ES2364850T3 - COMPOSITION UNDERSTANDING AN ANTIBODY THAT SELECTIVELY Binds TO SEGREGATED PROTEIN RICH IN CYSTEINE (CRSP). - Google Patents

Abstract

Composition formulated for parenteral administration, comprising a pharmaceutically acceptable carrier and an antibody that selectively binds to an isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 8, wherein the carrier comprises a sterile diluent for injection.

Description

Background of the invention

Segregated proteins play an integral role in the formation, differentiation and maintenance of cells in multicellular organisms. For example, it is known in the art that secretory proteins are involved in signaling between cells that are hot in direct contact. Such segregated signaling molecules are particularly important in the development of vertebrate tissue during embryogenesis as well as in the maintenance of the differentiated state of adult tissues. For example, the inductive interactions that occur between neighboring cell layers and tissues in the developing embryo are highly dependent on the existence and regulation of secreted signaling molecules. In inductive interactions, biochemical signals secreted by a cell population influence the fate of the development of a second cell population, normally altering the fate of the second cell population. For example, Wnt proteins are now recognized as one of the main families of signaling molecules important for development in organisms ranging from Drosophila to mice. For a review see Cadigan, K. M. and R. Nusse (1997) Genes & Development, 11: 3286-3305.

It is also well recognized today that the main families of signaling molecules have a double role to play both in the development of an organism as well as in the stimulation or maintenance of the differentiated state of tissues in the adult animal. In addition, the main families of signaling molecules have been implicated in the control of cell proliferation in mature adult tissues, for example, during normal cell renewal in the adult organism as well as in activated tissue regeneration as a result of tissue damage. adult.

Summary of the invention

The present invention is based, at least in part, on the discovery of nucleic acid molecules encoding a novel family of segregated proteins, referred to herein as cysteine-rich secreted proteins ("CRSP" or "CRSP proteins"). The CRSP molecules of the present invention are useful as modulating agents to regulate a variety of cellular processes. Consequently, isolated nucleic acid molecules encoding CRSP proteins or biologically active parts thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of nucleic acids encoding CRSP are disclosed.

A CRSP nucleic acid molecule may be 60% homologous to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or a complement thereof. An ACRSP nucleic acid molecule can be 80% homologous to the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the number of registration 98634 or a complement thereof. A CRSP nucleic acid molecule can be 60% homologous to the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 9, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the number No. 98633, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452 or a complement thereof. A CRSP nucleic acid molecule can be 85% homologous to the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 9, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the number No. 98633, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452 or a complement thereof. A CRSP nucleic acid molecule may be 70% homologous to the nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 12 or a complement thereof. An isolated CRSP nucleic acid molecule may have the nucleotide sequence shown in SEQ ID NO: 3 or a complement thereof. A CRSP nucleic acid molecule may further comprise nucleotides 1-37 of SEQ ID NO: 1. A CRSP nucleic acid molecule may further comprise nucleotides 1088-2479 of SEQ ID NO: 1. An isolated CRSP nucleic acid molecule may have the nucleotide sequence shown in SEQ ID NO: 1.

An isolated CRSP nucleic acid molecule may have the nucleotide sequence shown in SEQ ID NO: 6, or a complement thereof. A CRSP nucleic acid molecule may further comprise nucleotides 1-125 of SEQ ID NO: 4. A CRSP nucleic acid molecule may further comprise nucleotides 797-848 of SEQ ID NO: 4. An isolated CRSP nucleic acid molecule may have the nucleotide sequence shown in SEQ ID NO: 4.

An isolated CRSP nucleic acid molecule may have the nucleotide sequence shown in SEQ ID NO: 9, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, the nucleotide sequence of the insert Plasmid DNA deposited in the ATCC with the registration number 98452 or a complement thereof. A CRSP nucleic acid molecule may further comprise nucleotides 1-92 of SEQ ID NO: 7. A CRSP nucleic acid molecule may further comprise nucleotides 891-1529 of SEQ ID NO: 7. In another preferred embodiment, an isolated CRSP nucleic acid molecule may have the nucleotide sequence shown in SEQ ID NO: 7.

An isolated CRSP nucleic acid molecule may have the nucleotide sequence shown in SEQ ID NO: 12, or a complement thereof. A CRSP nucleic acid molecule may further comprise nucleotides 538-702 of SEQ ID NO: 10. An isolated CRSP nucleic acid molecule may have the nucleotide sequence shown in SEQ ID NO: 10.

A CRSP nucleic acid molecule may include a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID 5, SEQ ID NO: 8 or SEQ ID NO: 11. A CRSP nucleic acid molecule may include a nucleotide sequence that encodes a protein having an amino acid sequence homologous at least 60% to the amino acid sequence of SEQ ID NO: 2. A CRSP nucleic acid molecule may include a nucleotide sequence that encodes a protein having an amino acid sequence homologous at least 60% to the amino acid sequence of SEQ ID NO: 5. A CRSP nucleic acid molecule may include a nucleotide sequence that encodes a protein having an amino acid sequence homologous at least 60% to the amino acid sequence of SEQ ID NO: 8. A CRSP nucleic acid molecule may include a nucleotide sequence that encodes a protein having an amino acid sequence homologous at least 75% to the amino acid sequence of SEQ ID NO: 8. A CRSP nucleic acid molecule may include a nucleotide sequence that encodes a protein having an amino acid sequence homologous at least 65% to the amino acid sequence of SEQ ID NO: 11.

An isolated nucleic acid molecule can code for a CRSP protein that includes a signal sequence and at least one cysteine-rich domain, and can be secreted. An isolated nucleic acid molecule can code for a CRSP protein that includes a signal sequence and at least one cysteine-rich domain, preferably a cysteine-rich region, and can be secreted. A CRSP nucleic acid molecule can code for a CRSP protein and can be a naturally occurring nucleotide sequence.

CRSP nucleic acid molecules that specifically detect CRSP nucleic acid molecules in relation to nucleic acid molecules encoding proteins that are not CRSP are disclosed. For example, a CRSP nucleic acid molecule can hybridize under stringent conditions with a nucleic acid molecule comprising nucleotides 470-2479 of the nucleotide sequence shown in SEQ ID NO: 1, with nucleotides 1-475 of the sequence of nucleotides shown in SEQ ID NO: 4, or with nucleotides 1-600 of the nucleotide sequence shown in SEQ ID NO: 7, or hybridize under stringent conditions with the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, with the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633,

or with the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452. The CRSP nucleic acid molecule can be at least 500 nucleotides in length and hybridize under stringent conditions with a nucleic acid molecule that It comprises the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10 or a complement thereof.

An isolated nucleic acid molecule that is antisense with respect to the coding chain of a CRSP nucleic acid is also disclosed.

A vector comprising a CRSP nucleic acid molecule is also disclosed. The vector may be a recombinant expression vector. A host cell containing such a vector is disclosed. A method for producing a CRSP protein is disclosed by culturing in a suitable medium, such a host cell that contains a recombinant expression vector so that a CRSP protein is produced.

Isolated and recombinant CRSP polypeptides and proteins are disclosed. An isolated CRSP protein can have a signal sequence and at least one cysteine rich domain, preferably a cysteine rich region, and can be secreted. An isolated CRSP protein may be an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11. A CRSP protein may have an amino acid sequence homologous at least about 60% to the amino acid sequence of SEQ ID NO: 2. A CRSP protein may have an amino acid sequence homologous at least about 60% to the amino acid sequence of SEQ ID NO: 5. A CRSP protein may have an amino acid sequence homologous at least about 60% to the amino acid sequence of SEQ ID NO: 8. A CRSP protein may have an amino acid sequence homologous at least about 75% to the amino acid sequence of SEQ ID NO: 8. A CRSP protein may have an amino acid sequence homologous at least about 65% to the amino acid sequence of SEQ ID NO: 11. A CRSP protein may have the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11.

An isolated CRSP protein that is encoded by a nucleic acid molecule having a nucleotide sequence homologous at least about 60% to a nucleotide sequence of SEQ ID NO: 1, or a complement thereof is also disclosed. . An isolated CRSP protein that is encoded by a nucleic acid molecule having a nucleotide sequence homologous at least about 80% to a nucleotide sequence of SEQ ID NO: 4, or a complement thereof, is disclosed. An isolated CRSP protein that is encoded by a nucleic acid molecule having a nucleotide sequence homologous at least about 60% to a nucleotide sequence of SEQ ID NO: 7, or a complement thereof is disclosed. An isolated CRSP protein that is encoded by a nucleic acid molecule having a nucleotide sequence homologous at least about 85% to a nucleotide sequence of SEQ ID NO: 7, or a complement thereof is disclosed. An isolated CRSP protein that is encoded by a nucleic acid molecule having a nucleotide sequence homologous at least about 70% to a nucleotide sequence of SEQ ID NO: 10, or a complement thereof, is disclosed. An isolated CRSP protein that is encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes under stringent hybridization conditions with a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ is disclosed. ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or a complement thereof.

CRSP proteins or biologically active portions thereof, can be operably linked to a polypeptide other than CRSP to form CRSP fusion proteins. The invention characterizes antibodies that specifically bind to CRSP proteins, such as monoclonal or polyclonal antibodies. CRSP proteins

or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

A method for detecting the expression of CRSP in a biological sample by contacting the biological sample with an agent that can detect a polypeptide, a protein or a nucleic acid molecule of CRSP is disclosed, so that the presence of a polypeptide, a protein or a nucleic acid molecule of CRSP in the biological sample.

A method for detecting the presence of CRSP activity in a biological sample is disclosed by contacting the biological sample with an agent that can detect an indicator of CRSP activity so that the presence of CRSP activity in the sample is detected. biological

A method for modulating the activity of CRSP is disclosed which comprises contacting the cell with an agent that modulates the activity of CRSP in such a way that the activity of CRSP in the cell is modulated. The agent can inhibit CRSP activity or stimulate CRSP activity. The agent may be an antibody that specifically binds to a CRSP protein. The agent can modulate CRSP expression by modulating the transcription of a CRSP gene or the translation of a CRSP mRNA. The agent can be a nucleic acid molecule that has a nucleotide sequence that is antisense with respect to the coding chain of a CRSP mRNA or a CRSP gene.

The methods can be used to treat a subject who has a disorder characterized by activity or expression of aberrant nucleic acid or CRSP protein by administering an agent that is a CRSP modulator to the subject. The CRSP modulator can be a CRSP protein. The CRSP modulator may be a CRSP nucleic acid molecule. The CRSP modulator can be a peptide, peptidomimetic molecule or other small molecule. The disorder characterized by expression of aberrant nucleic acid or CRSP protein may be a developmental, differentiation or proliferative disorder.

A diagnostic test to identify the presence or absence of a genetic alteration characterized by at least one of (i) aberrant mutation or modification of a gene encoding a CRSP protein is also disclosed; (ii) erroneous regulation of said gene; and (iii) post-aberrant translation of a CRSP protein, in which a wild-type form of said gene encodes a protein with a CRSP activity.

A method for identifying a compound that binds to, or modulates the activity of, a CRSP protein is disclosed, providing an indicator composition comprising a CRSP protein that has CRSP activity, by contacting the indicator composition with a compound of test, and determining the effect of the test compound on the activity of CRSP in the indicator composition to identify a compound that modulates the activity of a CRSP protein.

The present invention provides a composition formulated for parenteral administration, comprising a pharmaceutically acceptable carrier and an antibody that selectively binds to an isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 (CRSP-3), in which The vehicle comprises a sterile injection diluent.

The antibody can be a monoclonal antibody. The antibody can be a recombinant antibody. The composition can be formulated for injection. The composition may be a sterile injectable solution, in which the antibody is incorporated into an appropriate solvent with one or a combination of components selected from the group consisting of physiological saline, bactariostatic water, phosphate buffered saline or a solvent containing water, ethanol, polyol or suitable mixtures thereof.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief description of the drawings

Figure 1 depicts the cDNA sequence and the predicted amino acid sequence of human CRSP-1. The nucleotide sequence corresponds to nucleic acids 1 to 2479 of SEQ ID NO: 1. The amino acid sequence corresponds to amino acids 1 to 350 of SEQ ID NO: 2.

Figure 2 depicts the cDNA sequence and predicted amino acid sequence of human CRSP-2. The nucleotide sequence corresponds to nucleic acids 1 to 848 of SEQ ID NO: 4. The amino acid sequence corresponds to amino acids 1 to 224 of SEQ ID NO: 5.

Figure 3 depicts the cDNA sequence and the predicted amino acid sequence of human CRSP-3. The nucleotide sequence corresponds to nucleic acids 1 to 1529 of SEQ ID NO: 7. The amino acid sequence corresponds to amino acids 1 to 266 of SEQ ID NO: 8.

Figure 4 depicts the cDNA sequence and the predicted amino acid sequence of human CRSP-4. The nucleotide sequence corresponds to nucleic acids 1 to 702 of SEQ ID NO: 10. The amino acid sequence corresponds to amino acids 1 to 179 of SEQ ID NO: 11.

Figure 5 depicts the cDNA sequence and the predicted amino acid sequence of human CRSP N-terminal. The nucleotide sequence corresponds to nucleic acids 1 to 928 of SEQ ID NO: 13. The amino acid sequence corresponds to amino acids 1 to 242 of SEQ ID NO: 14.

Figure 6 depicts the amino acid sequences of human CRSP-1, human CRSP-2, human CRSP-3 and human CRSP-4, as well as the consensus sequence generated from the alignment of these sequences. The figure details the relationship between the CRSP proteins of the present invention.

Figure 7 is a schematic diagram representing the relationship between CRSP proteins. The figure represents the biological and functional domains of human CRSP. Signal peptides are indicated by filled boxes. The cysteine-rich domains of a cysteine-rich region of CRSP are represented as CRD-1 and CRD-2.

Figure 8 depicts the cDNA sequence and the predicted amino acid sequence of murine CRSP-1. The nucleotide sequence corresponds to nucleic acids 1 to 928 of SEQ ID NO: 16. The amino acid sequence corresponds to amino acids 1 to 349 of SEQ ID NO: 17.

Figure 9 is a schematic diagram depicting the relationship between the nucleotide sequence of CRSP-1 and those of RIG and 7-1 of the RIG type (registration numbers U32331 and AF034208, respectively).

Detailed description of the invention

The present invention is based on the discovery of novel molecules, referred to herein as nucleic acid molecules and CRSP proteins, which comprise a family of molecules that have certain conserved structural and functional characteristics. The term "family" is intended when referring to the protein and nucleic acid molecules means two or more proteins or nucleic acid molecules that have a common structural domain and that have sufficient amino acid or nucleotide sequence homology as defined. in the present document. Such family members can occur naturally and can be either of the same species or of different species. For example, a family may contain a first protein of human origin, as well as other proteins other than human origin, or alternatively they may contain homologs of non-human origin. Family members may also have common functional characteristics.

A member of the CRSP family is identified based on the presence of at least one "cysteine rich domain" in the corresponding protein or nucleic acid molecule. As defined herein, a "cysteine rich domain" refers to a part of a CRSP protein (eg, CRSP-1) that is rich in cysteine residues, that is, at least about 6.7. , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids are cysteine residues. Preferably, a "cysteine rich domain" is a protein domain having an amino acid sequence of about 30-100 amino acids, preferably about 35-95 amino acids, more preferably about 40-90 amino acids, more preferably preferably of about 45-85 amino acids, even more preferably of about 50-80 amino acids, and even more preferably of about 55-75, 60-70 or 65 amino acids, of which at least about 3-20, of preferably about 5-15, or more preferably about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids are cysteine residues.

A preferred cysteine-rich domain has at least about 10, at least about 15, 16, 17, 18, 19 or preferably 20 cysteine residues located in the same relative amino acid position as cysteine residues in human CRSP-1 having SEQ ID NO: 2. For example, as shown in Figure 6, CRSP-2 has at least about 10 cysteine residues located in the same relative amino acid position as cysteine residues in human CRSP-1 that has SEQ ID NO: 2 (for example, cys151 in CRSP-2, SEQ ID NO: 5, is located in the same relative amino acid position as cys214 in CRSP-1, SEQ ID NO: 2; cys156 in CRSP-2, SEQ ID NO: 5, is located in the same relative amino acid position as cys219 in CRSP1, SEQ ID NO: 2; and cys157 in CRSP-2, SEQ ID NO: 5, is located in the same amino acid position as cys220 in CRSP- 1, SEQ ID NO: 2). Similarly, as shown in Figure 6, CRSP-3 has at least about 10 cysteine residues located in the same relative amino acid position as cysteine residues in human CRSP-1 having SEQ ID NO: 2. As also shown in Figure 6, CRSP-4 has at least about 10 cysteine residues located at the same relative amino acid position as cysteine residues in human CRSP-1 having SEQ ID NO: 2.

A preferred CRSP protein is a human protein (for example, encoded by a nucleotide sequence that corresponds to a naturally occurring human gene). For example, in one embodiment, a CRSP-1 protein (human CRSP-1) contains a first cysteine-rich domain that contains approximately amino acids 147-195 of SEQ ID NO: 2, which has 10 cysteine residues, and a second cysteine-rich domain that contains approximately amino acids 201-284 of SEQ ID NO: 2, which has 10 cysteine residues (the positions of cysteine residues are shown in Figure 6). A CRSP-2 protein (human CRSP-2) may contain a first cysteine-rich domain that contains approximately amino acids 41-90 of SEQ ID NO: 5, which has 10 cysteine residues, and a second cysteine-rich domain that contains approximately amino acids 41-218 of SEQ ID NO: 5, which has 10 cysteine residues (the positions of the cysteine residues are shown in Figure 6). A CRSP-3 protein (human CRSP-3) contains a first cysteine-rich domain that contains approximately amino acids 85-138 of SEQ ID NO: 8, which has 10 cysteine residues, and a second cysteine-rich domain that contains approximately amino acids 182-263 of SEQ ID NO: 8, which has 10 cysteine residues (the positions of the cysteine residues are shown in Figure 6). A CRSP-4 protein (human CRSP-4) may contain a first cysteine-rich domain that contains approximately amino acids 1-47 of SEQ ID NO: 11, which has 8 cysteine residues, and a second cysteine-rich domain that contains approximately amino acids 96-176 of SEQ ID NO: 11, which has 10 cysteine residues (the positions of the cysteine residues are shown in Figure 6).

Preferred CRSP proteins have more than one cysteine-rich domain, more preferably they have at least two cysteine-rich domains and, therefore, have a cysteine-rich region. As used herein, the term "cysteine-rich region" refers to a protein domain that includes at least two cysteine-rich domains and has an amino acid sequence of about 120-200, preferably about 130 -190, more preferably of about 140-180 amino acid residues, and even more preferably of at least about 135-175 amino acids of which at least about 10-30, preferably about 15-20, and of more preferably about 16, 17, 18, or 19 of the amino acids are cysteine residues. A cysteine-rich region may be located in the C-terminal region of a CRSP protein. For example, a CRSP-1 protein may contain a cysteine-rich region that contains approximately amino acids 147-284 of SEQ ID NO: 2, which has 20 cysteine residues at the positions indicated in Figure 6. A CRSP-2 protein may contain a cysteine-rich region containing approximately amino acids 41-218 of SEQ ID NO: 5, which has 20 cysteine residues at the positions indicated in Figure 6. A CRSP-3 protein may contain a cysteine-rich region that It contains approximately amino acids 85-263 of SEQ ID NO: 8, which has 20 cysteine residues at the positions indicated in Figure 6. A CRSP-4 protein may contain a cysteine-rich region containing approximately amino acids 1-176 of SEQ ID NO: 2, which has 18 cysteine residues in the positions indicated in Figure 6.

In addition to the cysteine-rich domains, the cysteine-rich region may contain a spacer region that separates the first and second cysteine-rich domain. As used herein, the "spacer region" refers to amino acid residues that are located between the first and second cysteine-rich domains of a cysteine-rich region and includes C-terminal amino acid residues with respect to to the first domain rich in cysteine and in N-terminal with respect to the second domain rich in cysteine. As defined herein, a "spacer region" refers to a protein domain of about 5-70, preferably about 10-65 amino acids, more preferably about 15-60 amino acids, even more preferably about 20-55, and even more preferably about 25-50, 30-45 or 35-40 amino acids. For example, the CRSP-1 protein contains a spacer region of approximately amino acids 196-200 of SEQ ID NO: 2; CRSP-2 protein contains a spacer region of approximately amino acids 91-137 of SEQ ID NO: 5; CRSP-3 protein contains a spacer region of approximately amino acids 139-181 of SEQ ID NO: 8; and the CRSP-4 protein contains a spacer region of approximately amino acids 48-95 of SEQ ID NO: 11.

The CRSP protein may have at least one cysteine rich domain, preferably a cysteine rich region, and a signal sequence. As used herein, a "signal sequence" refers to a peptide that contains approximately 20 amino acids that is produced at the N-terminal end of secretory and integral membrane proteins and that contains a large number of amino acid residues. hydrophobic For example, a signal sequence contains at least about 14-28 amino acid residues, preferably about 16-26 amino acid residues, more preferably about 18-24 amino acid residues, and more preferably about 20-22 residues. of amino acids, and has at least about 40-70%, preferably about 50-65%, and more preferably about 55-60% of hydrophobic amino acid residues (eg, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline). Such a "signal sequence," also referred to in the art as a "signal peptide," serves to direct a protein containing such a sequence to a lipid bilayer. For example, a CRSP-1 protein may contain a signal sequence of approximately amino acids 1-23 of SEQ ID NO: 2. A CRSP-2 protein may contain a signal sequence of approximately amino acids 1-19 of SEQ ID NO: 5 A CRSP-3 protein contains a signal sequence of approximately amino acids 1-20 of SEQ ID NO: 8.

CRSP proteins can be used to identify additional members of the CRSP family. For example, a protein that has homology to CRSP-1 was identified using the sequence that codes for the unique N-terminal region of CRSP-1 to search for a protein sequence database. A protein of this type, referred to herein as "CRSP type N terminal" is depicted in Figure 5. The CRSP type N terminal nucleotide sequence (SEQ ID NO: 13) encodes for a protein having 242 amino acids (SEQ ID NO: 14). The N-terminal nucleotide sequence of the CRSP type includes a 5'-untranslated region containing nucleotides 1-74 of SEQ ID NO: 13, a coding region containing nucleotides 75-800 of SEQ ID NO: 13 (which corresponds to nucleotides 1-726 of SEQ ID NO: 15), and a non-3 'translated region containing nucleotides 801-928 of SEQ ID NO: 13.

Consequently, a CRSP protein having at least one cysteine rich domain, preferably at least one cysteine rich region is disclosed. A CRSP protein having at least one region rich in cysteine is disclosed, in which the region rich in cysteine includes at least one domain rich in cysteine. A CRSP protein is disclosed that has at least one region rich in cysteine, in which the region rich in cysteine includes at least two domains rich in cysteine. A protein having homology of 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% is disclosed with respect to a cysteine-rich domain of a CRSP protein of the invention (e.g., CRSP-1, CRSP-2, CRSP-3, or CRSP-4).

A CRSP protein having at least one cysteine-rich domain, preferably at least one cysteine-rich region and a signal peptide is also disclosed. A CRSP protein having at least one cysteine-rich domain, preferably at least one cysteine-rich region and one signal peptide, in which the cysteine-rich region includes at least two cysteine-rich domains is disclosed. A CRSP protein having at least one cysteine-rich domain, preferably at least one cysteine-rich region and one signal peptide, in which the cysteine-rich region includes at least two cysteine-rich domains and a spacer is disclosed.

Preferred CRSP molecules have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO 11. As used herein, the term "Sufficiently homologous" refers to a first amino acid or nucleotide sequence containing a sufficient or minimum number of identical or equivalent amino acid residues or nucleotides (eg, an amino acid residue having a similar side chain) with respect to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains and / or a common functional activity. For example, the amino acid or nucleotide sequences that share common structural domains have at least about 40% homology, preferably 50% homology, more preferably 60% -70% homology along the amino acid sequence of domains and contain at least one, preferably two, more preferably three, and even more preferably four, five or six structural domains, are defined herein as sufficiently homologous. In addition, amino acid or nucleotide sequences that share at least 40%, preferably 50%, more preferably 60, 70, or 80% homology and share a functional activity are defined herein as sufficiently homologous.

As used interchangeably herein, a "CRSP activity", "CRSP biological activity"

or "CRSP functional activity", refers to an activity exerted by a nucleic acid molecule, polypeptide or CRSP protein on a CRSP sensitive cell as determined in vivo, or in vitro, according to usual techniques. A CRSP activity can be a direct activity, such as an association with a CRSP target molecule. As used herein, a "target molecule" is a molecule with which a CRSP protein binds or interacts in nature, so that CRSP-mediated function is achieved. A target molecule of CRSP can be a molecule other than CRSP or a polypeptide or CRSP protein. For example, a CRSP target molecule is a membrane-bound protein (for example, a cell surface receptor or "CRSP receptor") or a modified form of such a protein that has been altered so that the protein is soluble (for example, produced recombinantly so that the protein does not express a membrane binding domain). A CRSP target can be a second soluble protein molecule (for example, a "CRSP binding partner" or "CRSP substrate"). A CRSP binding partner can be a second protein other than soluble CRSP or a second CRSP protein molecule. Alternatively, a CRSP activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the CRSP protein with a second protein (eg, a CRSP receptor). As used herein, the term "CRSP receptor" refers to a protein or protein complex, to which a CRSP protein can be attached, for example, human CRSP. A receptor can be a cell surface receptor, for example, a nuclear hormone receptor. CRSP receptors can be isolated by methods known in the art and described further herein. The interaction of a CRSP protein with a CRSP receptor can result in the transduction of a signal from the cell surface to the nucleus. The transduced signal may be, an increase in intracellular calcium, an increase in phosphatidylinositol or another molecule, and may result in, for example, phosphorylation of specific proteins, a gene transcription modulation and any of the other biological activities set forth in the present document

A CRSP activity may be at least one or more of the following activities: (i) interaction of a CRSP protein with, and / or binding to, a second molecule, (eg, a protein, such as a CRSP receptor, a soluble form of a CRSP receptor, a receptor for a member of the wnt family of signaling proteins, or a signaling molecule other than CRSP); (ii) interaction of a CRSP protein with an intracellular protein through a membrane bound CRSP receptor; (iii) complex formation between a soluble CRSP protein and a second soluble CRSP binding partner (for example, a distinct CRSP protein molecule or a second CRSP protein molecule); (iv) interaction with other extracellular proteins (for example, regulation of wnt-dependent cell adhesion to extracellular matrix components); (v) binding to and elimination of an undesirable molecule (for example, a detoxifying activity or defense function); and / or (vi) an enzymatic activity. A CRSP activity may be at least one or more of the following activities: (1) cell signal transduction modulation, either in vitro or in vivo (eg, antagonism of the activity of members of the wnt family of segregated proteins or wnt dependent signal transduction suppression; (2) regulation of communication between cells (for example, regulation of cell-cell interactions dependent on wnt); (3) regulation of gene expression whose expression is modulated by the binding of CRSP (eg, CRSP-1) to a receptor; (4) regulation of gene transcription in a cell involved in development or differentiation, either in vitro or in vivo (for example, induction of cell differentiation); (5) regulation of gene transcription in a cell involved in development or differentiation, in which at least one gene codes for specific protein differentiation; (6) regulation of gene transcription in a cell involved in development or differentiation, in which at least one gene codes for a second segregated protein; (7) regulation of gene transcription in a cell involved in the development or differentiation, in which at least one gene codes for a signal transduction molecule; (8) regulation of cell proliferation, either in vitro or in vivo (for example, induction of cell proliferation or inhibition of proliferation as in the case of suppression of tumorigenesis (for example, suppression of glioblastoma formation)); (9) formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, both adult and embryonic (for example, induction of head formation during vertebrate development or maintenance of hematopoietic progenitor cells); (10) modulation of cell death, such as stimulation of cell survival; (11) regulation of cell migration; and / or (12) immune modulation.

As referred to herein, "specific differentiation proteins" include proteins involved in the transition of a cell from the undifferentiated to the differentiated phenotype. For example, such proteins can be specific structural differentiation proteins or specific transcription factors of differentiation. Such specific differentiation proteins are generally expressed at higher levels in cells that are transitioning from the undifferentiated phenotype to the differentiated phenotype (for example, during embryonic development or during the regeneration of mature tissue in the adult animal), or they are expressed at higher levels in fully differentiated or terminal differentiated cells compared to their undifferentiated counterparts. Also, as referred to herein, "specific differentiation genes" include nucleic acid molecules that code for specific differentiation proteins.

Accordingly, isolated CRSP proteins and polypeptides having a CRSP activity are also disclosed. Preferred CRSP proteins have at least one region rich in cysteine and a CRSP activity. The CRSP protein may have at least one cysteine-rich region, in which the cysteine-rich region comprises at least one cysteine-rich domain, and a CRSP activity. The CRSP protein may have at least one cysteine-rich region, in which the cysteine-rich region comprises at least two cysteine-rich domains, and a CRSP activity. A CRSP protein may further comprise a signal sequence. A CRSP protein may have a cysteine-rich region, a CRSP activity and an amino acid sequence sufficiently homologous to an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO 11 .

The human CRSP-1 cDNA, which is approximately 2479 nucleotides in length, codes for a protein that is approximately 350 amino acid residues in length. The human CRSP protein contains an N-terminal signal sequence and a cysteine-rich region comprising two cysteine-rich domains. A cysteine-rich region of CRSP can be found at least, for example, from about amino acids 147-284 of SEQ ID NO: 2. The cysteine-rich region of CRSP-1 comprises a first cysteine-rich domain from about amino acids 147 -195 of SEQ ID NO: 2 and a second cysteine-rich domain from approximately amino acids 201-284 of SEQ ID NO: 2. The human CRSP-1 protein is a secreted protein that additionally contains a signal sequence at approximately amino acids 1 -23 of SEQ ID NO: 2. The prediction of such a signal peptide can be made, for example, using the SIGNALP computer algorithm (Henrik, et al. (1997) Protein Engineering 10: 1-6).

The human CRSP-2 cDNA, which is approximately 848 nucleotides in length, codes for a protein that is approximately 224 amino acid residues in length. The human CRSP protein contains an N-terminal signal sequence and a cysteine-rich region comprising two cysteine-rich domains. A cysteine-rich region of CRSP can be found at least, for example, from about amino acids 41-218 of SEQ ID NO: 5. The cysteine-rich region of CRSP-2 comprises a first cysteine-rich domain from about amino acids 41 -90 of SEQ ID NO: 5 and a second cysteine-rich domain from about amino acids 138-218 of SEQ ID NO: 5. The human CRSP-2 protein is a secreted protein that additionally contains a signal sequence at about amino acids 1 -19 of SEQ ID NO: 5.

The CRSP-3 cDNA, which is approximately 1529 nucleotides in length, codes for a protein that is approximately 266 amino acid residues in length. The human CRSP protein contains an N-terminal signal sequence and a cysteine-rich region comprising two cysteine-rich domains. A cysteine-rich region of CRSP can be found at least, for example, from about amino acids 85-263 of SEQ ID NO: 8. The cysteine-rich region of CRSP-3 comprises a first cysteine-rich domain from about amino acids 85 -138 of SEQ ID NO: 8 and a second cysteine-rich domain from about amino acids 182-263 of SEQ ID NO: 2. The human CRSP-3 protein is a secreted protein that additionally contains a signal sequence at approximately amino acids. 1-20 of SEQ ID NO: 8.

The human CRSP-4 cDNA, which is approximately 702 nucleotides in length, encodes for a protein that is approximately 179 amino acid residues in length. The human CRSP protein contains a cysteine-rich region that comprises two domains rich in cysteine. A cysteine-rich region of CRSP can be found at least, for example, from about amino acids 1-176 of SEQ ID NO: 11. The cysteine-rich region of CRSP-4 comprises a first cysteine-rich domain from about amino acids 1 -47 of SEQ ID NO: 11 and a second cysteine-rich domain from approximately amino acids 96-176 of SEQ ID NO: 11.

Various aspects of the invention are described in more detail in the following subsections:

I. Isolated nucleic acid molecules

Isolated nucleic acid molecules encoding CRSP proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient to be used as hybridization probes to identify nucleic acids encoding CRSP (eg, CRSP mRNA, are disclosed. ) and fragments to be used as PCR primers for amplification or mutation of CRSP nucleic acid molecules. The term "nucleic acid molecule", as used herein, is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and DNA or RNA analogs. generated using nucleotide analogs. The nucleic acid molecule may be single stranded or double stranded, but preferably it is double stranded DNA.

An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (ie, sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA of the organism from which it is derived. nucleic acid For example, in various embodiments, the isolated CRSP nucleic acid molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of flanking nucleotide sequences naturally the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized

A nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452 or a part thereof, it can be isolated using usual molecular biology techniques and sequence information provided herein. Using all or part of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452 as a hybridization probe, CRSP nucleic acid molecules can be isolated using standard hybridization and cloning techniques (eg, as described in Sambrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

In addition, a nucleic acid molecule that encompasses all or part of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10, or the nucleotide sequence of the DNA insert of the deposited plasmid in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the number of Record 98452 can be isolated by polymerase chain reaction (PCR) using oligonucleotide primers designed based on the sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the insert nucleotide sequence of plasmid depos DNA located in the ATCC with the registration number 98452.

A nucleic acid can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to usual PCR amplification techniques. The nucleic acid thus amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. In addition, oligonucleotides corresponding to the nucleotide sequence of CRSP can be prepared by usual synthetic techniques, for example, using an automated DNA synthesizer.

An isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1. The sequence of SEQ ID NO: corresponds to the human CRSP-1 cDNA. This cDNA comprises sequences encoding the human CRSP-1 protein (ie, "the coding region", from nucleotides 38-1087), as well as sequences not translated into 5 '(nucleotides 1 to 37) and sequences not translated into 3 '(nucleotides 1088 to 2479). Alternatively, the nucleic acid molecule may comprise only the coding region of SEQ ID NO: 1, (for example, nucleotides 38 to 1087, corresponding to SEQ ID NO: 3). A plasmid containing the full-length nucleotide sequence encoding CRSP-1 was deposited in the American type culture collection (ATCC), Rockville, Maryland, on June 11, 1997, and registration number 98452 was assigned. deposited a plasmid containing the full-length nucleotide sequence encoding CRSP-1 in the American type culture collection (ATCC), Rockville, Maryland, on January 16, 1998, and registration number 98634 was assigned.

Another isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 4. The sequence of SEQ ID NO: 4 corresponds to the human CRSP-2 cDNA. This cDNA comprises sequences encoding the human CRSP-2 protein (ie, "the coding region", from nucleotides 126-796), as well as sequences not translated into 5 '(nucleotides 1 to 125) and sequences not translated into 3 '(nucleotides 797 to 848). Alternatively, the nucleic acid molecule may comprise only the coding region of SEQ ID NO: 4 (for example, nucleotides 126 to 796, corresponding to SEQ ID NO: 6).

Another isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 7. SEQ ID NO: 7 corresponds to the human CRSP-3 cDNA. This cDNA comprises sequences encoding the human CRSP-3 protein (ie, "the coding region", from nucleotides 93-890), as well as sequences not translated into 5 '(nucleotides 1 to 92) and untranslated sequences in 3 '(nucleotides 891 to 1529). Alternatively, the nucleic acid molecule may comprise only the coding region of SEQ ID NO: 7 (for example, nucleotides 93 to 890 corresponding to SEQ ID NO: 9). A plasmid containing the full length nucleotide sequence encoding CRSP-3 was deposited in the American type culture collection (ATCC), Rockville, Maryland, on January 16, 1998, and registration number 98633 was assigned.

Another isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 10. The sequence of SEQ ID NO: 10 corresponds to the human CRSP-4 cDNA. This cDNA comprises sequences encoding the human CRSP-4 protein (ie, "the coding region", from nucleotides 1-537), as well as sequences not translated into 3 '(nucleotides 538 to 702). Alternatively, the nucleic acid molecule may comprise only the coding region of SEQ ID NO: 10 (for example, nucleotides 1 to 537, corresponding to SEQ ID NO: 12).

Another isolated nucleic acid molecule comprises a nucleic acid molecule that is a complement to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10, the sequence of nucleotides of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, the nucleotide sequence of the deposited plasmid DNA insert in the ATCC with the registration number 98452, or a part of any of these nucleotide sequences. A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10, the nucleotide sequence of the DNA insert of the deposited plasmid in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the number of record 98452, is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10, or the nucleotide sequence of the DNA insert of the plasmid deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the number of record 98452, so that it can hybridize with the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the number register 98452, thereby forming a stable duplex.

Another invention of the isolated nucleic acid molecule comprises a nucleotide sequence that is homologous at least about 30-35%, preferably about 40-45%, more preferably about 50-55%, even of more preferably about 60-65%, and even more preferably at least about 70-75%, 80-85%, 90-95% to the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO : 4, SEQ ID NO: 7 or SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452 or a part of any of these nucleotide sequences.

In addition, the nucleic acid molecule may comprise a part of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the DNA insert of the plasmid deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with registration number 98452, for example a fragment that can be used as a probe or a fragment encoding a biologically active part of a CRSP protein. The nucleotide sequence determined from the cloning of human CRSP genes allows the generation of probes and primers designed for use in the identification and / or cloning of CRSP homologs into other cell types, for example, from other tissues. , as well as CRSP homologues of other mammals. The probe / primer typically comprises substantially a purified oligonucleotide. The oligonucleotide normally comprises a nucleotide sequence region that hybridizes under stringent conditions with at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the deposited plasmid DNA insert in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452, of an antisense sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in l to ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452, or of a naturally occurring mutant of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452. A nucleic acid molecule may comprise a nucleotide sequence that hybridizes under stringent hybridization conditions with a nucleic acid molecule comprising nucleotides 470-2479 of SEQ ID NO: 1 or with a nucleic acid molecule comprising nucleotides 1475 of SEQ ID NO: 4.

Probes based on the human CRSP nucleotide sequence can be used to detect transcripts or genomic sequences encoding identical or homologous proteins. For example, primers based on the nucleic acid depicted in SEQ ID NO: 1 or 3 can be used in PCR reactions to clone CRSP homologs (eg, CRSP-1 homologues). CRSP homologs can be cloned by PCR amplification (eg, RT-PCR) using primers that hybridize with a part of the nucleotide sequence encoding the cysteine-rich domain of CRSP. Similarly, probes based on the object CRSP sequences can be used to detect transcripts or genomic sequences encoding identical or homologous proteins. The probe may further comprise a marker group attached thereto, for example, the marker group may be a radioisotope, a fluorescent compound, an enzyme, or an enzymatic cofactor. Such probes can be used as part of a diagnostic test kit to identify cells or tissue that erroneously express a CRSP protein, such as measuring a level of a nucleic acid encoding CRSP in a sample of cells of a subject for example, detecting levels CRSP mRNA or by determining whether a genomic CRSP gene has mutated or has been deleted.

A nucleic acid fragment encoding a "biologically active part of a CRSP protein" can be prepared by isolating a part of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the sequence of nucleotides of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, or the nucleotide sequence of the DNA insert of the plasmid deposited in the ATCC under registration number 98452, which codes for a polypeptide having a biological activity of CRSP (the biological activities of CRSP proteins have been previously described), expressing the encoded part of the CRSP protein (for example, by recombinant expression in vitro) and evaluating the activity of the encoded part of the CRSP protein.

Nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the DNA insert are also disclosed. of the plasmid deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with Registration number 98452, due to the degeneracy of the genetic code and therefore encodes for the same CRSP proteins as those encoded by the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the sequence of nucleotides of the plasmid DNA insert deposited in the ATCC under registration number 98452. An isolated nucleic acid molecule of the invention may have a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO : 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11.

In addition to the human CRSP nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452 ,, Those skilled in the art will appreciate that DNA sequence polymorphisms that result in changes in the amino acid sequence of CRSP proteins may exist within a population (eg, the human population). Such genetic polymorphisms may exist in CRSP genes among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules that comprise an open reading frame encoding a CRSP protein, preferably a mammalian CRSP protein. Such natural allelic variations can normally result in a 1-5% variance in the nucleotide sequence of a CRSP gene. Each and every such nucleotide variation and amino acid polymorphism resulting in the CRSP genes that are the result of natural allelic variation and that do not alter the functional activity of a CRSP protein are disclosed.

In addition, nucleic acid molecules encoding CRSP proteins of other species are disclosed, and therefore have a nucleotide sequence that differs from the human sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO : 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633 , or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452. For example, a murine CRSP-1 cDNA has been identified based on the nucleotide sequence of human CRSP-1. The murine CRSP-1 nucleotide sequence (SEQ ID NO: 16) encodes a CRSP-1 protein that has 349 amino acids. The amino acid and nucleotide sequences of murine CRSP-1 are shown in Figure 8. The coding region of murine CRSP-1 is represented by SEQ ID NO: 18.

Nucleic acid molecules corresponding to natural and homologous allelic variants of the CRSP cDNAs can be isolated based on their homology with the human CRSP nucleic acids described herein using the human cDNA, or a part thereof, as a hybridization probe. according to the usual hybridization techniques under stringent hybridization conditions. Examples of tissues and / or libraries suitable for the isolation of subject nucleic acids include thymus, lymph nodes and inflammatory tissue. CDNA encoding a CRSP protein (for example, a CRSP-1 protein) can be obtained by isolating total mRNA from a cell, for example, a vertebrate cell, a mammalian cell, or a human cell, including embryonic cells. Then double stranded cDNA can be prepared from total mRNA and subsequently inserted into a suitable bacteriophage or plasmid vector using any one of a variety of known techniques. The gene encoding a CRSP-1 protein can also be cloned using polymerase chain reaction techniques established according to the nucleotide sequence information provided by the invention. The nucleic acid can be DNA or RNA or analogs thereof.

Accordingly, an isolated nucleic acid molecule of the invention can be at least 15 nucleotides in length and hybridizes under stringent conditions with the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the number No. 98633, or the nucleotide sequence of the plasmid DNA insert deposited with the ATCC under registration number 98452. The nucleic acid may be at least 30, 50, 100, 250 or 500 nucleotides in length. As used herein, it is intended that the term "hybrid under stringent conditions" describe the conditions of hybridization and washing in which the nucleotide sequence homologous at least 60% of each other normally remains hybridized to each other. Preferably, the conditions are such that homologous sequences in at least about 70%, more preferably in at least about 80%, even more preferably in at least about 85% or 90% of each other remain hybridized to each other. . Those skilled in the art know such stringent conditions and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred non-limiting example of stringent hybridization conditions is hybridization in 6X sodium chloride / sodium citrate (SSC) at approximately 45 ° C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50- 65 ° C. Preferably, an isolated nucleic acid molecule that hybridizes under stringent conditions with the sequence of SEQ ID NO: 1 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally occurring" nucleic acid molecule refers to an RNA or DNA molecule that has a naturally occurring nucleotide sequence (for example, encodes for a natural protein).

In addition to naturally occurring allelic variants of the CRSP sequences that may exist in the population, the person skilled in the art will further appreciate that changes can be made by mutation in the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO : 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452, thus leading to changes in the amino acid sequence of the encoded CRSP proteins, without altering the functional capacity of CRSP proteins. For example, nucleotide substitutions can be made that lead to amino acid substitutions (particularly conservative amino acid substitutions) in "non-essential" amino acid residues in the sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 , SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited with the ATCC under registration number 98452. A "non-essential" amino acid residue is a residue that can be altered from the wild-type CRSP sequence (eg, the sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11) without altering the biological activity, while an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among CRSP proteins (for example, cysteine residues within cysteine-rich domains), are expected to be particularly poorly susceptible to alteration. In addition, amino acid residues that are conserved between CRSP protein and other proteins that have a cysteine-rich domain are unlikely to be susceptible to alteration.

Accordingly, nucleic acid molecules encoding CRSP proteins that contain changes in amino acid residues that are not essential for activity are disclosed. Such CRSP proteins differ in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11 although they maintain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises a homologous amino acid sequence at least about 60% with the amino acid sequence of SEQ ID NO: 2 , SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11. Preferably, the protein encoded by the nucleic acid molecule is homologous at least about 65-70% with SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11, more preferably homologous at least about 75-80% with SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 , or SEQ ID NO: 11, even more preferably homologous in at least about 85-90% with SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11, and most preferably homologous at least about 95% with SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO:

eleven.

An isolated nucleic acid molecule encoding a protein homologous CRSP protein of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11 can be prepared by introducing one or more substitutions, additions or nucleotide deletions in the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452, so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452, by usual techniques, such as directed mutagenesis to the site and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged side chains (for example, glycine, asparagine, glutamine, serine, threonine , tyrosine, cysteine), non-polar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched beta side chains (for example, threonine, valine, isoleucine) and aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine). Therefore, a non-essential amino acid residue envisioned in a CRSP protein (for example, one not located in a cysteine-rich domain) is preferably replaced by another amino acid residue of the same side chain family. Alternatively, mutations may be introduced randomly throughout all or part of a sequence encoding CRSP, such as by saturation mutagenesis, and the resulting mutants can be examined to select biological activity of CRSP to identify mutants that retain activity. Following the mutagenesis of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98634, the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633, or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98452, the encoded protein can be recombinantly expressed and can protein activity determined.

A mutant CRSP protein may be tested for intracellular calcium, an increase in phosphatidylinositol or another molecule, and may result in, for example, the phosphorylation of specific proteins, a gene transcription modulation and any of the other biological activities set forth in This document.

A mutant CRSP protein can also be tested to determine the ability to (1) modulate cell signal transduction, either in vitro or in vivo; (2) regulate communication between cells; (3) regulate the expression of genes whose expression is modulated by the binding of CRSP (for example, CRSP-1) to a receptor; (4) regulate gene transcription in a cell involved in development or differentiation, either in vitro or in vivo;

(5)

 regulate cell proliferation, either in vitro or in vivo; (6) form and / or maintain ordered spatial arrangements of differentiated tissues in vertebrates; (7) modulate cell death (eg cell survival);

(8)

 regulate cell migration; and / or (9) modulate the function of the immune system.

In addition to the nucleic acid molecules encoding the CRSP proteins described above, isolated nucleic acid molecules that are antisense with respect thereto are disclosed. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, for example, complementary to the coding chain of a double-stranded cDNA molecule or complementary to an mRNA sequence. . Accordingly, an antisense nucleic acid can be linked by hydrogen bonding to a sense nucleic acid. The antisense nucleic acid may be complementary to a chain encoding whole CRSP, or only a part thereof. An antisense nucleic acid molecule may be antisense with respect to a "coding region" of the coding chain of a nucleotide sequence encoding CRSP. The term "coding region" refers to the region of the nucleotide sequence comprising codons that result in amino acid residues (for example, the coding region of human CRSP-1 corresponds to SEQ ID NO: 3, the coding region of Human CRSP-2 corresponds to SEQ ID NO: 6, the coding region of human CRSP-3 corresponds to SEQ ID NO: 9, the coding region of human CRSP-4 corresponds to SEQ ID NO: 12, and the coding region of N Human CRSP type terminal corresponds to SEQ ID NO: 15). The antisense nucleic acid molecule may be antisense with respect to a "non-coding region" of the coding chain of a nucleotide sequence encoding CRSP. The term “non-coding region” refers to sequences in 5 ’and 3’ that flank the coding region that do not translate into amino acids (that is, also called regions not translated into 5 ’and 3’).

Given the coding chain sequences encoding CRSP described herein (for example, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, or SEQ ID NO: 12), antisense nucleic acids can be designed according to the base mating rules of Watson and Crick. The antisense nucleic acid molecule may be complementary to the entire CRSP mRNA coding region, but more preferably it is an oligonucleotide that is antisense to only a part of the CRSP mRNA coding or non-coding region. For example, the antisense oligonucleotide may be complementary to the region surrounding the CRSP mRNA translation start site. An antisense oligonucleotide can be, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, for example, phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides that can be used to generate the antisense nucleic acid include 5fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5 (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl thiomethylidyl 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-6-adenytosine 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio N6-isopentenyladenine, uracil-5-oxyacetic (v), wxyabutoxy (v) , cheosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2 -thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (ac p3) w and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector in which a nucleic acid has been subcloned in an antisense orientation (ie, the RNA transcribed from the inserted nucleic acid will be antisense oriented with respect to a nucleic acid target of interest, described in detail a in the following subsection).

Antisense nucleic acid molecules are normally administered to a subject or generated in situ so that they hybridize with, or bind to, cellular mRNA and / or genomic DNA encoding a CRSP protein in order to inhibit protein expression, for example, inhibiting transcription and / or translation. Hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplex, by specific interactions in the double helix's main groove. An example route of administration of antisense nucleic acid molecules of the invention includes direct injection into a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then systematically administered. For example, for systemic administration, antisense molecules can be modified so that they specifically bind to receptors or antigens expressed on a selected cell surface, for example, by binding the antisense nucleic acid molecules to peptides or antibodies that bind to receptors or antigens of cell surface Antisense nucleic acid molecules can also be administered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

The antisense nucleic acid molecule can be a an-anomeric nucleic acid molecule. A an-anomeric nucleic acid molecule forms specific double stranded hybrids with complementary RNA in which, unlike the usual  units, the chains run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res .15: 6625-6641). The antisense nucleic acid molecule may also comprise a 2'-omethylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987) FEBS Lett. 215: 327-330).

An antisense nucleic acid of the invention can be a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that can cleave a single stranded nucleic acid, such as an mRNA, with which they have a complementary region. Thus, ribozymes (for example, hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591)) can be used to catalytically cleave transcripts of CRSP mRNAs so as to inhibit the translation of CRSP mRNA. A ribozyme having specificity for a nucleic acid encoding CRSP can be designed based on the CRSP cDNA nucleotide sequence described herein (ie, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the nucleotide sequence of the plasmid DNA insert deposited with the ATCC under registration number 98634,

or the nucleotide sequence of the plasmid DNA insert deposited in the ATCC with the registration number 98633). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence that must be cleaved into an mRNA encoding CRSP. See, for example, Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, CRSP mRNA can be used to select a catalytic RNA that has a specific ribonuclease activity from a combination of RNA molecules. See, for example, Bartel, D. and Szostak, J. W. (1993) Science 261: 1411-1418.

Alternatively, the expression of the CRSP gene can be inhibited by directing nucleotide sequences complementary to the regulatory region of the CRSP (e.g., the CRSP promoter and / or enhancer) to form triple helix structures that prevent transcription of the CRSP gene into target cells See generally, Helene, C. (1991) Anticancer Drug Des. 6 (6): 569-84; Helene, C. et al. (1992) Ann N. Y. Acad. Sci. 660: 27-36; and Maher, L. J. (1992) Bioassays 14 (12): 807-15.

The CRSP nucleic acid molecules can be modified in the basic moiety, sugar moiety or phosphate backbone to improve, for example, the stability, hybridization or solubility of the molecule. For example, the deoxyribose phosphate skeleton of nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms "peptide nucleic acids" or "PNA" refer to nucleic acid mimetic compounds, for example, mimetic DNA compounds, in which the deoxyribose phosphate skeleton is replaced by a skeleton. pseudopeptide and only the four natural nucleobases are conserved. The neutral skeleton of PNAs has been shown to allow specific hybridization with DNA and RNA under conditions of low ionic concentration. The synthesis of PNA oligomers can be performed using usual solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) cited above; Perry-O'Keefe et al. PNAS 93: 14670-675.

PNAs of CRSP nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNA can be used as antisense agents or antigens for the specific sequence modulation of gene expression by, for example, induction of transcription or translation stop or replication inhibition. CRSP nucleic acid molecule PNAs can also be used in the analysis of mutations of a single base pair in a gene, (for example, by addition of PNA-directed PCR binding structure); as "artificial restriction enzymes" when used in combination with other enzymes, (for example, S 1 nucleases (Hyrup B. (1996) cited above)); or as probes or primers for DNA hybridization or sequencing (Hyrup B. et al. (1996) cited above; Perry-O’Keefe cited above).

CRSP PNAs can be modified, (for example, to enhance their stability or cellular uptake), by linking lipophilic groups or other cooperating groups to PNA, by forming PNA-DNA chimeras, or by using liposomes or other administration techniques. of drugs known in the art. For example, PNA-DNA chimeras can be generated from CRSP nucleic acid molecules that can combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (for example, RNase H and DNA polymerases) to interact with the DNA part while the PNA part provides high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in time to base stacking, number of links between nucleobases and orientation (Hyrup B. (1996) cited above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) cited above and Finn P. J, et al. (1996) Nucleic Acids. Res. 24 (17): 3357-63. For example, a DNA chain can be synthesized on a solid support using the usual phosphoramidite coupling chemistry and modified nucleoside analogs, for example, 5 '- (4-methoxytrityl) amino-5'-deoxythymidine phosphoramidite can be used, as a between the PNA and the 5 'end of the DNA (Mag, M. et al. (1989) Nucleic Acids Res. 17: 5973-88). The PNA monomers are then coupled in a gradual manner to produce a chimeric molecule with a 5 ′ PNA segment and a 3 ′ DNA segment (Finn P.J. et al. (1996) cited above). Alternatively, chimeric molecules can be synthesized with a 5 'DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

The oligonucleotide may include other bound groups such as peptides (for example, to select as in vivo host cell receptors), or agents that facilitate transport across the cell membrane (see, for example, Letsinger et al. (1989 ) Proc. Natl. Acad. Sci. US. 86: 6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84: 648-652; PCT publication number WO88 / 09810, published on 15 December 1988) or the blood brain barrier (see, for example, PCT publication number WO89 / 10134, published April 25, 1988). In addition, oligonucleotides can be modified with hybridization activated cleavage agents (see, for example, Krol et al. (1988) BioTechniques 6: 958-976) or intercalating agents. (See, for example, Zon (1988) Pharm. Res. 5: 539-549). To this end, the oligonucleotide can be conjugated with another molecule, (for example, a peptide, a crosslinking agent activated by hybridization, a transport agent or cleavage agent activated by hybridization).

II. Isolated CRSP proteins and anti-CRSP antibodies

Isolated CRSP proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to create anti-CRSP antibodies are disclosed. In one embodiment, native CRSP proteins can be isolated from cellular or tissue sources by an appropriate purification scheme using usual protein purification techniques. CRSP proteins can be produced by recombinant DNA techniques. As an alternative to recombinant expression, a CRSP polypeptide or protein can be chemically synthesized using usual peptide synthesis techniques.

An "isolated" or "purified" protein or a biologically active part thereof is substantially free of cellular material or other contaminating proteins from the cellular or tissue source from which the CRSP protein is derived, or substantially free of chemical or other precursors. Chemicals when chemically synthesized. The term "substantially free of cellular material" includes CRSP protein preparations in which the protein is separated from cellular components of the cells from which it is recombinantly isolated or produced. The term "substantially free of cellular material" includes CRSP protein preparations that have less than about 30% (dry weight) of protein other than CRSP (also referred to herein as "contaminating protein"), more preferably less than about 20% protein other than CRSP, even more preferably less than about 10% protein other than CRSP, and most preferably less than about 5% protein other than CRSP. When the CRSP protein or biologically active part thereof is recombinantly produced, it is also preferably substantially free of culture medium, that is, the culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

The term "substantially free of chemical precursors or other chemicals" includes CRSP protein preparations in which the protein is separated from the chemical precursors or other chemicals that are involved in the synthesis of the protein. The term "substantially free of chemical precursors or other chemicals" includes CRSP protein preparations that have less than about 30% (dry weight) of chemical precursors or chemicals other than CRSP, more preferably less than about 20 % of chemical precursors or chemicals other than CRSP, even more preferably less than about 10% of chemical precursors or chemicals other than CRSP, and most preferably less than about 5% of chemical precursors or chemicals other than CRSP.

Biologically active portions of a CRSP protein include peptides comprising amino acid sequences sufficiently homologous to, or derived from, the amino acid sequence of the CRSP protein, for example, the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11, which includes fewer amino acids than full-length CRSP proteins, and show at least one activity of a CRSP protein. Normally, biologically active parts comprise a domain or motif with at least one activity of the CRSP protein. A biologically active part of a CRSP protein can be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acids in length.

A biologically active part of a CRSP protein may comprise at least one region rich in cysteine. A biologically active part of a CRSP protein may comprise at least one cysteine-rich region, in which the cysteine-rich region includes at least one cysteine-rich domain. A biologically active part of a CRSP protein may comprise at least one signal sequence.

A biologically active part of a CRSP protein may comprise an amino acid sequence of CRSP that lacks a signal sequence.

It should be understood that a biologically active part of a preferred CRSP protein may contain at least one of the structural domains identified above. A more preferred biologically active part of a CRSP protein may contain at least two of the structural domains identified above. An even more preferred biologically active part of a CRSP protein may contain at least three of the structural domains identified above. A particularly preferred biologically active part of a CRSP protein of the present invention may contain at least four of the structural domains identified above.

In addition, other biologically active parts, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated to determine one or more functional activities of a native CRSP protein.

The CRSP protein may have an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence homologous at least about 55% to SEQ ID NO: 2. The CRSP protein may have an amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence homologous at least about 35% to SEQ ID NO: 5. The CRSP protein may have an amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence homologous at least about 85% to SEQ ID NO: 8. The CRSP protein may have an amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence homologous at least about 35% to SEQ ID NO: 11. A CRSP protein may comprise an amino acid sequence that is homologous at least about 30-35%, preferably about 40-45%, more preferably about 50-55%, even more preferably about a 60-65%, and even more preferably at least about 70-75%, 80-85%, 90-95% or more to the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8

or SEQ ID NO: 11.

The CRSP protein may be substantially homologous to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11, and preferably retains the functional activity of the protein of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11, although it differs in the amino acid sequence due to mutagenesis or natural allelic variation, as described in detail in subsection I above. Accordingly, the CRSP protein may be a protein comprising an amino acid sequence homologous at least about 60% to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11 and preferably retains the functional activity of the CRSP proteins of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11, respectively. Preferably, the protein is homologous at least about 70% to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11, more preferably homologous at least about 80% a SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11, even more preferably at least approximately 90% homologous to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11, and most preferably homologous at least about 95% or more to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11.

To determine the homology percentage of two amino acid or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid or nucleotide residues are then compared at the corresponding amino acid positions or nucleotide positions. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as in the corresponding position in the second sequence, then the molecules are homologous in that position (ie, as used herein "homology" of amino acid or nucleic acid is equivalent to "identity" of amino acid or nucleic acid). The percentage of homology between the two sequences is a function of the number of identical positions shared by the sequences (ie,% homology = number of identical positions / total number of positions x 100). The determination of the percentage of homology between two sequences can be achieved using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm used for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl Acad. Sci. USA 87: 2264-68, modified as in Karlin and Altschul (1993) Proc. Natl Acad. Sci. USA 90: 5873-77. An algorithm of this type is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to CRSP nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to CRSP protein molecules of the invention. To obtain alignments with gaps for comparative purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Research 25 (17): 3389-3402. When the BLAST and Gapped BLAST programs are used, the default parameters of the respective programs (for example, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred non-limiting example of a mathematical algorithm used for sequence comparison is the Myers and Miller algorithm, CABIOS (1989). An algorithm of this type is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When the ALIGN program is used to compare amino acid sequences, a residue table of PAM120 weight, a gap length penalty of 12, and a gap penalty of 4 can be used.

Chimeric or CRSP fusion proteins are also disclosed. As used herein, a "chimeric protein" or "fusion protein" of CRSP comprises a CRSP polypeptide operably linked to a polypeptide other than CRSP. A "CRSP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to CRSP, while a "polypeptide other than CRSP" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to CRSP protein, for example, a protein that is different from the CRSP protein and that is derived from an identical or different organism. Within a CRSP fusion protein the CRSP polypeptide may correspond to all or a portion of a CRSP protein. A CRSP fusion protein may comprise at least a biologically active part of a CRSP protein. A CRSP fusion protein may comprise at least two biologically active parts of a CRSP protein. A CRSP fusion protein may comprise at least three biologically active parts of a CRSP protein. Within the fusion protein, the term "operably linked" is intended to indicate that the CRSP polypeptide and the non-CRSP polypeptide are fused together. The non-CRSP polypeptide can be fused to the N-terminal or C-terminal end of the CRSP polypeptide.

For example, the fusion protein is a GST-CRSP fusion protein in which the CRSP sequences are fused to the C-terminal end of the GST sequences. Such fusion proteins can facilitate the purification of recombinant CRSP.

The fusion protein may be a CRSP protein that contains a heterologous signal sequence at its N-terminal end. For example, the native CRSP signal sequence (ie, approximately amino acids 1 to 23 of SEQ ID NO: 2) can be deleted and replaced by a signal sequence from another protein. In certain host cells (eg, mammalian host cells), the expression and / or secretion of CRSP can be increased through the use of a heterologous signal sequence.

The fusion protein may be a CRSP-immunoglobulin fusion protein in which the CRSP sequences that primarily comprise the cysteine-rich regions of CRSP are fused to sequences derived from another member of the immunoglobulin family of proteins. Soluble derivatives of cell surface glycoproteins have also been prepared in the superfamily of immunoglobulin genes consisting of an extracellular domain of cell surface glycoprotein bound to a constant region (Fc) of immunoglobulin (see for example, Capon, DJ et al. . (1989) Nature 337: 525-531 and Capon U.S. Patents Nos. 5,116,964 and

5,428,130 [CD4-IgGI constructs]; Linsley, P.S. et al. (1991) J. Exp. Med. 173: 721-730 [a CD28-IgG1 construct and a B7-I-IgG1 construct]; and Linsley, P.S. et al. (1991) J. Exp. Med. 174: 561-569 and U.S. Patent Number

5,434,131 [a CTLA4-IgG1]). Such fusion proteins have proven useful for modulating receptor-ligand interactions. Soluble derivatives of cell surface protein receptor proteins from the tumor necrosis factor receptor (TNPR) superfamily consisting of an extracellular domain of the cell surface receptor fused with a constant region (Fc) of immunoglobulin have been prepared ( see for example Moreland et al. (1997) N. Engl. J. Med. 337 (3): 141-147; van der Poll et al. (1997) Blood 89 (10): 37273734; and Ammann et al. ( 1997) J. Clin. Invest. 99 (7): 1699-1703.)

CRSP-immunoglobulin fusion proteins can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a CRSP ligand and a CRSP receptor on the surface of a cell, thus suppressing CRSP-mediated signal transduction in vivo. . CRSP-immunoglobulin fusion proteins can be used to affect the bioavailability of a CRSP-related receptor. Inhibition of the CRSP / CRSP ligand interaction may be therapeutically useful both for the treatment of differentiation or proliferative disorders, as well as for the modulation (eg, stimulation or inhibition) of developmental responses, cell adhesion and / or cell fate. . In addition, CRSPimmunoglobulin fusion proteins can be used as immunogens to produce anti-CRSP antibodies in a subject, to purify CRSP ligands and in screening assays to identify molecules that inhibit the interaction of CRSP with a CRSP ligand.

Preferably, a CRSP chimeric or fusion protein is produced by usual recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences are linked together in frame according to usual techniques, for example using blunt or antler ends for ligation, restriction enzyme digestion to provide the appropriate ends, blocking cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable binding and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that give rise to complementary projections between two consecutive gene fragments that can subsequently be paired and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). In addition, there are many commercially available expression vectors that already encode for a fusion moiety (eg, a GST polypeptide). A nucleic acid encoding CRSP can be cloned into such an expression vector so that the fusion moiety is framework bound to the CRSP protein.

Variants of CRSP proteins that work either as CRSP agonists (mimetic compounds) or as CRSP antagonists are also disclosed. Variants of CRSP proteins can be generated by mutagenesis, for example, differentiated point mutation or truncation of a CRSP protein. An agonist of the CRSP proteins can conserve substantially the same, or a subset, of the biological activities of the naturally occurring form of a CRSP protein. An antagonist of a CRSP protein can inhibit one or more activities in the naturally occurring way of the CRSP protein by, for example, competitive binding to an element downstream or upstream of a cell signaling cascade that includes the protein CRSP Therefore, specific biological effects can be caused by treatment with a limited function variant. The treatment of a subject with a variant that has a subset of the biological activities of the naturally occurring form of the protein may have fewer side effects in a subject with respect to the treatment with the naturally occurring form of CRSP protein

Variants of a CRSP protein that function either as CRSP agonists (mimetic compounds) or as CRSP antagonists can be identified by examining combinatorial libraries of mutants, for example, truncation mutants, of a CRSP protein to select agonist or antagonist activity of a CRSP protein. A diverse library of CRSP variants can be generated by combinatorial mutagenesis at the nucleic acid level and encoded by a diverse gene library. A diverse library of CRSP variants can be produced by, for example, enzymatic ligation of a mixture of synthetic oligonucleotides into gene sequences so that a degenerate set of potential CRSP sequences can be expressed as individual polypeptides, or alternatively, as a set of proteins. major fusion (eg, for phage display) that contain the set of CRSP sequences therein. There are a variety of methods that can be used to produce libraries of potential CRSP variants from a degenerate oligonucleotide sequence. The chemical synthesis of a degenerated gene sequence can be performed in an automatic DNA synthesizer, and then the synthetic gene can be ligated into an appropriate expression vector. The use of a degenerate set of genes allows to provide, in a mixture, all the sequences encoding the desired set of potential CRSP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem 53: 323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res.

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In addition, fragment libraries of a sequence encoding a CRSP protein can be used to generate a diverse population of CRSP fragments to examine and subsequently select variants of a CRSP protein. A library of coding sequence fragments can be generated by treating a double-stranded PCR fragment of a sequence encoding CRSP with a nuclease under conditions where the creation of nicks occurs approximately only once per molecule, denaturing the chain DNA double, re-naturalizing the DNA to form double-stranded DNA that may include sense / antisense pairs of different products with a dent, eliminating single-chain portions of the reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into An expression vector. By this method, an expression library encoding N-terminal, C-terminal and internal fragments of various sizes of the CRSP protein can be derived.

Several techniques are known in the art to examine gene products from combinatorial libraries prepared by point mutations or truncation, and to examine cDNA libraries to select gene products that have a selected property. Such techniques are adaptable to quickly examine gene libraries generated by combinatorial mutagenesis of CRSP proteins. The most widely used techniques, which are susceptible to rigorous analysis, to examine large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting vector library and expressing combinatorial genes under conditions in which detection of a desired activity facilitates the isolation of the vector encoding the gene whose product was detected. Recursive set mutagenesis (REM - "Recrusive Ensemble Mutagenesis"), a new technique that enhances the frequency of functional mutants in libraries, can be used in combination with screening assays to identify variants of CRSP (Arkin and Yourvan (1992 ) PNAS 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

Cell-based assays can be exploited to analyze a diverse CRSP library. For example, an expression vector library can be transfected into a cell line that normally responds to a particular ligand in a CRSP-dependent manner. The cells transfected with the ligand are then contacted and the effect of mutant expression on the ligand signaling can be detected, for example, by measuring any of several immune cell responses. Plasmid DNA can then be recovered from the cells that score for inhibition, or alternatively, for potentiation of ligand induction, and further characterized individual clones.

An isolated CRSP protein, or a part or fragment thereof, can be used as an immunogen to generate antibodies that bind to CRSP using standard techniques for the preparation of polyclonal and monoclonal antibody. A full-length CRSP protein can be used or, alternatively, the invention provides fragments of CRSP antigenic peptides for use as immunogens. The CRSP antigenic peptide comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO. 2, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 11 and encompasses a CRSP epitope so that an antibody prepared against the peptide forms a specific immune complex with CRSP. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are CRSP regions that are located on the surface of the protein, for example, hydrophilic regions.

Normally a CRSP immunogen is used to prepare antibodies by immunization of a suitable subject, (eg, rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation may contain, for example, recombinantly expressed CRSP protein or a chemically synthesized CRSP polypeptide. The preparation may additionally include an adjuvant, such as a complete or incomplete Freund's adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic CRSP preparation induces a polyclonal anti-CRSP antibody response.

Accordingly, the invention relates to a composition according to claim 1 comprising anti-CRSP antibodies. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds to (immunoreacts with) a antigen, such as CRSP. Examples of immunologically active parts of immunoglobulin molecules include F (ab) and F (ab ’) 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind to CRSP. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only a kind of antigen binding site that can immunoreact with a particular epitope. of CRSP. Thus, a monoclonal antibody composition has a unique binding affinity for a particular CRSP protein with which it immunoreacts.

Polyclonal anti-CRSP antibodies can be prepared as described above by immunizing a suitable subject with a CRSP immunogen. The anti-CRSP antibody titer in the immunized subject can be monitored over time by usual techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using immobilized CRSP. If desired, antibody molecules directed against CRSP can be isolated from the mammal (eg, from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, for example, when the anti-CRSP antibody titers are the highest, antibody producing cells can be obtained from the subject and used to prepare antibodies by usual techniques, such as the hybridoma technique. originally described by Kohler and Milstein (1975) Nature 256: 495497) (see also, Brown et al. (1981) J. Immunol. 127: 539-46; Brown et al. (1980) J. Biol. Chem. .255 : 4980-83; Yeh et al. (1976) PNAS 76: 2927-31; and Yeh et al. (1982) Int. J. Cancer 29: 269-75), the most recent human B cell hybridoma technique ( Kozbor et al. (1983) Immunol Today 4:72), EBV hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or Trioma techniques The technology for producing monoclonal antibody hybridomas is well known (see generally RH Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyzes, Plenum Publishing Corp., New York, New York (1980); EA Lerner (1981) Yale J. Biol. Med., 54: 387-402; ML Gefter et al. (1977) Somatic Cell Genet. 3: 231-36). In summary, an immortal cell line (usually a myeloma) is fused with lymphocytes (usually splenocytes) of a mammal immunized with a CRSP immunogen as described above, and culture supernatants of the resulting hybridoma cells are examined to identify a hybridoma that produces a monoclonal antibody that binds to CRSP.

Any of the many well-known protocols used to fuse lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-CRSP monoclonal antibody (see, for example, G. Galfre et al. (1977) Nature 266: 55052; Gefter et al. Somatic Cell Genet., cited above; Lerner, Yale J Biol. Med., cited above; Kenneth, Monoclonal Antibodies, cited above). In addition, one skilled in the art will appreciate that there are many variations of such methods that may also be useful. Normally, the immortal cell line (for example, a myeloma cell line) is derived from the same mammalian species as lymphocytes. For example, murine hybridomas can be prepared by fusing lymphocytes of an immunized mouse with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a variety of myeloma cell lines can be used as a fusion partner according to usual techniques, for example, myeloma lines P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 / O-Ag14 . These myeloma lines are available from the ATCC. Normally, HAT-sensitive mouse myeloma cells are fused with mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills non-fused and fused myeloma cells in a non-productive manner (the non-fused splenocytes die after several days because they do not transform). Hybridoma cells that produce a monoclonal antibody of the invention are detected by examining the culture supernatants to select antibodies that bind to CRSP, for example, using a standard ELISA assay.

Alternatively to the preparation of hybridomas that secrete monoclonal antibody, a monoclonal anti-CRSP antibody can be identified and isolated by examining a recombinant combinatorial immunoglobulin library (eg, an antibody phage display library) with CRSP so as to isolate library members of immunoglobulin that bind to CRSP. Commercially available kits are available for generating and examining phage display libraries (eg, the recombinant phage antibody system ("Recombinant Phage Antibody System") from Pharmacia, catalog number 27-9400-0 1; and the presentation kit Phage Display Kit (Stratagene's SurfZAP ™), catalog number 240612). Additionally, examples of methods and reagents particularly useful for use in generating and examining antibody presentation libraries can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT international publication number WO 92/18619; Dower et al. PCT international publication number WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT international publication number WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT international publication number WO 92/01047; Garrard et al. PCT international publication number WO 92/09690; Ladner et al. PCT international publication number WO 90/02809; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; There et al. (1992) Hum. Antibod Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffiths et al. (1993) EMBO J 12: 725-734; Hawkins et al. (1992) J. Mol. Biol. 226: 889-896; Clarkson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19: 4133-4137; Barbas et al. (1991) PNAS 88: 7978-7982; and McCafferty et al. Nature (1990) 348: 552-554.

Additionally, recombinant anti-CRSP-3 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human parts, which can be prepared using usual recombinant DNA techniques, may be part of the composition of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. international application number PCT / US86 / 02269; Akira, et al. European patent application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European patent application 173,494; Neuberger et al. PCT international publication number WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European patent application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) PNAS 84: 3439-3443; Liu et al. (1987)

J. Immunol. 139: 3521-3526; Sun et al. (1987) PNAS 84: 214-218; Nishimura et al. (1987) Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J Natl. Cancer Inst. 80: 1553-1559); Morrison, S. L. (1985) Science 229: 1202-1207; Oi et al. (1986) BioTechniques 4: 214; Winter U.S. Patent No. 5,225,539; Jones et al. (1986) Nature 321: 552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J Immunol. 141: 4053-4060.

An anti-CRSP antibody (eg, monoclonal antibody) can be used to isolate CRSP by usual techniques, such as affinity chromatography or immunoprecipitation. An anti-CRSP antibody can facilitate the purification of natural CRSP from recombinantly produced cells and CRSP expressed in host cells. In addition, an anti-CRSP antibody can be used to detect CRSP protein (for example, in a cell lysate or cell supernatant) in order to assess the abundance and expression pattern of the CRSP protein. Anti-CRSP antibodies can be used diagnostically to monitor tissue protein levels, for example, as part of a clinical trial procedure, to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (ie, physically binding) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase; Examples of prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; An example of a luminescent material includes luminol; Examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125I, 131I, 35S or 3H.

III. Recombinant expression vectors and host cells

Vectors, preferably expression vectors, containing a nucleic acid encoding CRSP (or a part thereof) are disclosed. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop in which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors may undergo autonomous replication in a host cell into which they are introduced (for example, bacterial vectors that have an origin of bacterial replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of a host cell after introduction into the host cell, and therefore replicated together with the host genome. In addition, certain vectors can direct the expression of genes to which they are operatively linked.

Such vectors are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably since the plasmid is the most commonly used form of vector. However, it is intended that such other forms of expression vectors, such as viral vectors (eg, adeno-associated viruses, adenoviruses and retroviruses defective for replication), which have equivalent functions, be included in the term.

The recombinant expression vectors comprise a nucleic acid in a form suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include a

or more regulatory sequences, selected based on the host cells to be used for expression, that are operatively linked to the nucleic acid sequence sequence to be expressed. Within a recombinant expression vector, it is intended that "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence (s) in a manner that allows the expression of the nucleotide sequence. (for example, in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the level of desired protein expression, etc. Expression vectors can be introduced into host cells so as to produce proteins or peptides, including peptides or fusion proteins, encoded by nucleic acids as described herein (e.g., CRSP proteins, CRSP mutant forms, fusion proteins , etc.).

Recombinant expression vectors can be designed to express CRSP in prokaryotic or eukaryotic cells. For example, CRSP can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are further treated in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the in vitro recombinant expression vector can be transcribed and translated, for example using T7 promoter and T7 polymerase regulatory sequences.

Protein expression in prokaryotes is most often performed in E. coli with vectors containing constitutive or inducible promoters that direct the expression of either fusion or non-fusion proteins. Fusion vectors add a variety of amino acids to a protein encoded therein, usually at the amino-terminal end of the recombinant protein. Such fusion vectors typically serve three purposes: 1) increase recombinant protein expression; 2) increase the solubility of the recombinant protein; and 3) assist in the purification of the recombinant protein by acting as an affinity purification ligand. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to allow separation of the recombinant protein from the fusion moiety after purification of the fusion protein. Such enzymes, and their related recognition sequences, include factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ ) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant target protein.

Purified fusion proteins can be used in CRSP activity assays, in CRSP ligand binding (eg, direct assays or competitive assays described in detail below), to generate antibodies specific for CRSP proteins, as examples. In a preferred embodiment, a CRSP fusion expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells that are subsequently transplanted into the irradiated receptors. The pathology of the recipient subject is then examined after sufficient time has elapsed (for example six (6) weeks).

Examples of suitable inducible fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Target gene expression from the pTrc vector is based on the transcription of host RNA polymerase from a trp-lac hybrid fusion promoter. The expression of the target gene from the pET 11d vector is based on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174 (DE3) of a resident a prophage that harbors a T7 gn1 gene under transcriptional control of the lacUV promoter.

One strategy to maximize the expression of recombinant protein in E. coli is to express the protein in a host bacterium with an affected ability to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferably used in E. coli (Wada et al., (1992) Nucleic Acids Res .20: 2111-2118). Such alteration of nucleic acid sequences can be performed by usual DNA synthesis techniques.

The CRSP expression vector may be a yeast expression vector. Examples of vectors for S. cerivisae yeast expression include pYepSec1 (Baldari, et al., (1987) Embo J 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).

Alternatively, CRSP can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for protein expression in cultured insect cells (eg, Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).

A nucleic acid of the invention can be expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When used in mammalian cells, control functions of the expression vector are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells see Chapters 16 and 17 of Sambrook, J., Fritsh, EF, and Maniatis , T. Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

The recombinant mammalian expression vector may be able to direct the expression of the nucleic acid preferably in a particular cell type (for example, tissue specific regulatory elements are used to express the nucleic acid). Specific tissue regulatory elements are known in the art. Non-limiting examples of suitable tissue specific promoters include the albumin promoter (liver specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid specific promoters (Calame and Eaton (1988) Adv. Immunol 43: 235275), in particular T cell receptor promoters (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell 33: 741-748), specific neuron promoters (for example, the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477), specific pancreas promoters (Edlund et al. (1985) Science 230: 912-916), and specific mammary gland promoters (eg, whey promoter; U.S. Patent 4,873,316 and European Patent Application No. 264,166). Developmentally regulated promoters are also covered, for example the hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546 ).

A recombinant expression vector comprising a DNA molecule cloned in the expression vector in an antisense orientation is disclosed. That is, the DNA molecule is operably linked to a regulatory sequence in a manner that allows the expression (by transcription of the DNA molecule) of an RNA molecule that is antisense with respect to CRSP mRNA. Regulatory sequences operably linked to a cloned nucleic acid in the antisense orientation may be chosen that direct the continuous expression of an antisense RNA molecule in a variety of cell types, for example viral enhancers and / or promoters, or regulatory sequences may be chosen that direct tissue-specific or cell-specific expression constituting antisense RNA. The antisense expression vector may be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the type of cell in which The vector is introduced. For an evaluation of gene expression regulation using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1 (1) 1986.

Host cells are disclosed in which a recombinant expression vector has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular object cell but also to the progeny or potential progeny of such a cell. Since certain modifications may occur in later generations due to either mutation or environmental influences, such progeny may, in fact, not be identical to the original cell, but they are still included within the scope of the term as used in This document.

A host cell can be any prokaryotic or eukaryotic cell. For example, a CRSP protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary (CHO) cells or COS cells). Those skilled in the art know other suitable host cells.

Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transfection or transformation techniques. As used herein, it is intended that the terms "transformation" and "transfection" refer to a variety of techniques recognized in the art for introducing foreign nucleic acid (eg, DNA) into a host cell, including coprecipitation. of calcium phosphate or calcium chloride, transfection mediated by DEAE-dextran, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small fraction of cells can integrate the foreign DNA into their genome. In order to identify and select these members, a gene that codes for a selectable marker (eg, resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance against drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell in the same vector as that encoding CRSP or it can be introduced into a separate vector. Stably transfected cells can be identified with the nucleic acid introduced by drug selection (for example, cells that have incorporated the selectable marker gene will survive, while the other cells will die).

A host cell, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (ie, express) CRSP protein. Accordingly, methods for producing CRSP protein using host cells are disclosed. The method may comprise culturing the host cell (into which a recombinant expression vector encoding CRSP has been introduced) in a suitable medium so that the CRSP protein is produced. In another embodiment, the method further comprises isolating CRSP from the medium or from the host cell.

Host cells can also be used to produce non-human transgenic animals. For example, a host cell is a non-human fertilized oocyte or a non-human embryonic stem cell into which sequences encoding CRSP have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous CRSP sequences have been introduced into their genome or homologous recombinant animals in which endogenous CRSP sequences have been altered. Such animals are useful for studying the function and / or activity of CRSP and for identifying and / or evaluating modulators of CRSP activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the animal's cells include a transgene. Other examples of transgenic animals include nonhuman primates, sheep, dogs, cows, goats chickens, amphibians, etc. A transgene is exogenous DNA that integrates into the genome of a cell from which a transgenic animal develops and remains in the genome of the mature animal, thus directing the expression of a gene product encoded in one or more types of cells or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous CRSP gene has been altered by homologous recombination between the endogenous gene and the Exogenous DNA molecule introduced into a cell of the non-human animal, for example, an embryonic cell of the non-human animal, before the development of the animal.

A transgenic animal can be created by introducing a nucleic acid encoding CRSP into the male pronucleus of a non-human fertilized oocyte, for example, by microinjection, retroviral infection, and allowing the oocyte to develop into a pseudopregnant female host animal. The human CRSP cDNA sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homolog of a human CRSP gene, such as a mouse CRSP gene, can be isolated based on hybridization with the human CRSP cDNA (further described in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of transgene expression. A specific tissue regulatory sequence (s) can be operatively linked to the CRSP transgene to direct the expression of the CRSP protein to particular cells. Methods for generating non-human transgenic animals by embryonic manipulation and microinjection, particularly in animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both granted to Leder et al., U.S. Patent Number

4,873,191 issued to Wagner et al., And in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for the production of other transgenic animals. A transgenic founder animal can be identified based on the presence of the CRSP transgene in its genome and / or CRSP mRNA expression in animal tissues or cells. A non-human transgenic founding animal can then be used to breed additional animals carrying the transgene. In addition, non-human transgenic animals carrying a transgene encoding CRSP can be further bred to generate other transgenic animals carrying other transgenes.

To create a homologous recombinant non-human animal, a vector is prepared containing at least a part of a CRSP gene in which a deletion, addition or substitution has been introduced in order to alter, for example, functionally affect the CRSP gene. . The CRSP gene may be a human gene (for example, the cDNA of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9 or SEQ ID NO: 12), but more preferably, it is a non-human homolog of a human CRSP gene. For example, a mouse CRSP gene of SEQ ID NO: 16 can be used to construct a suitable homologous recombination vector to alter an endogenous CRSP gene in the mouse genome. The vector can be designed so that, after homologous recombination, the endogenous CRSP gene is functionally altered (ie, it no longer encodes for a functional protein; also referred to as a "deficient" vector). Alternatively, the vector can be designed so that, after homologous recombination, the endogenous CRSP gene is mutated or otherwise altered but will still encode for functional protein (for example, the regulatory region can be altered in a 5 'direction to thereby alter Endogenous CRSP protein expression). In the homologous recombination vector, the altered part of the CRSP gene is flanked at its 5 'and 3' ends by additional nucleic acid of the CRSP gene to allow homologous recombination to occur between the exogenous CRSP gene carried by the vector and an endogenous CRSP gene in a non-human embryonic stem cell. The additional flanking CRSP nucleic acid is of sufficient length for satisfactory homologous recombination with the endogenous gene. Normally, several kilobases of flanking DNA (both at the 5 'and 3' end) are included in the vector (see for example, Thomas, KR and Capecchi, MR (1987) Cell 51: 503 for a description of homologous recombination vectors ). The vector is introduced into a non-human embryonic stem cell line (for example, by electroporation) and cells in which the introduced CRSP gene has been homologously recombined with the endogenous CRSP gene are selected (see for example, Li ,

E. et al., (1992) Cell 69: 915). The selected cells are then injected into a blastocyte of a non-human animal (eg, a mouse) to form aggregation chimeras (see for example, Bradley, A. in Teratocarcinomas and Embryonic Stem Cells A Practical Approach, EJ Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A non-human chimeric embryo can then be implanted in a suitable pseudopregnated female non-human host animal and carry the embryo to term. The progeny that houses homologously recombined DNA in its germ cells can be used to breed animals in which all cells of the animal contain homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are further described in Bradley, A. (1991) Current Opinion in Biotechnology 2: 823-829 and in PCT international publications numbers: WO 90/11354 by Le Mouellec et al .; WO 91/01140 by Smithies et al; WO 92/0968 by Zijlstra et al .; and WO 93/04169 by Berns et al.

Transgenic non-human animals that contain selected systems that allow regulated expression of the transgene can be produced. An example of such a system is the cre / loxP recombinase system of bacteriophage P1. for a description of the cre / loxP recombinase system, see, for example, Lakso et al. (1992) PNAS 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251: 1351-1355). If a cre / loxP recombinase system is used to regulate transgene expression, animals containing transgenes encoding both Cre recombinase and a selected protein are required. Such animals can be provided by the construction of non-human "double" transgenic animals, for example, by reproduction of two non-human transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385: 810-813. In summary, a cell, for example, a somatic cell, of the transgenic animal can be isolated and induced to exit the growth cycle and enter the Go phase. The quiescent cell can then be fused, for example, by the use of electrical pulses, to a non-human endonucleated oocyte of an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured so that it develops to give a morula or blastocyte and is then transferred to a pseudopregnant female host animal. The offspring born of this female host animal will be a clone of the animal from which the cell is isolated, for example, the somatic cell.

IV. Pharmaceutical compositions

CRSP nucleic acid molecules, CRSP proteins, and anti-CRSP antibodies (also referred to herein as "active ingredients") can be incorporated into pharmaceutical compositions suitable for administration. Such compositions normally comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption and isotonic retarding agents, and the like, compatible with administration. Pharmaceutical The use of such media and agents for pharmaceutically active principles is well known in the art. Except insofar as any conventional media or agent is incompatible with the active substance, its use in the compositions is contemplated. Complementary active ingredients can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its desired route of administration. Examples of routes of administration include parenteral administration, for example, intravenously, intradermally, subcutaneously, orally (eg, inhalation), transdermally (topically), transmucosal, and rectal. The solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparation can be included in ampoules, disposable syringes or multi-dose vials made of glass or plastic. The present invention provides a composition formulated for parenteral administration, comprising a pharmaceutically acceptable carrier and an antibody that selectively binds to an isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 (CRSP3), wherein the vehicle It comprises a sterile diluent for injection. The antibody can be a monoclonal antibody. The antibody can be a recombinant antibody. The composition can be formulated for injection. The composition may be a sterile injectable solution, in which the antibody is incorporated into an appropriate solvent with one or a combination of components selected from the group consisting of physiological saline, bacteriostatic water, phosphate buffered saline or a solvent containing water, ethanol, polyol or suitable mixtures thereof.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (which are soluble in water) or sterile dispersions and powders for improvised preparation of the dispersion or sterile injectable solutions. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, NJ) phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that there is an easy injection capacity. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of the injectable compositions can be caused by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active ingredient (anti-CRSP antibody) in the required amount in an appropriate solvent with one or a combination of the components listed above, as necessary, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active ingredient in a sterile vehicle containing a basic dispersion medium and other components of those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying, and lyophilization, which produces a powder of the active ingredient plus any additional desired component from a previously filtered solution for sterility thereof.

In one embodiment, the active ingredients are prepared with vehicles that will protect the compound against rapid elimination of the organism, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylacic acid can be used. The methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeting cells infected with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Patent No. 4,522,811.

It is especially advantageous to formulate parenteral compositions in unit dosage forms to facilitate administration and uniformity of dosage. The unit dosage form, as used herein, refers to physically differentiated units suitable as unit dosages for the subject to be treated; each unit containing a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The specification for the unit dosage forms of the invention are dictated by and depend directly on the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and on the limitations inherent in the art of formulating an active ingredient thereof. type for the treatment of individuals.

The toxicity and therapeutic efficacy of compounds of this type can be determined by usual pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the lethal dose for 50% of the population) and the ED50 (the therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / DE50. Compounds showing high therapeutic indices are preferred. Although compounds that show toxic side effects can be used, care should be taken to design an administration system that directs such compounds to the site of the affected tissue in order to minimize possible damage to uninfected cells and thus reduce side effects. .

Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range, depending on the pharmaceutical form used and the route of administration used. For any compound used in the invention, the therapeutically effective dose can be calculated initially from cell culture assays. A dose can be formulated in animal models to achieve a range of circulating plasma concentrations that include IC50 (i.e., the concentration of the test compound that achieves half of the maximum inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

It is known that nucleic acid molecules can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be administered to a subject by, for example, intravenous injection, local administration (see US Patent 5,328,470) or by stereotactic injection (see for example, Chen et al. (1994) PNAS 91: 3054- 3057). The pharmaceutical preparation of the gene therapy vector may include the gene therapy vector in an acceptable diluent, or it may comprise a slow release matrix in which the gene delivery vehicle is included. Alternatively, when the intact complete gene delivery vector can be produced from recombinant cells, for example, retroviral vectors, the pharmaceutical preparation may include one or more cells that produce the gene delivery system.

Pharmaceutical compositions may include a package, package, or dispenser, together with instructions for administration.

V. Uses and methods

The molecules of the present invention (for example, nucleic acid molecules, proteins, protein homologs, and antibodies described herein) can be used in one or more of the following methods: a) screening assays; b) diagnostic medicine (for example, diagnostic tests, prognostic tests, clinical monitoring tests, and pharmacogenetic tests); and c) treatment methods (for example, therapeutic and prophylactic). As described herein, a CRSP protein has a

or more of the following activities: intracellular calcium, an increase in phosphatidylinositol or another molecule, and may result, for example, the phosphorylation of specific proteins, a modulation of gene transcription and any of the other biological activities explained in the present document

Preferably, a CRSP activity is at least one or more of the following activities: (i) interaction of a CRSP protein with and / or binding to a second molecule, (eg, a protein, such as a CRSP receptor (for example, CRSP-1), a soluble form of a CRSP receptor, a receptor for a member of the wnt family of signaling proteins, or a signaling molecule other than CRSP); (ii) interaction of a CRSP protein with an intracellular protein through a membrane bound CRSP receptor; (iii) complex formation between a soluble CRSP protein and a second soluble CRSP binding partner (for example, a non-CRSP protein molecule or a second CRSP protein molecule); (iv) interaction with other extracellular proteins (for example, regulation of wnt-dependent cell adhesion to extracellular matrix components); (v) binding to and elimination of an unwanted molecule (for example, a function of detoxifying or defense activity); and / or (vi) an enzymatic activity, and therefore can be used in, for example, (1) cell signal transduction modulation, either in vitro or in vivo (for example, antagonism of the activity of the members of the wnt family of segregated proteins or suppression of wnt dependent signal transduction); (2) regulation of communication between cells (for example, regulation of cell-cell interactions dependent on wnt); (3) regulation of gene expression whose expression is modulated by binding CRSP (for example, CRSP-1) to a receptor; (4) regulation of gene transcription in a cell that participates in development or differentiation, either in vitro or in vivo (for example, induction of cell differentiation); (5) regulation of gene transcription in a cell that participates in development or differentiation, in which at least one gene encodes a specific protein for differentiation; (6) regulation of gene transcription in a cell that participates in development or differentiation, in which at least one gene codes for a second segregated protein; (7) regulation of gene transcription in a cell that participates in development or differentiation, in which at least one gene encodes a signal transduction molecule; (8) regulation of cell proliferation, either in vitro or in vivo (for example, induction of cell proliferation or inhibition of proliferation as in the case of suppression of tumorigenesis (for example, suppression of glioblastoma formation) ); (9) formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, both adult and embryonic (for example, induction of head formation during vertebrate development or maintenance of hematopoietic progenitor cells); (10) modulation of cell death, such as stimulation of cell survival; (11) migration of regulation cells; I

(12) immune modulation.

Accordingly, a method of use is disclosed (for example, a diagnostic test, a prognostic test, or a prophylactic / therapeutic method of treatment) in which a molecule is used (for example, a CRSP protein, a nucleic acid CRSP or a CRSP modulator), for example, to diagnose, predict and / or treat a disease and / or condition in which any of the aforementioned activities (i.e. activities (i) - (vi) and (1) - (12) in the previous paragraph). A method of use is also disclosed (for example, a diagnostic test, a prognostic test, or a prophylactic / therapeutic treatment method) in which a molecule is used (for example, a CRSP protein, CRSP nucleic acid or a CRSP modulator), for example, for the diagnosis, prognosis and / or treatment of subjects, preferably of a human subject, in which any of the aforementioned activities is pathologically altered. The methods of use (for example, diagnostic tests, prognostic tests, or prophylactic / therapeutic treatment methods) involve the administration to a subject, preferably a human subject, of a composition of the present invention for diagnosis, prognosis and / or therapeutic treatment. The methods of use (for example, diagnostic tests, prognostic tests, or prophylactic / therapeutic treatment methods) may involve the administration of a composition of the present invention to a human subject.

It is disclosed the use of isolated nucleic acid molecules of the invention that can be used, for example, to express the CRSP protein (for example, by a recombinant expression vector in a host cell in gene therapy applications), to detect CRSP mRNA (for example, in a biological sample) or a genetic alteration in a CRSP gene, and to modulate CRSP activity, as described further below. In addition, CRSP proteins can be used as drugs or selection compounds that modulate CRSP activity, as well as to treat disorders characterized by insufficient or excessive CRSP protein production or production of CRSP protein forms that have diminished or aberrant activity compared to the wild-type CRSP protein (for example, developmental disorders, proliferative diseases such as cancer as well as diseases, conditions or conditions characterized by abnormal cell differentiation and / or survival, an abnormal extracellular structure, or an abnormality in a defense mechanism ). In addition, anti-CRSP antibodies can be used to detect and isolate CRSP proteins, regulate the bioavailability of CRSP proteins, and modulate CRSP activity. The term "an aberrant activity", as applied to an activity of a protein such as CRSP (for example, CRSP-1), refers to an activity that differs from the activity of the natural or native protein or that differs from Protein activity in a healthy subject. An activity of a protein can be aberrant because it is stronger than the activity of its native counterpart. Alternatively, an activity can be aberrant because it is weaker or because it is absent from the activity of its native counterpart. An aberrant activity can also be a change in an activity. For example, an aberrant protein can interact with a different protein with respect to its native counterpart. A cell may have an aberrant CRSP activity (for example, CRSP-1) due to the higher or lower than normal expression of the gene encoding CRSP.

A. Selection tests:

A method (also referred to herein as a "screening assay") for identifying modulators, that is, compounds or test agents or candidates (eg, peptides, peptidomimetics, small molecules or other drugs) that is disclosed is disclosed. bind to CRSP proteins or have a stimulating or inhibiting effect on, for example, the expression of CRSP or the activity of CRSP. Modulators may include, for example, CRSP agonists and / or CRSP antagonists. The term "agonist", as used herein, is intended to refer to an agent that mimics or regulates by increment (for example, potency or supplements) a bioactivity of CRSP (eg, CRSP-1). A CRSP agonist can be a compound that mimics a bioactivity of a CRSP protein, such as the transduction of a signal from a CRSP receptor, for example, by interaction with a CRSP-1 receptor. A CRSP agonist can also be a compound that regulates by increasing the expression of a CRSP gene. A CRSP agonist can also be a compound that modulates the expression or activity of a protein that is subsequently located to a CRSP receptor, thereby mimicking or enhancing the effect of CRSP binding to a CRSP receptor.

"Antagonist" as used herein is intended to refer to an agent that inhibits, decreases or suppresses a bioactivity of CRSP (eg, CRSP-1). An antagonist can be a compound that decreases the signaling of a CRSP protein, for example, a compound that can cause binding to CRSP-1 or a CRSP-1 receptor. A preferred CRSP antagonist inhibits the interaction between a CRSP protein and another molecule, such as a CRSP receptor. Alternatively, a CRSP antagonist may be a compound that regulates by decreasing the expression of a CRSP gene. A CRSP antagonist can also be a compound that modulates the expression or activity of a protein that is subsequently located to a CRSP receptor, thereby antagonizing the effect of CRSP binding to a CRSP receptor.

Assays are provided to select test compounds or candidates that bind to or modulate the activity of a CRSP protein or polypeptide or biologically active part thereof. Trials to select test compounds or candidates that bind to or modulate the activity of a CRSP receptor are disclosed. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel phase or solid phase libraries; synthetic library methods that require convolution; the método one pearl one compound ’(‘ one-bead onecompound) library method; and synthetic library methods using selection by affinity chromatography. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to libraries of peptide compounds, non-peptide oligomers or small molecules (Lam, KS (1997) Anticancer Drug Des. 12: 145 ).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl Acad. Sci. U.S.A. 90: 6909; Erb et al. (1994) Proc. Natl Acad. Sci. USA 91: 11422; Zuckermann et al. (1994). J Med. Chem. 37: 2678; Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; and in Gallop et al. (1994) J Med. Chem. 37: 1233.

Compound libraries can be presented in solution (for example, Houghten (1992) Biotechniques 13: 412-421), or in beads (Lam (1991) Nature 354: 82-84), chips (Fodor (1993) Nature 364: 555 -556), bacteria (US Patent 5,223,409 to Ladner), spores (US Patent USP'409 to Ladner), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89: 1865-1869) or phage ( Scott and Smith (1990) Science 249: 386-390); (Devlin (1990) Science 249: 404-406); (Cwirla et al. (1990) Proc. Natl. Acad Sci. 87: 6378-6382); (Felici (1991) J Mol. Biol. 222: 301-310); (Ladner cited above).

An assay may be a cell-based assay in which a cell that expresses a CRSP receptor on the cell surface is in contact with a test compound and the ability of the test compound to bind to a CRSP receptor is determined. The cell, for example, can be of mammalian origin or a yeast cell. The determination of the ability of the test compound to bind to a CRSP receptor can be carried out, for example, by coupling the test compound with a radioisotope or an enzymatic label, so that the binding of the test compound to the receptor CRSP can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 125I, 35S, 14C,

or 3H, either directly or indirectly, and the radioisotope can be detected by direct radio emission counting or by scintillation counting. Alternatively, the test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase or luciferase, and the enzyme marker can be detected by determining the conversion of an appropriate substrate into product.

It is also possible to determine the ability of a test compound to interact with a CRSP receptor without marking any of the interacting compounds. For example, a microphysiometer can be used to detect the interaction of a test compound with a CRSP receptor without labeling the test compound or the receptor. McConnell, H. M. et al. (1992) Science 257: 1906-1912. As used herein, a "microphysiometer" (for example, Cytosensor ™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-directed potentiometric detector (LAPS). Changes in this acidification rate can be used as indicators of the interaction between the ligand and the receptor.

The assay may comprise contacting a cell that expresses a CRSP receptor on the cell surface with a CRSP protein or a biologically active part thereof, to form a test mixture, contacting the test mixture with a compound of test, and determine the ability of the test compound to interact with a CRSP receptor, wherein determining the ability of the test compound to interact with a CRSP receptor comprises determining the ability of the test compound to preferentially bind to the receptor of CRSP compared to the ability of CRSP, or a biologically active part thereof, to bind to the receptor.

An assay may be a cell-based assay that comprises contacting a cell that expresses a CRSP target molecule with a test compound and determining the ability of the test compound to modulate (eg stimulate or inhibit) the activity of the molecule. CRSP target. The determination of the ability of the test compound to modulate the activity of a CRSP target molecule can be carried out, for example, by determining the ability of the CRSP protein to bind to or interact with the CRSP target molecule.

The determination of the ability of the CRSP protein to bind or interact with a CRSP target molecule can be carried out by one of the methods described above to determine direct binding. The determination of the ability of the CRSP protein to bind or interact with a CRSP target molecule can be carried out by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by the detection of the induction of a second cellular messenger of the target molecule (i.e. intracellular Ca2 +, diacylglycerol, IP3, etc.), detection of the catalytic / enzymatic activity of the molecule target of an appropriate substrate, detection of the induction of an indicator gene (comprising a CRSP sensitive regulatory element operably linked to a nucleic acid encoding a detectable label, for example, luciferase), or detection of a cellular response, by example, development, differentiation or proliferation rate.

An assay of the present invention may be a cell-free assay in which a CRSP protein or biologically active part thereof is contacted with a test compound and the ability of the test compound to bind to the CRSP protein is determined. or biologically active part thereof. The binding of the test compound to the CRSP protein can be determined directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the CRSP protein or biologically active part thereof with a known compound that binds to CRSP to form a test mixture, contacting the test mixture with a test compound, and determining the ability of the test compound to interact with a CRSP protein, wherein determining the ability of the test compound to interact with a CRSP protein comprises determining the ability of the test compound to preferentially bind to the CRSP or biologically part. active of the same ace compared to the known compound.

The assay may be a cell-free assay in which a CRSP protein or biologically active part thereof is contacted with a test compound and the ability of the test compound to modulate (eg, stimulate or inhibit) is determined. the activity of the CRSP protein or biologically active part thereof. The determination of the ability of the test compound to modulate the activity of a CRSP protein can be carried out, for example, by determining the ability of the CRSP protein to bind to a CRSP target molecule by one of the methods described above. to determine direct union. The determination of the ability of the CRSP protein to bind to a CRSP target molecule can also be carried out using a technology such as Biomolecular Interaction Analysis (BIA) in real time. Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63: 2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5: 699

705. As used herein, "BIA" is a technology for the study of biospecific interactions in real time, without marking any of the interacting compounds (for example, BIAcore ™). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

The determination of the ability of the test compound to modulate the activity of a CRSP protein can be carried out by determining the ability of the CRSP protein to further modulate the activity of a CRSP target molecule. For example, the catalytic / enzymatic activity of the target molecule on an appropriate substrate can be determined, as described above.

The cell-free assay may involve contacting a CRSP protein or biologically active part thereof with a known compound that binds to the CRSP protein to form a test mixture, contacting the test mixture with a test compound. , and determining the ability of the test compound to interact with the CRSP protein, wherein determining the ability of the test compound to interact with the CRSP protein comprises determining the ability of the CRSP protein to preferentially bind to or to modulate activity. of a CRSP target molecule.

In many drug selection programs that test libraries of natural compounds and extracts, high performance assays are desirable in order to maximize the number of compounds examined in a given period of time. Often, tests performed on cell-free systems, such as those that can be derived from purified or semi-purified proteins, are preferred as "primary" selections because they can be generated to allow rapid development and relatively easy detection of an alteration in a molecular target that is mediated by a test compound. In addition, the effects of the toxicity and / or cellular bioavailability of the test compound can generally be ignored in the in vitro system, however the test focusing mainly on the effect of the drug on the molecular target, since it can manifest itself in an alteration in the affinity of union with elements upstream or downstream. Accordingly, in an example selection assay, the compound of interest is contacted with a CRSP protein (for example, CRSP-1) or a CRSP binding partner (for example, CRSP-1), for example, a receiver. The receptor may be soluble or the receptor may be present on a cell surface. A composition containing a CRSP binding partner or a CRSP protein, respectively, is then added to the mixture of the compound and to the CRSP protein or CRSP binding partner. The detection and quantification of CRSP protein complexes and CRSP binding partners provide a means to determine the efficacy of a compound to inhibit (or enhance) the formation of the complex between CRSP and a binding partner. The efficacy of the compound can be evaluated by generating dose response curves from the data obtained using various concentrations of the test compound. In addition, a control test can also be performed to provide a baseline for comparison. In the control assay, the isolated and purified CRSP polypeptide or binding partner is added to a composition containing the binding partner CRSP or the CRSP polypeptide, and the formation of a complex in the absence of the test compound is quantified.

Cell-free assays are susceptible to use with soluble and / or membrane bound forms of isolated proteins (for example CRSP proteins or biologically active parts thereof or CRSP target molecules). In the case of cell-free assays in which an isolated membrane bound form of protein is used (eg, a target molecule or CRSP receptor) it may be desirable to use a solubilizing agent so that the form bound to the Isolated protein membrane remains in solution. Examples of solubilizing agents of this type include non-ionic detergents such as n-octylglucoside, ndodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecipoli (ethylene glycol ether) n, 3 - [(3-colamidopropyl) dimethylamino] -1-propane (CHAPS) sulfonate, 3 - [(3-colamidopropyl) dimethylamino] -2-hydroxy-1-propane (CHAPSO) sulfonate , or N-dodecyl = N, N-dimethyl-3-ammonium-1-propane sulfonate.

In more than one alternative of the above test methods, it may be desirable to immobilize either CRSP or its target molecule to facilitate separation of the complexed from the uncomplexed forms of one or both proteins, as well as adapt the automation of the assay. The binding of a test compound to a CRSP protein, or the interaction of a CRSP protein with a target molecule in the presence and absence of a candidate compound, can be carried out in any suitable container to contain the reagents. Examples of such containers include microtiter plates, test tubes and microcentrifuge tubes. A fusion protein can be provided that adds a domain that allows one or both proteins to bind to a matrix. For example, glutathione-S-transferase / CRSP fusion proteins or glutathione-Stransferase / target protein fusion proteins can be adsorbed on glutathione-Sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates , which are then combined with the test compound or test compound, either the non-adsorbed target protein or the CRSP protein, and the mixture is incubated in conductive states for complex formation (for example, in physiological states for salt and pH). After incubation, the beads or wells of the microtiter plates are washed to remove any unbound components, the matrix is immobilized in the case of the beads, the complex is determined directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix and the level of binding or activity of the CRSP can be determined using standard techniques.

Other techniques for immobilizing proteins in matrices can also be used in the selection assays. For example, either a CRSP protein or a CRSP target molecule can be immobilized using conjugation of biotin and streptavidin. The CRSP protein or biotinylated target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (eg, biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the 96-well plates coated with streptavidin (Pierce Chemical). Alternatively, antibodies reactive with CRSP protein or target molecules may be derivatized but that do not interfere with the binding of the CRSP protein to its target molecule, plate wells, and unbound target molecule or CRSP protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for complexes immobilized to GST, include immunodetection of complexes using antibodies reactive with the CRSP protein

or the target molecule, as well as enzyme linked assays that are based on the detection of an enzymatic activity associated with the CRSP protein or the target molecule.

Modulators of CRSP expression can be identified in a method in which a cell is contacted with a candidate compound and the expression of the CRSP mRNA or CRSP protein in the cell is determined. The expression level of the CRSP mRNA or the CRSP protein in the presence of the candidate compound is compared with the expression level of the CRSP mRNA or the CRSP protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of CRSP expression based on this comparison. For example, when the expression of the CRSP mRNA or the CRSP protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the expression of the CRSP mRNA or CRSP protein Alternatively, when the expression of the CRSP mRNA or the CRSP protein is lower (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the expression of the CRSP mRNA or protein. CRSP The level of expression of the CRSP mRNA or the CRSP protein in the cells can be determined by the methods described herein to detect the CRSP mRNA or the CRSP protein.

CRSP proteins can be used as "bait proteins" in a two-hybrid assay or a three-hybrid assay (see, for example, U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054; Bartel et al. (1993) Biotechniques 14: 920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and WO94 / 10300 of Brent), to identify other proteins, which bind to

or interact with CRSP ("CRSP binding proteins" or "CRSP-pb") and modulate CRSP activity. Such CRSP binding proteins are also likely to participate in the propagation of the signals produced by CRSP proteins as, for example, to downstream elements of a CRSP-mediated signaling path. Alternatively, such CRSP binding proteins are likely to be cell surface molecules associated with cells that do not express CRSP, in which such CRSP binding proteins participate in signal transduction.

The two hybrid system is based on the modular nature of most transcription factors, which consists of activation and DNA binding domains that can be separated. In summary, the assay uses two different DNA constructs. In a construct, the gene that codes for a CRSP protein is fused with a gene that codes for the DNA binding domain of a known transcription factor (eg, GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that codes for an unidentified protein ("prey" or "sample") is fused with a gene that codes for the activation factor of the known transcription. If the "bait" and "prey" proteins can interact, in vivo, forming a CRSP-dependent complex, the activation and DNA binding domains of the transcription factor are transported in close proximity. This proximity allows the transcription of an indicator gene (for example, LacZ) that is operatively linked to a transcription regulation site that responds to the transcription factor. The expression of the reporter gene can be detected and colonies of cells containing the functional transcription factor can be isolated and used to obtain the cloned gene encoding the protein that interacts with the CRSP protein.

Novel agents identified by the screening assays described above and methods for producing such agents by using these assays are disclosed. Accordingly, a compound or agent that can be obtained by a method comprising the steps of any one of the above-mentioned selection assays (for example, cell-based assays or cell-free assays) is disclosed. For example, a compound or agent that can be obtained by a method comprising contacting a cell expressing a CRSP target molecule with a test compound and determining the ability of the test compound to bind to, or modulate is disclosed. the activity of, the target molecule of CRSP. A compound or agent that can be obtained by a method comprising contacting a cell that expresses a CRSP target molecule with a CRSP protein or a biologically active part thereof is disclosed to form a test mixture, put in Contact the test mixture with a test compound and determine the ability of the test compound to interact with, or modulate the activity of, the CRSP target molecule. A compound or agent that can be obtained by a method comprising contacting a CRSP protein or a biologically active part thereof with a test compound and determining the ability of the test compound to bind to, or modulate () is disclosed. for example, stimulate or inhibit) the activity of, the CRSP protein or the biologically active part thereof. A compound or agent that can be obtained by a method comprising contacting a CRSP protein or a biologically active part thereof is disclosed with a known compound that binds to the CRSP protein to form a test mixture, put in Contact the test mixture with a test compound and determine the ability of the test compound to interact with, or modulate the activity of the CRSP protein.

Accordingly, the additional use of an identified agent as disclosed herein is disclosed in an appropriate animal model. For example, an identified agent can be used as described herein (for example, a CRSP modulating agent, an antisense CRSP nucleic acid molecule, a CRSP specific antibody, or a CRSP binding partner) in an animal model to determine the efficacy, toxicity or side effects of treatment with such agent. Alternatively, an identified agent as described herein can be used in an animal model to determine the mechanism of action of such an agent.

Uses of novel agents identified by the screening assays described above for diagnoses, prognoses and treatments are disclosed, as described herein. Accordingly, the use of such agents in the design, formulation, synthesis, manufacture, and / or production of a drug or pharmaceutical composition for use in diagnosis, prognosis or treatment, as described herein is disclosed. . For example, a method for synthesizing or producing a drug or pharmaceutical composition is disclosed by referring to the structure and / or properties of a compound that can be obtained by one of the selection assays described above. For example, a drug or pharmaceutical composition can be synthesized based on the structure and / or properties of a compound obtained by a method in which a cell expressing a CRSP target molecule is contacted with a test compound and the test is determined. ability of the test compound to bind to, or modulate the activity of, the CRSP target molecule. A method for synthesizing or producing a drug or pharmaceutical composition based on the structure and / or properties of a compound that can be obtained by a method in which a CRSP protein or a biologically active part thereof is placed is disclosed. in contact with a test compound and the ability of the test compound to bind to, or modulate (for example, stimulate or inhibit) the activity of, the CRSP protein or the biologically active part thereof is determined.

B. Screening tests

Parts or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, therefore, locate gene regions associated with a genetic disease; (ii) identify an individual from a minimum biological sample (tissue typing); and (iii) help in forensic identification of a biological sample. These applications are described in the subsections below.

1. Chromosome mapping

Once the sequence (or a part of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This procedure is called chromosomal mapping. Accordingly, parts or fragments of the CRSP nucleotide sequence, described herein, can be used to map the location of the CRSP genes on a chromosome. The mapping of the CRSP sequences on chromosomes is an important first step in correlating these sequences with genes associated with disease.

5

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55

In summary, CRSP genes can be mapped onto chromosomes to prepare PCR primers (preferably 15-25 bp in length) from the nucleotide sequence of CRSP. A computer analysis of the CRSP sequences can be used to predict primers that do not expand more than one exon in the genomic DNA, thus complicating the amplification procedure. These primers can then be used for PCR examination of somatic cell hybrids containing individual human chromosomes. Only hybrids containing the human gene corresponding to the CRSP sequences will result in an amplified fragment.

Somatic cell hybrids are prepared by fusion of somatic cells of different mammals (eg, human and mouse cells). As human and mouse cell hybrids grow and divide, they gradually lose human chromosomes randomly, but retain mouse chromosomes. By using means in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the chromosome that contains the gene that codes for the necessary enzyme will be conserved. Through the use of various means, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a complete set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D’Eustachio P. et al. (1983) Science 220: 919-924). Somatic cell hybrids can also be produced that contain only fragments of human chromosomes using human chromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a quick procedure to assign a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a simple thermal cycler. Using the nucleotide sequence of CRSP to design oligonucleotide primers, sublocalization with panels of specific chromosome fragments can be achieved. Other mapping strategies that can be similarly used to map a 90, 1p, or 1v sequence for your chromosome include in situ hybridization (described in Fan, Y. et al. (1990) PNAS, 87: 6223-27), preselection with chromosomes separated by labeled flow cytometry and preselection by hybridization with specific cDNA libraries of chromosomes.

Fluorescent in situ hybridization (FISH) of a DNA sequence with an extended chromosomal metaphase can additionally be used to provide accurate one-stage chromosomal localization. Chromosome extensions can be prepared using cells whose division has been metaphase blocked by a chemical such as colcemid that alters the mitotic achromatic spindle. Chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands is revealed on each chromosome, so that the chromosomes can be individually identified. The FISH technique can be used with a DNA sequence of only 500 or 600 bases. However, clones greater than 1,000 bases are more likely to bind to a single chromosomal location with sufficient signal strength for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will be sufficient to obtain good results in a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).

Reagents for mapping individual chromosomes can be used to mark a single chromosome or a single site on that chromosome, or reagent panels can be used to mark multiple sites and / or multiple chromosomes. In fact, reagents corresponding to non-coding regions of the genes for mapping purposes are preferred. Coding sequences are more likely to be conserved within gene families, thereby increasing the possibility of cross hybridization during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with the genetic map data. (Such data is found, for example, in

V. McKusick, Mendelian Inheritance in Man, available online through the Welch Medical Library of Johns Hopkins University. The relationship between a gene and a disease, mapped on the same chromosomal region, can then be identified through binding analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325: 783

787.

In addition, differences in DNA sequences between affected and unaffected individuals with a disease associated with the CRSP gene can be determined. If a mutation is observed in any or all affected individuals but not in any unaffected individual, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves looking first to detect structural alterations in chromosomes, such as deletions, translocations that are visible from chromosome extensions or detectable using PCR based on that DNA sequence. Finally, complete sequencing of the genes of several individuals can be performed to confirm the presence of a mutation to distinguish mutations of polymorphisms.

2. Tissue typing

CRSP sequences can also be used to identify individuals from minimal biological samples. The United States Army, for example, is considering the use of polymorphisms in the length of restriction fragments (RFLP) for the identification of its personnel. In this technique, the genomic DNA of an individual with one or more restriction enzymes is digested, and examined with probes in a Southern blot to give rise to unique bands for identification. This method does not suffer from the current limitations of "Dog Tags" that can be lost, changed, or stolen, making positive identification difficult. The sequences are useful as additional DNA markers for RFLP (described in US Patent No. 5,272,057).

In addition, the sequences can be used to provide an alternative technique that determines the actual base DNA sequence based on selected parts of an individual's genome. Therefore, the CRSP nucleotide sequence described herein can be used to prepare two PCR primers from the 5 ′ and 3 ′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

The corresponding DNA sequence panels of individuals, prepared in this way, can provide unique individual identifications, since each individual will have a unique set of such DNA sequences due to allelic differences. The sequences can be used to obtain such identification sequences from individuals and tissues. The nucleotide sequence of CRSP only represents parts of the human genome. Allelic variation occurs to some extent in the coding regions of these sequences, and to a greater extent in the non-coding regions. It is estimated that allelic variation between human individuals occurs with a frequency of approximately once per 500 bases. Each of the sequences described herein can be used, to some extent, as a pattern against which an individual's DNA can be compared for identification purposes. Since higher numbers of polymorphisms occur in non-coding regions, fewer sequences are needed to differentiate individuals. The non-coding sequences of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10, can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers, each of them. which results in an amplified non-coding sequence of 100 bases. If the intended coding sequences are used, such as those of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, or SEQ ID NO: 12, a more appropriate number of primers for the identification of positive individual will be of 500-2,000.

If a panel of reagents from a CRSP nucleotide sequence described herein is used to generate a unique identification database, those same reagents can be used later to identify that individual's tissue. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

3. Use of partial CRSP sequences in forensic biology

DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field that uses genetic typing of biological evidence found at the scene of a crime as a means of positively identifying, for example, the principal author of a crime. To perform such identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, for example, hair or skin, or body fluids, for example, blood, saliva, or semen found in the body. Crime scene The amplified sequence can then be compared with a pattern, thus allowing identification of the origin of the biological sample.

The sequences can be used to provide polynucleotide reagents, for example, PCR primers, directed towards specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications, for example, by providing another "identification marker" ( that is another DNA sequence that is unique to a particular individual). As mentioned earlier, the actual basic sequence information can be used for identification as a precise alternative to patterns formed by fragments generated by restriction enzymes. Sequences directed towards non-coding regions of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10 are particularly suitable for this use, since a greater number of polymorphisms occur in the regions non-coding, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the nucleotide sequences of CRSP or a portion thereof, for example, fragments derived from non-coding regions of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10 having a length of at least 20 bases, preferably at least 30 bases.

The CRSP nucleotide sequences described herein can be further used to provide polynucleotide reagents, for example, labeled or labeled probes that can be used in, for example, an in situ hybridization technique, to identify a specific tissue, for example, brain tissue. This can be very useful in cases where a forensic pathologist presents with a tissue of unknown origin. The panels of such CRSP probes can be used to identify tissues by species and / or by type of organ.

Similarly, these reagents, for example, CRSP probes or primers, can be used to examine tissue culture for contamination (i.e., examine for the presence of a mixture of different cell types in a culture).

C. Diagnostic medicine:

In the field of diagnostic medicine, diagnostic tests, prognostic tests and clinical monitoring trials are used for prognostic (predictive) purposes to treat an individual prophylactically. Accordingly, diagnostic assays for determining CRSP protein and / or nucleic acid expression, as well as CRSP activity, are disclosed in the context of a biological sample (eg, blood, serum, cells, tissue) to determine if an individual is affected by a disease or disorder or is at risk of developing a disorder associated with the expression or activity of aberrant CRSP, such as proliferation, differentiation, and / or aberrant cell survival, resulting in for example a neurodegenerative disease or cancer. Prognostic (or predictive) tests are disclosed to determine if an individual is at risk of developing a disorder associated with activity, nucleic acid expression or CRSP protein. For example, mutations in a CRSP gene in a biological sample can be tested. Such assays can be used for prognostic or predictive purposes to prophylactically treat an individual before the onset of a disorder characterized by or associated with the activity, expression of nucleic acid or CRSP protein.

Monitoring of the influence of agents (eg, drugs, compounds) on the expression or activity of CRSP in clinical trials is disclosed.

These and other agents are described in detail below in the following sections.

1. Diagnostic tests

An exemplary method for detecting the presence or absence of the CRSP protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and placing the biological sample with a compound or agent that can detect the CRSP protein. or the nucleic acid (eg, mRNA, genomic DNA) encoding the CRSP protein, so that the presence of CRSP protein or nucleic acid in the biological sample is detected. A preferred agent for the detection of genomic DNA or mRNA of CRSP is a labeled nucleic acid probe that can hybridize with genomic DNA or mRNA of CRSP. The nucleic acid probe may be, for example, a full-length CRSP nucleic acid, such as the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, the plasmid DNA insert deposited in the ATCC with the registration number 98634, the plasmid DNA insert deposited in the ATCC with the registration number 98633, the plasmid DNA insert deposited in the ATCC with the registration number 98452 , or a part thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions with genomic DNA or CRSP mRNA. Other probes suitable for use in diagnostic tests are described herein.

A preferred agent for detecting the CRSP protein is an antibody that can bind to the CRSP protein, preferably an antibody with a detectable label. The antibodies may be polyclonal, or more preferably, monoclonal. An intact antibody or a fragment thereof (eg, Fab or F (ab ’) 2) can be used. It is intended that the term "labeled", in relation to the probe or antibody, encompasses the direct labeling of the probe or antibody by coupling (ie, physically binding) a detectable substance to the probe or antibody, as well as the indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody and the end labeling of a biotin DNA probe so that it can be detected with fluorescence labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present in a subject. That is, the detection method can be used to detect genomic DNA, protein or mRNA of CRSP in a biological sample in vitro as well as in vivo. For example, in vitro techniques for CRSP mRNA detection include Northern-type hybridizations and in situ hybridizations. In vitro techniques for CRSP protein detection include enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detecting genomic DNA from CRSP include Southern hybridizations. In addition, in vitro techniques for the detection of CRSP protein include introducing a labeled anti-CRSP antibody into a subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by usual imaging techniques.

The biological sample may contain protein molecules from the test subject. Alternatively, the biological sample may contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a serum sample isolated by conventional means from a subject.

The methods may further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent that can detect genomic DNA, mRNA or CRSP protein, so as to detect the presence of genomic DNA, mRNA or CRSP protein in the biological sample and the presence of genomic DNA, mRNA or CRSP protein in the control sample is compared with the presence of genomic DNA, mRNA or CRSP protein in the test sample.

Kits for detecting the presence of CRSP in a biological sample are disclosed. For example, the kit may comprise a labeled agent or compound that can detect the mRNA or CRSP protein in a biological sample; means to determine the amount of CRSP in the sample; and means to compare the amount of CRSP in the sample with a standard. The compound or agent may be packaged in a suitable container. Additionally, the kit may comprise instructions for using the kit to detect nucleic acid or CRSP protein.

2. Forecasting tests

The diagnostic methods described herein may also be used to identify subjects who have or are at risk of developing a disease or disorder associated with aberrant CRSP activity or expression. For example, the tests described herein, such as the previous diagnostic tests or the following tests, can be used to identify a subject who has or is at risk of developing a disorder associated with activity, nucleic acid expression or the CRSP protein, such as a proliferative disorder, a developmental or differentiation disorder, a disorder as well as hematopoietic diseases, conditions or conditions characterized by abnormal cell survival, abnormal extracellular structure or an abnormality in a defense mechanism. Alternatively, prognostic assays can be used to identify a subject who has or is at risk of developing a proliferative or differentiation disease (for example, cancer). Therefore, a method for identifying a disease or disorder associated with the activity or expression of aberrant CRSP is disclosed, in which the test sample is obtained from a subject and the nucleic acid or CRSP protein is detected. (eg, mRNA, genomic DNA), in which the presence of nucleic acid or CRSP protein is diagnostic for a subject who has or is at risk of developing a disease or disorder associated with the activity or expression of aberrant CRSP. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample may be a biological fluid (eg, serum), cell sample or tissue.

In addition, the prognostic assays described herein can be used to determine whether or not an agent can be administered to a subject (eg, an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant CRSP activity or expression. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder, such as proliferative disorder, differentiation or developmental disorder, a hematopoietic disorder, as well as disorders characterized by abnormal cell survival, abnormal extracellular structure. or an anomaly in a defense mechanism. Alternatively, such methods can be used to determine whether or not a subject can be effectively treated with an agent for a differentiation or proliferative disease (eg, cancer). Thus, methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant CRSP activity or expression are given, in which a test sample is obtained and the activity or expression of nucleic acid is detected. or CRSP protein (for example, in which the abundance of activity or expression of nucleic acid or CRSP protein is diagnostic for a subject to whom the agent can be administered to treat a disorder associated with aberrant CRSP activity or expression.)

The methods can also be used to detect genetic alterations in a CRSP gene, thus determining if a subject with the altered gene is at risk of suffering from a disorder characterized by aberrant development, aberrant cell differentiation, aberrant cell proliferation or an aberrant hematopoietic response. The methods may include detecting, in a sample of cells of the subject, the presence or absence of a genetic alteration by at least one of an alteration that affects the integrity of a gene encoding a CRSP protein, or the failed expression of the gene of CRSP. For example, such genetic alterations can be detected by determining the existence of at least one of 1) a deletion of one or more nucleotides of a CRSP gene; 2) an addition of one or more nucleotides to a CRSP gene; 3) a substitution of one or more nucleotides of a CRSP gene, 4) a chromosomal rearrangement of a CRSP gene; 5) an alteration in the level of a messenger RNA transcript of a CRSP gene, 6) aberrant modification of a CRSP gene, such as the genomic DNA methylation pattern, 7) the presence of a splicing pattern unnatural type of a messenger RNA transcript of a CRSP gene, 8) an unnatural type level of a CRSP protein, 9) allelic loss of a CRSP gene, and 10) post-translational modification of an inappropriate CRSP protein As described herein, there are a large number of assay techniques known in the art that can be used to detect alterations in a CRSP gene. A preferred biological sample is a tissue or serum sample isolated by conventional means of a subject.

Alteration detection may involve the use of a probe / primer in a polymerase chain reaction (PCR) (see, for example, U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligase chain reaction (CSF) (see, for example, Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1994) PNAS 91: 360 -364), the latter of which may be particularly useful for detecting point mutations in the CRSP gene (see Abravaya et al. (1995) Nucleic Acids Res. 23: 675-682). This method may include the steps of taking a sample of cells from a patient, isolating the nucleic acid (eg, genomic, mRNA or both) from the cells in the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize with a CRSP gene under conditions such that hybridization and amplification of the CRSP gene occurs (if present), and detect the presence or absence of an amplification product, or detect the size of the amplification product and Compare the length with a control sample. It is envisioned that it may be desirable to use PCR and / or CSF as a preliminary amplification step together with any of the techniques used to detect mutations described herein.

Alternative amplification methods include: self-sustained sequence replication (Guatelli, JC et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), translation amplification system (Kwoh, DY et al., 1989 , Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta replicase (Lizardi, PM et al, 1988, Bio / Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of amplified molecules using techniques well known to those skilled in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are found in a very small amount.

Mutations in a CRSP gene of a sample cell can be identified by alterations in the cleavage pathways of restriction enzymes. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicate mutations in the sample DNA. In addition, the use of sequence specific ribozymes (see, for example, U.S. Patent No. 5,498,531) can be used to rate the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in CRSP can be identified by hybridizing sample and control nucleic acids, for example, DNA or RNA, to high density assays containing hundreds or thousands of oligonucleotide probes (Cronin, MT et al. (1996) Human Mutation 7: 244-255; Kozal, MJ et al. (1996) Nature Medicine

2: 753-759). For example, genetic mutations in CRSP can be identified in two-dimensional matrices containing DNA probes generated by light as described in Cronin, M.T. et al. above. In summary, a first probe hybridization matrix can be used to scan through long extensions of DNA in a sample and control to identify base changes between the sequences by making linear arrays of overlapping probes sequentially. This stage allows the identification of point mutations. This stage is followed by a second series of hybridization that allows the characterization of specific mutations using matrices of smaller specialized probes complementary to all the variants or mutations detected. Each mutation series is composed of sets of parallel probes, one complementary to the wild type gene and the other complementary to the mutant gene.

Any of a variety of sequencing reactions known in the art can be used to directly sequence the CRSP gene and detect mutations by comparing the sequence of the sample CRSP with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) PNAS 74: 560) or Sanger ((1977) PNAS 74: 5463). It is also contemplated that any of a variety of automated sequencing procedures can be used when performing diagnostic tests ((1995) Biotechniques 19: 448), including sequencing by mass spectrometry (see, for example, International PCT Publication No. WO 94 / 16101; Cohen et al. (1996) Adv. Chromatogr. 36: 127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38: 147-159).

Other methods to detect mutations in the CRSP gene include methods in which protection from cleavage agents is used to detect bases with erroneous mating in RNA / RNA or RNA / DNA heterodux (Myers et al. (1985) Science 230 : 1242). In general, the "mismatch cleavage" technique begins by providing RNA or DNA heterodux (labeled) by hybridization, which contain the wild-type CRSP sequence with potentially mutant RNA or DNA obtained from a tissue sample. Double-chain duplexes are treated with an agent that cleaves the single-chain regions of the duplex as it will exist due to erroneous pairings of base pairs between the control and sample chains. For example, RNA / DNA duplexes can be treated with RNase and DNA / DNA hybrids can be treated with S1 nuclease to enzymatically digest the mismatched regions. In other embodiments, the duplexes may either be DNA / DNA or RNA / DNA with hydroxylamine or osmium tetroxide and with piperidine in order to digest the mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the mutation site. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85: 4397; Saleeba et al. (1992) Methods Enzymol. 217: 286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

The mismatch reaction may employ one or more proteins that recognize mismatched base pairs in double stranded DNA (called "DNA mismatch repair enzymes") in defined systems to detect and map point mutations in cDNA of CRSP obtained from cell samples. For example, the E. coli mutY enzyme cleaves A in mismatched G / A and thymidine DNA glycosylase from HeLa T cells in mismatches G / T (Hsu et al. (1994) Carcinogenesis 15: 1657-1662 ). According to an example embodiment, a probe based on a CRSP sequence, for example, a wild-type CRSP sequence, hybridized with a cDNA or other DNA product of a test cell (s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.

Alterations in electrophoretic mobility can be used to identify mutations in CRSP genes. For example, single chain conformation polymorphism (SSCP) can be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86: 2766, see also Cotton (1993) Mutat Res 285: 125-144; and Hayashi (1992) Genet Anal Tech Appl 9: 7379). Single-stranded DNA fragments of sample and control CRSP nucleic acids will be denatured and their renaturation allowed. The secondary structure of single chain nucleic acids varies according to the sequence, the resulting alteration in electrophoretic mobility allows the detection of even a single base change. DNA fragments can be labeled or detected with labeled probes. The sensitivity of the assay can be increased using RNA (instead of DNA), in which the secondary structure is more sensitive to a change in sequence. The object method may use heteroduplex analysis to separate double chain heteroduplex molecules based on electrophoretic mobility changes (Keen et al. (1991) Trends Genet 7: 5).

The movement of mutant or wild-type fragments in polyacrylamide gels containing a denaturing agent gradient can be tested using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313: 495). When DGGE is used as the method of analysis, the DNA will be modified to ensure that it is not completely denatured, for example by adding a GC binding structure ("GC clamp") of approximately 40 bp of high-point GC-rich DNA. PCR fusion. In a further embodiment, a temperature gradient is used instead of a denaturation gradient to identify differences in sample and control DNA mobility (Rosenbaum and Reissner (1987) Biophys Chem 265: 12753).

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers can be prepared in which the known mutation is placed in the center and then hybridized with target DNA under conditions that allow hybridization only if perfect pairing is found (Saiki et al. (1986) Nature 324: 163) ; Saiki et al. (1989) Proc. Natl Acad. Sci USA 86: 6230). Such allele-specific oligonucleotides hybridize with PCR amplified target DNA or several different mutations when the oligonucleotides are bound to the hybridization membrane and hybridized with the labeled target DNA.

Alternatively, together with the present invention, allele specific amplification technology that depends on selective PCR amplification can be used. Oligonucleotides used as primers for specific amplification can carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17: 2437-2448 ) or at the 3 'terminal end of a primer when, under appropriate conditions, mismatch can prevent, or reduce the extent of the polymerase (Prossner (1993) Tibtech 11: 238). In addition, it may be desirable to introduce a novel restriction site in the mutation region to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6: 1). It is envisioned that in certain cases amplification can also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88: 189). In such cases, linkage will only occur only if there is perfect pairing at the 3 ’end of the 5’ sequence making it possible to detect the presence of a known mutation at a specific site looking for the presence or absence of amplification.

The methods described herein can be carried out, for example, using pre-packaged diagnostic kits comprising at least one antibody reagent or probe nucleic acid described herein, which can be conveniently used, for example, in the clinical practice to diagnose patients who show symptoms or family history of a disease or condition that involves a CRSP gene.

In addition, any type of cell or tissue in which CRSP is expressed can be used in the prognostic tests described herein.

3. Effects monitoring during clinical trials

The monitoring of the influence of agents (for example, drugs, compounds) on the expression or activity of CRSP (for example, modulation of cell signal transduction, regulation of gene transcription in a cell involved in development or differentiation, regulation of cell proliferation) can be applied not only in the selection of basic drug, but also in clinical trials. For example, the efficacy of an agent determined by a screening assay, as described herein, to increase CRSP gene expression, protein levels, or to regulate by increasing CRSP activity, can be monitored. in clinical trials of subjects showing decreased CRSP gene expression, protein levels or decreased CRSP activity. Alternatively, the efficacy of an agent determined by a screening assay to decrease CRSP gene expression, protein levels, or regulate by decreasing CRSP activity, can be monitored in clinical trials of subjects showing increased expression of the CRSP gene, protein levels, or increased CRSP activity regulated. In such clinical trials, the expression or activity of CRSP and, preferably, of other genes that have been implicated in, for example, a proliferative disorder, can be used as a "reader" or markers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including CRSP, that are modulated in cells can be identified by treatment with an agent (e.g., compound, drug or small molecule) that modulates CRSP activity (e.g., identified in a screening test as described herein). Therefore, to study the effect of agents on proliferative disorders, developmental or differentiation disorders, hematopoietic disorders as well as disorders characterized by abnormal cell survival and / or differentiation, an abnormal extracellular structure, or an abnormality in a defense mechanism, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed to detect levels of CRSP expression and other genes involved in proliferative disorder, developmental or differentiation disorder, hematopoietic disorder as well as disorders characterized by survival and / or abnormal cell differentiation, an abnormal extracellular structure or an abnormality in a defense mechanism, respectively. Gene expression levels (i.e., a gene expression pattern) can be quantified by Northern or RT-PCR transfer analysis, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring activity levels of CRSP or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells against the agent. Therefore, this response state can be determined before, and at different points during the individual's treatment with the agent.

A method for monitoring the efficacy of treating a subject with an agent (for example, an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule or other drug candidate identified by the screening assays described is disclosed. herein) comprising the steps of (i) obtaining a sample before administration of a subject before administration of the agent, (ii) detecting the level of expression of a genomic DNA, mRNA or CRSP protein in the sample before administration; (iii) obtain one or more samples after administration of the subject; (iv) detect the level of expression or activity of genomic DNA, mRNA or CRSP protein, in post-administration samples; (v) comparing the level of expression or activity of genomic DNA, mRNA or CRSP protein, in the sample before administration with genomic DNA, mRNA or CRSP protein in the sample or samples after administration; and (vi) modify the administration of the agent to the subject accordingly. For example, increasing the administration of the agent may be desirable to increase the expression or activity of CRSP to levels greater than those detected, that is, to increase the effectiveness of the agent. Alternatively, a decrease in the administration of the agent to decrease the expression or activity of CRSP to levels lower than those detected, that is to decrease the effectiveness of the agent, may be desirable. According to such an embodiment, the expression or activity of CRSP can be used as an indicator of the efficacy of an agent, even in the absence of an observable phenotype response.

C. Treatment:

The present invention provides both prophylactic and therapeutic methods for treating a subject at risk of (or prone to) suffering from a disorder or having a disorder associated with aberrant CRSP activity or expression with a composition according to claim 1. In relation to the treatment both prophylactic and therapeutic, such treatments can be specifically designed or modified, based on knowledge obtained from the pharmacogenomic field. "Pharmacogenomics," as used herein, refers to the application of genomic technologies such as gene sequencing, statistical genetics and gene expression analysis for drugs in clinical development and in the market. More specifically, the term refers to the study of how a patient's genes determine their response to a drug (for example, the "drug response phenotype," or "drug response genotype" of a patient). Therefore, methods for designing a therapeutic or prophylactic treatment of an individual with any of the CRSP molecules, or with CRSP modulators according to the drug response genotype of that individual are disclosed. Pharmacogenomics allows an internist or doctor to direct prophylactic or therapeutic treatments to patients who will benefit most from the treatment and to avoid treating patients who will experience side effects related to toxic drugs.

1. Prophylactic methods

In one aspect, a method is disclosed for avoiding in a subject, a disease or condition associated with an aberrant CRSP activity or expression, by administering to the subject an agent that modulates the expression of CRSP or at least one activity of CRSP Subjects who are at risk of suffering from a disease that is caused by or that contributes to the activity or expression of aberrant CRSP can be identified by, for example, any or a combination of diagnostic or prognostic tests as described herein. . Administration of a prophylactic agent may occur before the manifestation of the characteristic symptoms of CRSP aberration, so as to avoid a disease or disorder or, alternatively, delay its progression. Depending on the type of CRSP aberration, for example, a CRSP agonist or CRSP antagonist agent can be used to treat the subject. The appropriate agent can be determined based on screening assays described herein. Prophylactic methods are discussed in more detail in the following subsections.

2. Therapeutic methods

Another aspect of the invention relates to methods for modulating the activity or expression of CRSP for therapeutic purposes. The modulator method involves contacting a cell with an agent that modulates one or more of the activities of the activity of the CRSP protein associated with the cell. An agent that modulates the activity of the CRSP protein may be an agent as described herein, such as a nucleic acid or a protein, a naturally occurring target molecule of a CRSP protein, a peptide, a CRSP peptidomimetic, or other small molecule. The agent can stimulate one or more CRSP protein activities. Examples of such stimulants include CRSP protein and a nucleic acid molecule encoding CRSP that has been introduced into the cell. The agent may inhibit one or more activities of the CRSP protein. Examples of such inhibitory agents include antisense CRSP nucleic acid molecules and anti-CRSP antibodies. These modulating methods can be performed in vitro (for example, by culturing the cell with the agent) or, alternatively, in vivo, (for example, by administering the agent to a subject). As such, methods for treating an individual afflicted with a disease or disorder characterized by aberrant CRSP activity or expression of a CRSP protein or nucleic acid molecule are disclosed. The method may involve the administration of an agent (for example, an agent identified by a screening assay described herein), or combination of modulating agents (for example, regulate by increase or regulate by decrease) activity or activity. CRSP expression. The method may involve the administration of a CRSP protein or a nucleic acid molecule as a treatment to compensate for the activity or expression of aberrant or reduced CRSP.

Stimulation of CRSP activity is desirable in situations where CRSP is abnormally reduced by decrease and / or in which the increase in CRSP activity is likely to have a beneficial effect. Similarly, inhibition of CRSP activity is desirable in situations where CRSP is abnormally regulated by increase and / or where the decrease in CRSP activity is likely to have beneficial effects. An example of such a situation is when a subject has a disorder characterized by cellular differentiation or aberrant development. Another example of such a situation is when the subject has a proliferative disease (for example, cancer) or a disorder characterized by an aberrant hematopoietic response. Yet another example of such a situation is when it is desirable to achieve tissue regeneration in a subject (for example, when a subject has experienced brain or spinal cord damage and it is desirable to regenerate neuronal tissue in a regulated manner).

Therefore, the disease can be a disease characterized by abnormal proliferation, differentiation, and / or cell survival. For example, the disease can be a hyper or hypoproliferative disease. A treatment is disclosed for diseases characterized by abnormal proliferation, differentiation, and / or cell survival in a subject, which are not characterized by abnormal CRSP activity (eg, CRSP-1 activity). In fact, since it is likely that CRSP can modulate the proliferative state of a cell (i.e., proliferation, differentiation, and / or survival status of a cell), CRSP can regulate a disease in which the proliferative state of a cell results from a defect other than the anomalous CRSP activity.

Hyperproliferative diseases can be treated with therapeutic agents of CRSP (for example, CRSP-1) including neoplastic and hyperplastic diseases, such as various forms of cancer and leukemia and fibroproliferative disorders. Other hyperproliferative diseases that can be treated or avoided with the subject CRSP therapeutic agents (for example CRSP-1 therapeutic agents) include cancerous states, precancerous states and benign states. The condition to be treated or avoided may be a solid tumor, such as a tumor that arises in an epithelial tissue. Accordingly, the treatment of such cancer could comprise administration to the subject of a CRSP therapeutic agent that decreases the interaction of CRSP with a CRSP receptor. Other cancers that can be treated or avoided with a CRSP protein include sarcomas and carcinomas, for example, lung cancer, colon cancer, prostate cancer, breast, ovarian, esophagus, lung, melanoma, seminoma and squamous cell adenocarcinoma. Other solid tumors include those that can be found in a medical textbook.

The condition to be treated or avoided may also be a soluble tumor, such as leukemia, either chronic or acute, including chronic or acute myelogenous leukemia, chronic or acute lymphocytic leukemia, promyelocytic leukemia, monocytic leukemia, myelomonocytic leukemia, and erythrolecucemia . Still other proliferative disorders that can be treated with a CRSP therapeutic agent of the invention include heavy chain disease, multiple myeloma, lymphoma, for example, Hodgkin lymphoma and non-Hodgkin lymphoma and Waldenstroem macroglobulemia.

Diseases or conditions characterized by a solid or soluble tumor can be treated by administering a therapeutic agent of CRSP either locally or systematically, so that aberrant cell proliferation is inhibited or decreased. Methods for administering the composition of the invention are described in detail below.

The invention also provides the composition according to claim 1 to prevent the formation and / or development of tumors. For example, the development of a tumor can be anticipated by the presence of a specific lesion, such as a preneoplastic lesion, for example, hyperplasia, metaplasia, and dysplasia, which can be detected, for example, by cytological methods. Such injuries can be found; for example, in epithelial tissue. The inhibition of the progression of such a lesion in a neoplastic lesion, comprising administration to the subject having a preneoplastic lesion of an amount of CRSP-1 therapeutic agent sufficient to inhibit the progression of the preneoplastic lesion in a lesion is disclosed. neoplastic

The invention also provides the treatment or prevention of diseases or conditions in which cell proliferation is desired. For example, CRSP therapeutic agents can be used to stimulate tissue repair or wound healing, such as after surgery or to stimulate tissue healing of burns. Other diseases in which cell proliferation is desirable are hypoproliferative diseases, that is, diseases characterized by abnormally low proliferation of certain cells.

In yet another embodiment, the invention provides the treatment or prevention of diseases or conditions characterized by aberrant cell differentiation. Accordingly, the invention provides stimulation of cell differentiation in conditions characterized by an inhibition of normal cell differentiation that may or may not be accompanied by excessive proliferation. Alternatively, CRSP therapeutic agents can be used to inhibit the differentiation of specific cells.

In a preferred method, the cell that proliferates and / or differs in an aberrant manner is a cell present in the nervous system. A role for CRSP in the nervous system is suggested at least in part from the fact that human CRSP-1 is expressed in human fetal brain. Accordingly, the invention provides the treatment of diseases or conditions associated with the central or peripheral nervous system. For example, the invention provides the treatment of lesions of the nervous system associated with an aberrant proliferation, differentiation or survival of any of the following cells: neurons, Schwann cells, glial cells and other types of neural cells. Nervous system disorders include, but are not limited to: spinal cord damage, brain damage, injuries associated with surgery, ischemic lesions, cancerous lesions, infectious lesions, degenerative lesions (Parkinson's disease, Alzheimer's disease, Huntington's chorea , amyotrophic lateral sclerosis), demyelinating diseases (multiple sclerosis, myelopathy associated with human immunodeficiency, transverse myelopathy, progressive multifocal leukoencephalopathy, pontine myelinolysis), motor neuron damage, progressive spinal muscular atrophy, progressive bulbar paralysis, primary lateral sclerosis, childhood muscular atrophy and juvenile, progressive child bulbar paralysis (Fazio-Londe syndrome), poliomyelitis, and hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease).

In another embodiment, the invention provides for the enhancement of survival and / or stimulation of proliferation and / or differentiation of cells and tissues in vitro. In a preferred embodiment, the therapeutic agents of CRSP according to claim 1 are used to stimulate tissue regeneration and / or repair (for example, to treat nerve injury). For example, tissues of a subject can be obtained and grown in vitro in the presence of a therapeutic agent of CRSP, so that the tissue cells stimulate to proliferate and / or differentiate. Then the tissue can be re-administered to the subject.

Among the approaches that can be used to alleviate the symptoms that involve aberrant CRSP activity and / or abnormal cell proliferation, differentiation, and / or survival, are, for example, the antisense, ribozyme and triple helix molecules described above. . Examples of suitable compounds include the antagonists, agonists or homologs described in detail above.

Still other CRSP therapeutic agents disclosed herein consist of a first peptide comprising a CRSP peptide that can bind to a CRSP receptor, and a second peptide that is cytotoxic. Such therapeutic agents can be used to specifically target and lyse cells that express or overexpress a receptor for CRSP.

3. Pharmacogenomics

CRSP molecules, as well as agents, or modulators that have a stimulating or inhibiting effect on CRSP activity (eg, expression of the CRSP gene) as identified by a screening assay described herein, may administered to individuals to treat (prophylactically or therapeutically) disorders (eg, proliferative or developmental disorders) associated with aberrant CRSP activity. Along with such treatment, pharmacogenomics can be considered (i.e., the study of the relationship between the genotype of an individual and the response of this individual to a foreign compound or drug). Differences in the metabolism of therapeutic agents may result in severe toxicity or therapeutic failure by altering the relationship between the dose and the blood concentration of the pharmacologically active drug. Therefore, a doctor or internist may consider applying the knowledge obtained in relevant pharmacogenomic studies to determine whether or not to administer a CRSP molecule or CRSP modulator, as well as to design the dosage and / or therapeutic regimen of treatment with a CRSP molecule. or CRSP modulator.

Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected people. See for example, Eichelbaum, M., Clin Exp Pharmacol Physiol, 1996, 23 (10-11): 983-985 and Linder, M.W., Clin Chem, 1997, 43 (2): 254266. In general, two types of pharmacogenetic states can be distinguished. Genetic states transmitted as a single factor that alters the way in which drugs act in the body (altered drug action) or transmitted genetic states as unique factors that alter the way the body acts on drugs (altered drug metabolism) . These pharmacogenetic states can occur either as rare genetic defects or as naturally occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common hereditary enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidizing drugs (antimalarials, sulfonamides, analgesics, nitrofurans), and consumption of broad beans.

A pharmacogenomic approach to identify genes that predict the response to the drug, known as a "broad genome association", is based primarily on a high resolution map of the human genome consisting of markers related to known genes (for example, a "biallelic" gene marker map consisting of 60,000-100,000 polymorphic sites or variables of the human genome, each of which has two variants). Such a high-resolution genetic map can be compared with a genome map of each of a statistically significant number of patients taking part in a phase II / III drug trial to identify markers associated with a particular observed drug response or side effects. . Alternatively, such a high resolution map can be generated from a combination of some of the ten million single nucleotide polymorphisms (SNPs) known in the human genome. As used herein, an "SNP" is a common alteration that occurs on a single nucleotide base in a DNA extension. For example, an SNP can be produced once every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority will not be associated with disease. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular SNP pattern in their individual genome. Thus, treatment regimens can be designed, for groups of genetically similar individuals, taking into account characteristics that may be common among such genetically similar individuals.

Alternatively, a method called the "candidate gene approach" can be used to identify genes that predict drug responses. According to this method, if a gene encoding a drug target is known (for example, a CRSP protein or CRSP receptor of the present invention), all common variants of that gene can be fairly easily identified in the population and can be determined If having one version of the gene versus another is associated with a response to the particular drug.

As an illustrative embodiment, the activity of enzymes that metabolize the drug is a major determinant of both the intensity and duration of the drug's action. The discovery of the genetic polymorphisms of the enzymes that metabolize the drug (for example, N-acetyltransferase 2 (NAT 2) and CYP2D6 and CYP2C 19 enzymes of cytochrome P450) has provided an explanation of why some patients do not obtain the effects of the drug expected or show exaggerated drug response and severe toxicity after taking the usual and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the rapid metabolizer (MS) and the slow metabolizer (PM, pour metabolizer). The prevalence of PM is different among different populations. For example, the gene encoding CYP2D6 is highly polymorphic and several mutations in PM have been identified, all of which result in the absence of functional CYP2D6. The slow metabolizers of CYP2D6 and CYP2C19 quite often experience side effects and exaggerated drug response when they receive the usual doses. If a metabolite is the active therapeutic moiety, the PM show no therapeutic response, as demonstrated by the analgesic effect of codeine mediated by its morphine metabolite formed by CYP2D6. On the other end are the so-called ultrafast metabolizers that do not respond to the usual doses. Recently, it has been identified that the molecular basis of ultrafast metabolism is due to the amplification of the CYP2D6 gene.

Alternatively, a method called "gene expression profile elaboration" can be used to identify genes that predict the response to the drug. For example, gene expression of an animal to which a drug is dosed (for example, a CRSP molecule or CRSP modulator of the present invention) can give an indication of whether or not toxicity-related gene pathways have been activated. .

The information generated from more than one of the above pharmacogenomic approaches can be used to determine the dosage and treatment regimens suitable for the prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosage or drug selection, can prevent adverse reactions or therapeutic failures and therefore, enhance therapeutic or prophylactic efficacy when treating a subject with a CRSP molecule or a CRSP modulator, such as a modulator identified by one of the exemplary selection tests described herein.

This invention is further illustrated by the following examples that should not be considered as limiting.

EXAMPLES

Example 1: Isolation and characterization of human CRSP-1 cDNA

In this example, the isolation and characterization of the gene encoding CRSP-1 (also called "CRISPY-1" or "TANGO 59") is described.

Isolation of human CRSP-1 cDNA

The invention is based at least in part on the discovery of a human gene encoding a segregated protein, referred to herein as cysteine-1-rich segregated protein (CRSP-1). A partial cDNA was isolated using a method to trap the signal sequence. This methodology takes advantage of the fact that molecules such as CRSP have an amino terminal signal sequence that directs certain secreted and membrane bound proteins through the cellular secretory apparatus.

In summary, a random cDNA library was prepared using mRNA prepared from human fetal brain tissue (Clontech, Palo Alto CA) using the Stratagene-ZAP-cDNA Synthesis ™ kit (catalog number 20041). The cDNA in the pTrap mammalian expression vector adjacent to a cDNA encoding placental alkaline phosphatase lacks a secretory signal. Plasmids were transformed into E. coli and DNA was prepared using the Wizard ™ DNA purification kit (Promega). DNA was transfected into COS-7 cells with Lipofectamine ™ (Gibco-BRL). After a 48-hour incubation, supernatants from COS cells were tested for alkaline phosphatase in a Wallac Micro-Beta scintillation counter using the Phospha-Light ™ kit (Tropix Inc., catalog number BP300). Individual plasmid DNAs that score positive in the COS cell alkaline phosphatase secretion assay were further analyzed by DNA sequencing using standard procedures.

Using a partial cDNA isolated by the method described above, a full length cDNA encoding human CRSP-1 was cloned. The nucleotide sequence encoding the full-length human CRSP-1 protein is shown in Figure 1 and is set forth as SEQ ID NO: 1. The full length protein encoded by this nucleic acid is comprised of approximately 350 amino acids and has The amino acid sequence shown in Figure 1 and set forth as SEQ ID NO: 2. The coding part (open reading frame) of SEQ ID NO: 1 is set forth as SEQ ID NO: 3. The DNA for clone jthKb075a10 was deposited. in the ATCC with the registration number 98634.

Analysis of human CRSP-1

The determination of the hydrophobic profile of human CRSP-1 having the amino acid sequence set forth in SEQ ID NO: 2 indicates the presence of a hydrophobic region from about amino acid 1 to about amino acid 23 of SEQ ID NO: 2. The analysis Additional amino acid sequence SEQ ID NO: 2 using signal peptide prediction programs predicted the presence of a signal peptide from about amino acid 1 to about amino acid 19, 21, or 23 of SEQ ID NO: 2. Accordingly , the mature CRSP-1 protein includes about 327, 329, or 331 amino acids ranging from about amino acid 20, 22, or 24 to about amino acid 350 of SEQ ID NO: 2. The presence of the signal sequence, in addition to the The fact that CRSP-1 was identified using a signal sequence trapping system indicates that CRSP-1 is a secreted protein. Moreover, the prediction of such a signal peptide and such a signal peptide cleavage site can be performed, for example, using the SIGNALP computer algorithm (Henrik, et al. (1997) Protein Engineering 10: 1-6).

In addition, when cDNA encoding human CRSP-1 was expressed in bacterial cells with a FLAG C-terminal marker, the FLAG-labeled CRSP-1 protein product was found to be secreted into the cellular medium.

Examination of the cDNA sequence depicted in Figure 1 shows that human CRSP-1 is particularly rich in cysteine residues. As shown in Figure 1, CRSP-1 contains 20 cysteine residues located between amino acid 147 and amino acid 284 of SEQ ID NO: 2. These cysteine residues can possibly form 10 disulfide bridges.

A BLAST search (Altschul et al. (1990) J. Mol. Biol. 215: 403) of the nucleotide and amino acid sequences of CRSP-1 has revealed that CRSP-1 is significantly similar to chicken cDNA encoding a protein of unknown function, GenBank registration number D26311. This cDNA was isolated from a chicken lens cDNA library and showed that it was expressed in crystalline fibers and lens epithelium, but not in the neural retina or liver cells. (Sawada et al. (1996) Int. J. Dev. Biol. 40: 531). CRSP-1 and chicken protein have an amino acid sequence identity of 56% and an amino acid sequence similarity of 72%. The amino acid sequence similarity between chicken protein and human CRSP-1 is particularly high in the cysteine-rich domain of CRSP-1 that is located between amino acids 147 and 284 of SEQ ID NO: 2. In particular, the 20 CRSP-1 cysteine residues located in this region are present in chicken protein (see Figure 3).

Two genes recently identified in a test to detect suppressors of glioblastoma formation (Ligon et al., 1997 Oncogene 14 1075-1081) also show significant homology with hCRSP-1. These genes, RIG (“Regulated in Glioblastoma”) and 7-1 of RIG type (GenBank registration numbers U32331 and AF034208, respectively), were identified in a differential test to detect mRNA regulated by the introduction of a normal copy of the chromosome 10 in a glioblastoma cell line that houses a deletion on chromosome 10 that stimulates tumorigenesis. A schematic diagram is presented that summarizes the relationship between the sequences of the CRSP-1 and RIG genes as in Figure 9. The indicated region of identity between CRSP-1, RIG and 7-1 of the RIG type comprises a region of the UTR in 3 'of human CRSP-1 mRNA. Initially this cDNA was isolated in the differential test described and subsequently used to isolate RIG and 7-1 full-length RIG messengers as described. RIG type 7-1 is extremely homologous (96.8% similar as determined using the Lipman-Pearson protein alignment program, Ktuple (Tupla K): 2; Gap penalty (gap penalty): 4, Gap lenght penalty (gap length penalty): 12; and 64.7% homologue using the DNA alignment program Wilbur Lipman, Ktuple: 3; Gap penalty: 3; Window (interval): 20) to CRSP- 1, although the encoded protein lacks the N-terminal signal sequence and therefore, it is not expected to be a secreted protein. The full-length RIG mRNA only exhibits homology with CRSP-1 in the region of the initial cDNA. These data associate CRSP-1 with human glioblastoma and suggest that CRSP-1 may be important in suppressing the tumor phenotype. A role in glioblastoma is also consistent with the high level of CRSP-1 mRNA expression observed in human brain tissue. In addition, the co-localization of the CRSP-1, RIG and RIG type genes in a region of chromosome 11 (11p15.1) involved in the development of human malignant astrocytoma (Ligon et al. 1997 Oncogene 14 1075-1081) additionally indicates a role for these genes in tumorigenesis.

The human CRSP-1 protein also has some similarity of amino acid sequence with metallothionein, particularly in the cysteine-rich domain.

CRSP-1 mRNA tissue distribution

This example describes the tissue distribution of mRNA of CRSP-1, as determined by Northern blot hybridization.

Northern blot hybridizations with the various RNA samples were performed under usual conditions and washed under stringent conditions, i.e. 0.2 x SSC at 65 ° C. In each sample, the probe was hybridized with a single RNA of approximately 2.5 kb. The results of hybridization of the probe with various mRNA samples are described below.

Hybridization of a Fetal Multiple-tissue Northern (MTN) transfer from Clontech (Clontech, LaJolla, CA) containing kidney, liver, lung and fetal brain RNA indicated the presence of high levels of CRSP-1 mRNA in lung and fetal brain and slightly lower levels of GRSP-1 mRNA in fetal kidney. However, no significant level of CRSP-1 mRNA was found in the fetal liver.

Hybridization of a human-type Northern tissue transfer (MTN) from Clontech (Clontech, LaJolla, CA) containing RNA from the pancreas, kidney, skeletal muscle, liver, lung, placenta, brain and adult heart with a CRSP probe -1 human indicated the presence of high levels of CRSP-1 mRNA in the heart, slightly lower levels in the brain and much lower levels in the placenta and lung. Some mRNA of CRSP-1 was also found in adult skeletal muscle. However, no significant levels of CRSP-1 mRNA were observed in the adult pancreas, kidney or liver. Interestingly, the chicken gene that was homologous to CRSP-1 was not expressed at detectable levels in any of the livers (Sawada et al. (1996) Int. J. Dev. Biol. 40: 531).

Additional hybridization of a human-type Northern tissue-multiple (MTN) transfer from Clontech (Clontech, LaJolla, CA) that includes adult spinal cord RNA revealed elevated CRSP-1 expression levels in isolated adult spinal cord mRNA.

An in situ analysis further revealed the following expression patterns when hybridizing tissue sections with CRSP-1 probes. CRSP-1 mRNA was expressed in the heart, lung, aorta, eye (retina and lens) and brain (cortex) in 14.5-day mouse embryos. CRSP-1 mRNA was expressed in the heart, lung, eye (retina and lens), brain (cortical), and cartilage (foot, trachea, larynx, head, sternum and spinal cord) in tissues of 1-post-natal mice ,5 days. CRSP-1 mRNA was expressed in the brain (cortex, hippocampus, brainstem), heart (atrium only), eye (retina and outer lens layer) in adult mouse tissues.

Therefore, CRSP-1 is specifically expressed in tissue, with the highest expression in the brain, heart and spinal cord.

CRSP-1 protein expression data

Constructs

Expression constructs were prepared for two forms of CRSP-1 using the mammalian pMET-stop expression vector. Form 1 comprised a cDNA incorporating the 350 aa CRSP-1 protein coding sequence (CRSP-1 long flag) and form 2 comprised the complete CRSP-1 coding sequence except for the last 18 amino acids (CRSP-1 flag.corta). A C-terminal sequence encoding the FLAG epitope (DYKDDDDK) was added to both forms of CRSP-1 to facilitate detection and purification. CRSP1flag cDNAs were generated by PCR from a full length CRSP-1 cDNA template and ligated to pMET-stop using EcoR1 and Sal1 restriction sites.

Test transfection - small-scale expression

Expression constructs were transfected for CRSP-1flag.larga and CRSP-1flag.corta in 293T cells using 10 µl of lipofectamine (GIBCO / BRL) and 2 µg of DNA per well of a 6-well plate of confluent cells 70-80% After 5 hours at 37 ° C, the cells were fed with 1 ml of 20% FCS / DMEM. After overnight incubation at 37 ° C, the cells were conditioned in 1 ml of OptiMEM for 48 hours at 37 ° C. Samples of supernatant and cell pellets were solubilized in boiling SDS-PAGE gel buffer, eluted on a 4-20% SDS-PAGE gel, transferred to a nylon membrane and screened with the anti-FLAG antibody M2 monoclonal. Samples from both the supernatant and sediment showed significant immunoreactivity in a molecular weight range of 40-65 kDa on autoradiographic film using a HRP conjugated secondary antibody and ECL detection reagents. Therefore, both forms of CRSP-1 under test are secreted from 293T cells thus experimentally confirming that CRSP-1 is a secreted protein. It should be noted that the molecular weights of both forms of CRSP-1 tested are higher than expected from the amino acid sequence, suggesting that CRSP-1 proteins secreted by 293T cells can be glycosylated. This is consistent with the presence of four potential sites for N-linked glycosylation in the CRSP-1 protein.

Large-scale CRSP-1 protein production

For large-scale CRSP-1flag expression, 293T cells with a 70-80% confluence with 27 µg of DNA, 100 µl of lipofectamine in 18 ml of OptiMEM were transfected at 30 x 150 mM for 5 hours at 37 ° C. 18 ml of 10% FCS / DMEM was added to each plate and incubated overnight at 37 ° C. Twenty-four hours after the start of transfection, the transfection supernatant was aspirated and 35 ml of OptiMEM was added to each plate and the plates were incubated at 37 ° C for 72 hours. The conditioned medium was collected, centrifuged at 4000 rpm for 30 min at 4 ° C, and filtered through a 0.45 micron filter unit. 1100 ml were passed through a 1.6 x 10 cm anti-FLAG M2 affinity column prebalanced in PBS buffer, pH 7.4 at a flow rate of 2.0 ml per minute. After washing with 200 ml of PBS buffer pH 7.4, the bound material was eluted by a step of 200 mM Glycine buffer, pH 3.0 and 0.5 ml fractions were collected. After elution, a significant protein peak was detected by absorbance at 280 nm. The samples corresponding to the conditioned medium, the flow-through and eluted fractions were analyzed by SDS-PAGE stained with Coomassie blue and blue and by Western blot analysis as described above. Significant immunoreactivity was detected in a molecular weight range of 40-65 kDa in conditioned medium and in eluted fractions but not in the through-flow sample, indicating that the CRSP-1 segregated flag.large protein bound specifically to the affinity column and was eluted effectively by the conditions described. Staining of SDS-PAGE gels with Coomassie blue suggested that the predominant immunoreactive protein constituted> 90% of the protein present in the bound and eluted protein peak. The peak fractions of the eluted protein were pooled and dialyzed against phosphate buffered saline resulting in a volume of 4 ml of CRSP-1 flag long protein at a concentration of approximately 1 mg / ml.

Example 2: Isolation and characterization of cDNA from human CRSP-2, CRSP-3, and CRSP-4

To identify novel proteins related to CRSP-1, the amino acid sequence of human CRSP-1 was used to search the private and dbEST databases using TBLASTN (WashUversion, 2.0, BLOSUM62 search matrix). From this search, four different partial protein sequences were identified that showed significant homology with human CRSP-1. These sequences were designed as partial sequences for the novel CRSP-h; CRSP-2-h, CRSP-3-h, CRSP-4-h and N-type terminal h-CRSP.

The hCRSP-2 partial protein sequence was derived from a single dbEST clone with registration number AA565546. This clone was subsequently obtained from the IMAGE collection and fully sequenced to define the complete hCRSP-2 sequence depicted in Figure 2.

The hCRSP-3 partial protein sequence was derived from a clone of jthKb075a10 with a sequence identification code jthKb075a10. This clone was further sequenced and this sequence information was combined with additional dbEST sequences using the Phrap meeting program (P. Green, U. Washington) to define the complete hCRSP-3 sequence depicted in Figure 3. The DNA for the clone of jthKb075a10 in the

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ATCC with registration number 98633. Northern blot analysis of various tissues using a hCRSP-3 specific probe revealed that CRSP-3 mRNA was limited to the placenta. CRSP-3 mRNA expression has not yet been demonstrated in other normal adult human tissues.

Recently a murine and Xenopus homologue of CRSP-3 called dkk-1 ("dickkopf", German, hardhead) has been described which is proposed to code for a segregated signaling molecule (Nature, 391, 357-368). Dkk-1 is described as a potent inducer of head formation during the early development of Xenopus, its mechanism of action apparently assuming the inhibition of the wnt family of segregated factors.

Given the homology between CRSP-3 and dkk-1, it is suggested that hCRSP-3 may exhibit profound head induction activity during early human embryonic development. In addition, it is thought that the mechanisms of action of dkk-1 involve antagonism of members of the wnt family of secreted proteins. Wnt protein overexpression has been associated with certain types of cancer. For example, activation of the expression of wnt-1 by proviral MMTV insertion causes breast cancer in mice (see Cadigan and Nusse, Genes and Dev., 1997 11 3286-3305). Therefore, at least CRSP-3, and potentially other members of the CRSP family, may also have a role in suppressing wnt signaling in cancer.

The partial protein sequence CRSP-4-h was derived from a single dbEST clone with registration number W55979. This clone from the IMAGE collection was subsequently obtained and fully sequenced to define a CRSP-4-h sequence shown in Figure 4. Northern blot analysis of various tissues using a specific probe for CRSP-4-h revealed that CRSP-4 mRNA was expressed in various adult human tissues. CRSP-4 mRNA expression was superior in the heart, brain, placenta, lung and skeletal muscle.

The human CRSP N-terminal partial protein sequence was derived from a single dbEST clone with registration number AA397836. This clone was subsequently obtained from the IMAGE collection and fully sequenced to define the N-terminal sequence of the CRSP type shown in Figure 5.

Structure of the CRSP family proteins

An alignment of the amino acid sequences of human CRSP-1, human CRSP-2, human CRSP-3 and human CRSP-4 is shown in Figure 6. The amino acid residues that are conserved among members of the CRSP family are boxed. The 20 conserved cysteine residues are indicated by an asterisk. Cysteine-rich domains are indicated as CRD-1 and CRD-2. The domain structure of the full length CRSP proteins of the present invention is shown in Figure 7. The homology of amino acids and nucleotides between members of the CRSP family is as follows in Tables 1 and 2.

Table 1

CRSP-1

CRSP-2 CRSP-3 CRSP-4 mdkk-1 xdkk-1 CLFEST

CRSP-1

100 16.0  18.6  15.1  18.5 16.5  53.0

CRSP-2

100 33.7  35.2 32.6  33.7 16.2

CRSP-3

100 33.1  80.2 53.5  17.4

CRSP-4

100 30.5 33.7 12.5

The amino acid homology percentages were determined using the ALIGN program, (version 2.0) (GCG software package) using a residue table in weight PAM120, gap lenght penalty (gap length penalty) of 12, and gap penalty (penalty of hole) of 4.

Table 2

CRSP-1

CRSP-2 CRSP-3 CRSP-4 mdkk-1 xdkk-1 CLFEST

CRSP-1

100 30.0  37.2  34.7  31.5 45.4  58.8

CRSP-2

100 43.0  35.9 38.8  38.4 36.7

CRSP-3

100 59.3  66.4 53.7  32.1

CRSP-4

100 38.8 38.4 36.7

Nucleotide homology percentages were determined using the Wilbur Lipman DNA alignment program, Ktuple: 3; Gap Penalty (gap penalty): 3; Window (interval): 20. LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: MILLENNIUM BIOTHERAPEUTICS, INC. (B) STREET: 620 MEMORY DRIVE (C) CITY: CAMBRIDGE (D) STATE: MASSACHUSETTS (E) COUNTRY: USA (F) ZIP CODE: 02319 (G) PHONE:

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(B) FAX: (617)742-4214

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 2479 base pairs

15 (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

20 (A) NAME / KEY: CDS

(B) LOCATION: 38..1087

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

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(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 350 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULA: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

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(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 1050 base pairs

5 (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

(ii)

TYPE OF MOLECULA: cDNA

(ix)

 CHARACTERISTIC:

10 (A) NAME / KEY: CDS (B) LOCATION: 1..1050 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

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(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 848 base pairs

5 (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA (ix) FEATURE:

10 (A) NAME / KEY: CDS (B) LOCATION: 125..796 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

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(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 224 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

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(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 672 base pairs

5 (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

10 (A) NAME / KEY: CDS

  (B) LOCATION: 1,672

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

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(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1529 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULA: cDNA

(ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 93..890

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

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(2) INFORMATION FOR SEQ. ID NO: 8: (i) SEQUENCE CHARACTERISTICS

(TO)

 LENGTH: 266 amino acids

(B)

 TYPE: amino acids

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

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(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 798 base pairs

5 (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

10 (A) NAME / KEY: CDS (B) LOCATION: 1.988 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

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(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 702 base pairs

5 (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

10 (A) NAME / KEY: CDS (B) LOCATION: 1.537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

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(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS

(TO)

 LENGTH: 179 amino acids

(B)

 TYPE: amino acid

(D)

 TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

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(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 537 base pairs 5 (B) TYPE: nucleic acid

(C) CHAIN TYPE: simple

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) CHARACTERISTICS:

10 (A) NAME / KEY: CDS

(B) LOCATION: 1.537

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

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(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: 5 (A) LENGTH: 928 base pairs

(B)

TYPE: nucleic acid

(C)

 CHAIN TYPE: simple

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

10 (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 75..800

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

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(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 242 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

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(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 726 base pairs

5 (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

10 (A) NAME / KEY: CDS (B) LOCATION: 1.726 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

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(2) INFORMATION FOR SEQ. ID NO: 16:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2381 base pairs

70

  (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA

 (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 110..1156

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

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(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS (A) LENGTH: 349 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

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(2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1047 base pairs

5 (B) TYPE: nucleic acid (C) CHAIN TYPE: simple  (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

10 (A) NAME / KEY: CDS (B) LOCATION: 1..1047 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

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SEQUENCE LIST

<110> Millennium Pharmaceuticals, Inc

<120> Novel CRSP protein and nucleic acid molecules and their uses 5 <130> Document P59744L-EP

<140> EP06021943.3

<141> 04-16-1998

<150> Document PCT / US98 / 07894

<151> 04-16-1998 10 <150> Document EP98918453

<151> 1998-04-16

<160> 18

<170> PatentIn version 3.1

<210> 1

15 <211> 2479

<212> DNA

<213> human

<220>

<221> misc_feature

20 <222> (1146) .. (1146)

<223> unknown nucleotide

<220>

<221> CDS

<222> (38) .. (1087)

<223>

<400> 1

image 1

image 1

image 1

<270> 2

<211> 350

<212> PRT

<213> human

<220>

<221> misc_feature

<222> (1196) .. (1196)

<223> unknown nucleotide

<400> 2

image 1

image 1

<210> 3

<211> 1050

<212> DNA

<213> human

<400> 3

image 1

image 1

<210> 4

<211> 848

<212> DNA 5 <213> human

<220>

<221> CDS

<222> (125) .. (796)

<223>

10 <400> 4

image 1

image 1

<210> 5

<211> 224

<212> PRT

<213> human

<400> 5

image 1

image 1

<210> 6

<211> 672

<212> DNA

<213> human

<400> 6

image 1

<210> 7

5 <211> 1529

<212> DNA

<213> human

<220>

<221> misc_feature

10 <222> (1300) .. (1300)

<223> unknown nucleotide

<220>

<221> CDS

<222> (93) .. (890)

15 <223>

<220>

<221> misc_feature

<222> (1312) .. (1312)

<223> unknown nucleotide

20 <400> 7

image 1

image 1

image 1

<210> 8

<211> 266

<212> PRT 5 <213> human

<220>

<221> misc_feature

<222> (1300) .. (1300)

<223> unknown nucleotide

10 <220>

<221> misc_feature

<222> (1312) .. (1312)

<223> unknown nucleotide

<400> 8 <210> 9

image 1

image 1

<211> 798

<212> DNA

<213> human

<400> 9

image 1

<210> 10

<211> 702

<212>

DNA 5 <213> human

<220>

<221> CDS

<222> (1) .. (537)

<223>

<210> 11

<211> 179

<212> PRT

<213> human

<400> 11

<210> 12

<211> 537

<212> DNA

<213> human

<400> 12

<210> 13

<211> 928

<212>

DNA 5 <213> human

<220>

<221> CDS

<222> (75) .. (800)

<223>

<210> 14

<211> 242

<212> PRT

<213> human

<400> 14

<210> 15

<211> 726

<212> DNA

<213> human

<400> 15

<210> 16

<211> 2381

<212>

DNA 5 <213> murine

10 <400> 10

image 1

image 1

image 1

image 1

image 1

image 1

10 <400> 13

image 1

image 1

image 1

image 1

image 1

image 1

<220>

<221> misc_feature

<222> (1) .. (1)

<223> unknown base - shown as “F” in the application as originally submitted

10 <220>

<221> CDS

<222> (10) .. (1156)

<223>

<400> 16

image 1

image 1

image 1

<210> 17

<211> 349

<212> PRT

<213> murine <220>

<221> misc_feature

<222> (1) .. (1)

<223> unknown base - shown as “F” in the application as originally submitted

<400> 17

image 1

image 1

<210> 18

<211> 1047

<212> DNA

<213> murine

<400> 18

image 1

image 1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the convenience of the reader. It is not part of the European Patent document. Although great care has been taken in the compilation of references, errors or omissions cannot be excluded and the EPO declines responsibilities for this matter.

Patent documents cited in the description

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Non-patent documents mentioned in the description

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Claims (4)

1. Composition formulated for parenteral administration, comprising a pharmaceutically acceptable carrier and an antibody that selectively binds to an isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 8, wherein the carrier comprises a sterile diluent for injection . Composition according to claim 1, wherein the antibody is a monoclonal antibody.

3.

Composition according to claim 1, wherein the antibody is a recombinant antibody.

Four.

Composition according to claim 1, the composition being formulated for injection.

5.

Composition according to claim 1, the composition being a sterile injectable solution, wherein the antibody is incorporated into an appropriate solvent with one or a combination of components selected from the

A group consisting of physiological saline solution, bacteriostatic water, phosphate buffered saline solution or a solvent containing water, ethanol, polyol or suitable mixtures thereof.