in either SEQ ID NO: 3 or SEQ ID NO: 6, optionally further comprising an amino terminal methionine; (B) an amino acid sequence for the ortholog of either SEQ ID NO: 2 or SEQ ID NO: 5; (c) an amino acid sequence that is at least about 70% identical to the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5, wherein And the polypeptide has an amino acid sequence having the activity of the polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 5; (d) any of SEQ ID NO: 2 or SEQ ID NO: 5 containing at least about 25 amino acid residues. A fragment of the amino acid sequence set forth in, wherein the fragment is set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Or having the activity of Ripepuchido, or antigenic fragments; and (e) the amino acid sequence shown in any of SEQ ID NO: 2 or SEQ ID NO: 5, A
DN in either TCC Deposit Number PTA-1479 or PTA-1480
An amino acid sequence encoded by the A insert, or an amino acid sequence of an allelic variant or splice variant of the amino acid sequence described in any of (a) to (c), Isolated polypeptide. 15. An isolated polypeptide comprising: (a) an amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 having at least one conservative amino acid substitution, wherein: , The polypeptide is
An amino acid sequence having the activity of the polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 5; (b) an amino acid sequence shown in either SEQ ID NO: 2 or SEQ ID NO: 5 having at least one amino acid insertion Here, the polypeptide has an amino acid sequence having the activity of the polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 5; (c) SEQ ID NO: 2 or SEQ ID NO: having at least one amino acid deletion Amino acid sequence shown in any of 5, wherein the polypeptide has the activity of the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5; (d) C-terminal truncation And / or an amino acid sequence as set forth in either SEQ ID NO: 2 or N-terminal truncations, wherein the po Peptides,
An amino acid sequence having the activity of the polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 5; and (e) selected from the group consisting of amino acid substitution, amino acid insertion, amino acid deletion, C-terminal truncation, and N-terminal truncation Amino acid sequence shown in either SEQ ID NO: 2 or SEQ ID NO: 5 having at least one modification, wherein said polypeptide is the polypeptide shown in either SEQ ID NO: 2 or SEQ ID NO: 5. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: having the activity of. 16. An isolated polypeptide encoded by the nucleic acid molecule of any of claims 1, 2 or 3, wherein the polypeptide is SEQ ID NO: 2. Alternatively, an isolated polypeptide having the activity of the polypeptide set forth in SEQ ID NO: 5. 17. The isolated polypeptide of claim 14, wherein the percent identity is GAP, BLASTP, FASTA, BLASTA.
An isolated polypeptide determined using a computer program selected from the group consisting of the BLASTX, BestFit, and Smith-Waterman algorithms. 18. A selective binding agent or a fragment thereof that specifically binds to the polypeptide of claim 13, 14, or 15. 19. The selective binding agent or a fragment thereof according to claim 18, which specifically binds to a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5 or a fragment thereof. 20. The selective binding agent according to claim 18, which is an antibody or a fragment thereof. 21. The selective binding agent of claim 18, which is a humanized antibody. 22. The selective binding agent according to claim 18, which is a human antibody or a fragment thereof. 23. The selective binding agent according to claim 18, which is a polyclonal antibody or a fragment thereof. 24. The selective binding agent according to claim 18, which is a monoclonal antibody or a fragment thereof. 25. The selective binding agent of claim 18, which is a chimeric antibody or fragment thereof. 26. The CDR-grafted antibody or a fragment thereof,
8. The selective binding agent according to item 8. 27. The selective binding agent according to claim 18, which is an anti-idiotype antibody or a fragment thereof. 28. The selective binding agent of claim 18, which is a variable region fragment. 29. A Fab fragment or a Fab ′ fragment,
A variable region fragment according to claim 28. 30. At least one complementarity determining region having specificity for a polypeptide having the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5.
Selective binding agents or fragments thereof. 31. The selective binding agent of claim 18, which is bound to a detectable label. 32. The selective binding agent of claim 18, which antagonizes the biological activity of the CHL2 polypeptide. 33. A CHL2 polypeptide-related disease, condition, or disorder.
19. A method for treating, preventing, or ameliorating, comprising administering to a patient an effective amount of a selective binding agent according to claim 18. 34. A selective binding agent produced by immunizing an animal with a polypeptide comprising the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 5. 35. A hybridoma that produces a selective binding agent capable of binding the polypeptide of any of claims 1, 2 or 3. 36. A method for detecting or quantifying the amount of CHL2 polypeptide using the selective binding agent or fragment according to claim 18. 37. A composition comprising the polypeptide of any of claims 13, 14, or 15 and a pharmaceutically acceptable formulation agent. 38. The composition of claim 37, wherein the pharmaceutically acceptable formulation is a carrier, adjuvant, solubilizer, stabilizer, or antioxidant. 39. The composition of claim 37, wherein the polypeptide comprises the amino acid sequence set forth in either SEQ ID NO: 3 or SEQ ID NO: 6. 40. A polypeptide comprising a derivative of the polypeptide of any one of claims 13, 14 or 15. 41. The polypeptide of claim 40, which has been covalently modified with a water soluble polymer. 42. The polypeptide of claim 41, wherein the water soluble polymer is polyethylene glycol, monomethoxy polyethylene glycol, dextran, cellulose, poly- (N-vinylpyrrolidone) polyethylene glycol, propylene. Glycol homopolymer, polypropylene oxide /
A polypeptide selected from the group consisting of ethylene oxide copolymers, polyoxyethylated polyols, and polyvinyl alcohol. 43. A composition comprising the nucleic acid molecule of any of claims 1, 2 or 3 and a pharmaceutically acceptable formulation agent. 44. The composition of claim 43, wherein the nucleic acid molecule is contained in a viral vector. 45. A viral vector comprising the nucleic acid molecule according to any one of claims 1, 2 and 3. 46. The method of claim 13 or 14 fused to a heterologous amino acid sequence.
Or a fusion polypeptide comprising the polypeptide of any one of claims 15 to 20. 47. The fusion polypeptide of claim 46, wherein the heterologous amino acid sequence is an IgG constant domain or fragment thereof. 48. A method for treating, preventing or ameliorating a medical condition, the polypeptide according to any of claims 13, 14 or 15, or 1. A method comprising the step of administering to a patient a polypeptide encoded by the nucleic acid of either claim 2 or claim 3. 49. A method of diagnosing a pathological condition or susceptibility to a pathological condition in a subject, comprising: (a) any of claim 13, 14, or 15 in the sample. Determining the presence or amount of expression of the polypeptide of claim 1 or of the polypeptide encoded by the nucleic acid molecule of any of claims 1, 2 or 3; and (b) said Diagnosing a pathological condition or susceptibility to a pathological condition based on the presence or expression level of expression of the polypeptide. 50. A device comprising: (a) a membrane suitable for implantation; and (b) a cell encapsulated within said membrane, said cell comprising: Alternatively, a cell that secretes the protein according to claim 15.
A device, wherein the membrane is permeable to the protein and impermeable to substances harmful to the cells. 51. A method of identifying a compound that binds to a CHL2 polypeptide, the method comprising: (a) contacting a polypeptide according to any one of claims 13, 14 or 15 with a compound. And (b) determining the degree of binding of the CHL2 polypeptide to the compound. 52. The method of claim 51, further comprising the step of determining the activity of said polypeptide when bound to said compound. 53. A method of modulating the level of a polypeptide in an animal, comprising the step of administering to the animal the nucleic acid molecule of any of claim 1, claim 2 or claim 3. Method. 54. A transgenic non-human mammal comprising a nucleic acid molecule according to any of claims 1, 2 or 3. 55. A process for determining whether a compound inhibits CHL2 polypeptide activity or CHL2 polypeptide production, exposing a transgenic mammal of claim 54 to the compound. And measuring CHL2 polypeptide activity or CHL2 polypeptide production in the mammal. 56. An isolated polypeptide comprising: leucine or methionine at position 2; methionine at position 5; lysine at position 6; alanine at position 7; isoleucine at position 8; phenylalanine at position 14; Leucine at position;
Threonine at position 23; Leucine at position 25; Valine at position 27; Glutamic acid at position 30; Tyrosine at position 32; Methionine at position 34; Glutamine at position 36; Lysine at position 39; Alanine at position 41; Alanine at position 41; Threonine at position 45; 55th Valine; 59th Valine; 60th Asparagine; 66th Proline; 68th Asparagine; 72th Serine or Threonine; 74th Valine; 75th Arginine; 94th Arginine; 99th Arginine Asparagine; Serine at position 100; Lysine at position 105; 10
6th Valine; 113th Tyrosine; 116th Serine; 118th Serine; 1
Arginine at position 20; Leucine at position 123; Alanine at position 125; Alanine at position 129; Alanine at position 139; Threonine at position 142; Serine at position 144; 14
7th asparagine; 148th valine; 149th serine; 159th alanine; 160th alanine; 161st alanine; 164th valine; 166th valine; 173rd valine; 175th arginine; Aspartic acid at position 177; Alanine at position 190; Phenylalanine at position 191; Arginine at position 192;
Leucine at position 194; Asparagine at position 196; Leucine at position 205; Alanine at position 210; Alanine at position 212; Serine at position 213; Alanine at position 216; 2
Serine at position 17; Alanine at position 218; Isoleucine at position 219; Alanine at position 222; Leucine at position 225; Phenylalanine at position 226; Leucine at position 230; Glutamine or arginine at position 233; Glutamine at position 241; Glutamine at position 242; Leucine; isoleucine at position 244; glutamine or asparagine at position 245; glutamine at position 249; leucine or valine at position 251; alanine at position 256; asparagine at position 257; serine at position 259; alanine at position 260; 26
Glutamine at position 1; phenylalanine at position 265; valine at position 268; leucine at position 269; leucine at position 272; valine at position 275; valine at position 278; 28
Glutamic acid at position 4; Glutamic acid at position 288; Alanine or isoleucine at position 292; Serine at position 300; Isoleucine at position 306; Valine at position 313; 3
Serine at position 14; Leucine at position 319; Glutamine at position 323; Threonine at position 324; Alanine at position 326; Alanine at position 327; Serine at position 329; 331
Serine at position 334; Leucine at position 334; Alparagine at position 337; Valine at position 339; Leucine at position 340; Serine at position 342; Phenylalanine at position 344; 34
Glutamic acid at position 9; isoleucine or valine at position 354; methionine at position 356; valine at position 366; methionine or valine at position 368; isoleucine at position 371; leucine at position 375; leucine at position 376; glutamine at position 377;
Phenylalanine at position 381; Asparagine at position 383; Isoleucine at position 384; Leucine at position 393; Arginine at position 395; Valine at position 398; Alanine or valine at position 399; Tyrosine at position 403; Asparagine at position 406; 40
A isoleucine at position 9; an alanine or valine at position 415; an isoleucine at position 417; and a leucine at position 421; and a single amino acid sequence represented by SEQ ID NO: 5 having at least one conservative amino acid substitution. An isolated polypeptide, wherein the polypeptide has the activity of the polypeptide set forth in SEQ ID NO: 5.