WO2007057444A1

WO2007057444A1 - Insl3/rlf polypeptides and uses thereof

Info

Publication number: WO2007057444A1
Application number: PCT/EP2006/068622
Authority: WO
Inventors: Gregg Mcallister; Jadwiga Bienkowska; Melanie Yorke-Smith; Mireille Guerrier
Original assignee: Laboratoires Serono S.A.
Priority date: 2005-11-21
Filing date: 2006-11-17
Publication date: 2007-05-24

Abstract

The present invention relates to INSL3 polypeptides and their uses, particularly for therapeutic or prophylactic treatment in human subjects. The invention also relates to nucleic acids encoding said polypeptides, vectors comprising such nucleic acids and recombinant cells containing the same. The invention further discloses methods of producing such polypeptides, as well as methods and tools for detecting or dosing these polypeptides in any sample.

Description

INSL3/RLF POLYPEPTIDES AND USES THEREOF

The present invention relates to new INSL3 (Insulin-like 3 peptide) polypeptides and their uses, particularly for therapeutic or prophylactic treatment in human subjects. The invention also relates to nucleic acids encoding said polypeptides, vectors comprising such nucleic acids and recombinant cells containing the same, as well as corresponding pharmaceutical compositions. The invention further discloses methods of producing such polypeptides, as well as methods and tools for detecting or dosing these polypeptides in any sample.

BACKGROUND

Insulin-like 3 peptide (INSL3), also designated as Leydig insulin-like hormone or relaxin like factor (RLF) is a peptide factor expressed in testicular Leydig cells and in thecal and lutheal cells of the ovary in a sexually dimorphic pattern in mammals. INSL3 belongs to the insulin-like hormone superfamily, which comprises insulin, relaxin, and insulin-like growth factors I (IGFl) and II (IGF2). The members of this family are characterized by a signal peptide, a B-chain, a connecting C-peptide, and an A-chain (Ivell R and Bathgate RA, 2002, Biol Reprod, 67(3): 699-705). The biological actions of the relaxin superfamily members are mediated through hormone I receptors. In particular, the insulin receptor, the IGF- I and IGF-II receptors, GPCR135, GPCR142, as well as LGR7 and LGR8. LGR7 is the relaxin receptor, which binds all human relaxin peptides with high affinity, but has very low affinity for INSL3 (Bathgate et al, Trends in Endocrinology and Metabolism 2003, 14: 207-213). LGR8 is the INSL3 receptor, which binds INSL3 with high affinity. Knockout mice lacking a LGR8 gene have identical phenotype to the INSL3 knockout mice. However in the human, LGR8 will also bind relaxin, although it has a higher affinity for INSL3 (Bathgate et al., Trends in Endocrinology and Metabolism 2003, 14: 207-213). The LGR8 receptor is expressed in the gubernaculum, ovary and testis, where in the latter organ it mediates INSL3's actions on testes descent (Bathgate et al., Trends in Endocrinology and Metabolism 2003, 14: 207- 213). In the ovary, LGR8 mediates follicular development and in both the testes and ovaries, it is implicated in male and female germ-cell maturation (Kawamura et al., 2004, PNAS 101, 7323-7328). LGR8 is also expressed in the kidney, bone, peripheral-blood leucocytes, muscle, brain, thyroid, and uterus (Hsu et al, Science, 295: 671-674).

The human INSL3 gene has been described as containing 2 exons and 1 intron and was assigned to bands pl3.2-pl2 of the short arm of chromosome 19 (Burkhardt E et al., 1994, Genomics: 20(1): 13-9). INSL3 is a secreted protein. It is translated as a pre-propeptide, initially containing a signal peptide, B-chain, C-peptide and A-chain. Upon hormone maturation, signal sequence and C-peptide are excised, while A- and B- chains are assembled with two inter-chain and one intra-chain disulphide bonds. In human, INSL3 (pre-propeptide) is 131 amino acids and amino acid residues 1 to 20 represent the signal peptide, amino acids 21 to 55 the B-chain, amino acids 58 to 104 the C peptide, amino acids 106 to 131 the A-chain (SwissProt entry: P51460). The interchain disulfide bonds (between B and A chains) occur between Cys-34 and Cys- 116, and between Cys-46 and Cys-129, and the intra-chain between Cys-115 and Cys- 120. Mutations and polymorphisms have been described in the human gene coding for INSL3 (Bogatcheva NV, Agoulnik AI. Reprod Biomed Online. 2005;10(l):49-54). The vast majority of the mutations described so far lead to a single amino acid substitution in the pre-propeptide structure; the only exception is represented by the mutation R73X resulting in the termination of the translation. The most variable part of INSL3 is represented by the C-peptide (mutations or polymorphisms describe in this part of INSL3 include A60T, P93L, R102C and Rl 02H). Other mutations or polymorphisms described include: A24G, V43L, P49S and Nl 10K.

Considering the biological activities of INSL3, it would be highly valuable to obtain biologically active INSL3 variants, which bind the biological receptors. Ideally, such variants would include ligands, such as agonists, reverse agonists, partial agonists, mixed agonists/antagonists and full antagonists, which bind at the LGR8 receptor and initiate, inhibit, activate, or otherwise control, the biological activities of members of this protein superfamily. It would be of particular interest to obtain new agonists and antagonists of human INSL3. SUMMARY OF THE PRESENT INVENTION

The present invention relates to novel INSL3 polypeptides and their uses, particularly for therapeutic or prophylactic treatment in human subjects. The invention further discloses methods of producing such polypeptides, as well as methods and tools for detecting or dosing these polypeptides in a sample. The invention also discloses nucleic acids encoding said polypeptides, vectors comprising such nucleic acids, in particular expression vectors, and recombinant cells containing the same, as well as corresponding pharmaceutical compositions. Further included are antibodies specific for the novel INSL3 polypeptides of the present invention.

The present invention results in part from the identification, isolation and characterization of a novel exon in the human gene INSL3. This novel exon leads to the identification of a novel transcriptional variant of INSL3 having particular structural and biological properties. This transcriptional variant and derivatives thereof represent valuable pharmaceutical products.

An object of this invention thus resides in an isolated polypeptide comprising or consisting of the sequence set forth at SEQ ID NO: 1 or a variant of said polypeptide.

The polypeptide having the sequence set forth at SEQ ID NO: 1 corresponds to the C- terminal part of the novel transcriptional variant of INSL3 encoded by a novel exon of the human gene INSL3 disclosed here for the first time (named hereafter exon IA).

In a further aspect, the invention resides in an isolated polypeptide comprising or consisting of the sequence set forth at SEQ ID NO: 2 (named hereafter INSL3v) or a variant of said polypeptide. The polypeptide of SEQ ID NO: 2 is a novel transcriptional variant of INSL3, which is encoded by exon 1 of the human gene INSL3 and the novel exon IA. In a preferred embodiment, these peptides are mature peptide lacking the N- terminal signal peptide.

Another object of the present invention resides in a fusion protein comprising a human INSL3 polypeptide variant as defined above operably linked to an additional amino acid domain. A further object of this invention resides in a nucleic acid encoding a human

INSL3 polypeptide variant or a fusion protein as defined above, as well as any cloning or expression vector comprising such a nucleic acid. The invention also relates to recombinant host cells comprising a vector or nucleic acid as defined above, as well as to methods of producing a human INSL3 polypeptide variant as defined above using such recombinant cells.

Another object of the present invention resides in a polypeptide as defined above in the form of active conjugates or complex.

A further object of this invention also relates to an antibody, or a fragment or derivative of such an antibody, which selectively binds to a polypeptide as defined above.

The invention also relates to an immunoconjugate comprising an antibody as defined above conjugated to a heterologous moiety.

A further object of this invention also resides in a pharmaceutical composition comprising a polypeptide, nucleic acid, vector or recombinant cell as defined above and a pharmaceutically acceptable carrier, excipient, or stabilizer.

The invention further relates to a method of treating, preventing or ameliorating the symptoms of a disorder in a patient, the disorder involving disregulation of INSL3 expression or activity, the method comprising administering to the patient a pharmaceutical composition as defined above.

The invention also relates to a method of treating, preventing or ameliorating the symptoms of a INSL3 -mediated disorder in a patient, wherein the disorder is selected from the group consisting of cancers, scleroderma, uncontrolled or abnormal collagen or fibronectin formation or breakdown, neurological disorders, angiogenic disorders, cardiovascular disorders, female reproductive disorders, conditions associated with pregnancy, renal disease, inflammatory bowel disease, cryptorchidism, disregulation of spermatogenesis and reproductive development including descent of the gonads, female or male infertility, the method comprising administering to the patient an effective amount of a polypeptide as defined above.

The invention further resides in the use of a polypeptide as defined above or of a pharmaceutical composition as defined above in the manufacture of a medicament for the treatment of a INSL3 -mediated disorder in a patient, the disorder being selected from the group consisting of : cancers, scleroderma, uncontrolled or abnormal collagen or fibronectin formation or breakdown, neurological disorders, angiogenic disorders, cardiovascular disorders, female reproductive disorders, conditions associated with pregnancy, renal disease, inflammatory bowel disease, cryptorchidism, disregulation of spermatogenesis and reproductive development including descent of the gonads, female or male infertility. A further object of this invention also resides in a pharmaceutical composition comprising an antibody, or a fragment or a derivative thereof as described here above, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

The invention also relates to a method of treating, preventing or ameliorating the symptoms of a cardiovascular disease or a cancer in a subject, the method comprising administering to the patient an effective amount of an antibody, or a fragment or a derivative thereof as described here above. The invention also resides in the use of an antibody, or a fragment or a derivative thereof as described here above, in the manufacture of a medicament for the treatment of a cardiovascular disease or a cancer.

Other aspects of this invention include primers and probes specific for a nucleic acid as defined above, as well as their uses to detect or diagnose the presence of such a nucleic acid in a sample.

LEGEND TO THE FIGURES

Figure 1 : DNA and protein sequence of a variant of INSL3 (INSL3v) comprising a human B-chain, and lacking the connecting C-peptide and the A-chain.

Figure 2 : INSL3v coding exon organization in human genomic DNA and position of PCR primers.

Figure 3 : Nucleotide sequence and translation of cloned INSL3 v PCR product.

DETAILED DESCRIPTION OF THE INVENTION

1. INSL3 polypeptides and variants of the present invention: In a first aspect, the invention resides in an isolated polypeptide comprising or consisting of the sequence set forth at SEQ ID NO: 1. The polypeptide having the sequence set forth at SEQ ID NO: 1 corresponds to the C-terminal part of the novel transcriptional variant of INSL3 encoded by a novel exon of the human gene INSL3 disclosed here for the first time (named hereafter exon IA, see figures 1 and 2).

The term "isolated" when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Isolated products of this invention may thus be contained in a culture supernatant, partially enriched or purified, produced from heterologous sources, cloned in a vector or formulated with a vehicle, etc.

In a further aspect, the present invention resides in an isolated polypeptide comprising or consisting of a variant of the polypeptide set forth at SEQ ID NO: 1. A variant being defined according to the present invention as a polypeptide having at least 80% amino acid sequence identity with the sequence SEQ ID NO: 1, preferably at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, more preferably at least 98% amino acid sequence identity and most preferably at least 99% amino acid sequence identity. Ordinarily, the variant polypeptides are at least 8 amino acids in length, often at least 10 amino acids in length, more often at least 12 amino acids in length. More preferably, the variant are deferring from SEQ ID NO: 1 by two and even more preferably by one amino acid.

"Percent (%) amino acid sequence identity" with respect to the INSL3 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific INSL3 polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. J MoI Biol. (1990). 215 (3) : 403-410). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

In a further aspect, the invention resides in an isolated polypeptide comprising or consisting of the sequence set forth at SEQ ID NO: 2 (named hereafter INSL3v). The polypeptide of SEQ ID NO: 2 is a novel transcriptional variant of INSL3, which is encoded by exon 1 of the human gene INSL3 and the novel ex on IA (see figures 1 and 2). This transcriptional variant has particular structural properties. In figure 1, nucleotides 1 to 190 of the human transcriptional variant presented represents the 3' end of exon 1 and nucleotides 191 to 228 represents the 5' end of the novel exon IA. It can be concluded that the polypeptide encoded and presented in figure 1 comprises a complete B-chain (which is represented by amino acids 21 to 55) but lacks a large portion of the connecting C-peptide and all the A-chain. Moreover this polypeptide bears a new C-terminal part encoded by the new exon IA (rshsdaqagvqw (SEQ ID NO: I)). It can also be concluded that the interchain disulfide bonds (between B and A chains) which occur between Cys-34 and Cys-116, and between Cys-46 and Cys-129, and the intra-chain between Cys-115 and Cys-120 of the INSL3 long variant are not present in the new variant.

In a further aspect, the present invention resides in an isolated polypeptide comprising or consisting of a variant of the polypeptide set forth at SEQ ID NO 2. A variant of the polypeptide set forth at SEQ ID NO: 2 being defined according to the present invention as a active polypeptide having at least 80% amino acid sequence identity with the sequence SEQ ID NO: 2, preferably at least 90% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and most preferably at least

99% amino acid sequence identity. Ordinarily, the variant polypeptides are at least 50 amino acids in length, often at least about 65 amino acids in length, more often at least

75 amino acids in length. More preferably, the variants of the present invention resides in polypeptide comprising one or several amino acid substitutions as compared to the SEQ ID NO: 2, typically from 0 to 10 amino acid substitutions, even more typically from 0 to 5, 4, 3, 2 or 1 amino acid substitutions. More particularly, the variant polypeptide differs from the sequence set forth at SEQ ID NO: 2 by one, two, three or four mutations chosen in the group consisting of: A24G, A60T, V43L and P49S.

In a preferred embodiment, these peptides are mature peptide lacking the N- terminal signal peptide. More particularly, the polypeptides of the present invention lack the signal peptide consisting of amino acids 1 to 20 of SEQ ID NO: 2. Therefore, in a particular aspect, the invention resides in a polypeptide comprising or consisting of the sequence of amino acids 21 to 75 of SEQ ID NO: 2 or variants of said sequence as defined hereabove. Preferably any of the above or below described INSL3 polypeptides and variants retain at least some biological activity. More preferably said biological activity is at least one of the following: binding to the LGR8 receptor, modulation of the cAMP levels of LGR8 expressing cells, induction of the descent of the gonads in a mammal in particular in human, maturation and/or descent of the testes in a mammal in particular in human, enhancement of the survival of sperm cells in vitro and/or in vivo in a mammal in particular in human, male and/or female germ-cell maturation in a mammal in particular in human, development of ovarian follicles and/or induction of the maturation of the oocyte in vitro and/or in vivo in a mammal in particular in human, regulation of relaxin activity in the heart in a mammal in particular in human, stimulation of the proliferation of cancer cells (preferably of cells originating from the thyroid gland or prostate), action on kidney and/or thyroid function, central action on the brain, modulation of muscle function, effect on bone and/or blood cell function, reproductive action on the uterus. Even more preferably said biological activity is at least one of the following: induction of the descent of the gonads, maturation and/or descent of the testes, development of ovarian follicles and/or maturation of the oocyte.

These biological activities can be verified using several biological assays that are known per se in the art (see for example the biological assays described at example 4). More preferably any of the above or below described EPO polypeptides and variants retain at least (or at least about) 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the biological activity as compared to the biological activity of the native/unmodified/wild-type INSL3 protein. In some embodiments of this aspect of the invention, the protein can have higher biological activity than the native/unmodified/wild-type protein.

2. Fusion proteins :

The present invention also relates to fusion proteins comprising an INSL3 polypeptide or variant as disclosed above, operably linked to an additional amino acid domain. The additional amino acid domain may be located upstream (N-ter) or downstream (C-ter) from the sequence of the polypeptides described here above. The additional domain may comprise any functional region, providing for instance an increased stability, targeting or bioavailability of the fusion protein ; facilitating purification or production, or conferring on the molecule additional biological activity. Specific examples of such additional amino acid sequences include a GST sequence, a His tag sequence, a multimerication domain, the constant region of an immunoglobulin molecule or a heterodimeric protein hormone such as human chorionic gonadotropin (hCG) as described in US 6,193,972. The term "operably linked" indicates that the polypeptide and additional amino acid domain are associated through peptide linkage, either directly or via spacer residues. In this manner, the fusion protein can be produced recombinant Iy, by direct expression in a host cell of a nucleic acid molecule encoding the same, as will be discussed below. Also, if needed, the additional amino acid sequence included in the fusion proteins may be eliminated, either at the end of the production/purification process or in vivo, e.g., by means of an appropriate endo-/ exopeptidase. For example, a spacer sequence included in the fusion protein may comprise a recognition site for an endopeptidase (such as a caspase) that can be used to separate by enzymatic cleavage the desired polypeptide variant from the additional amino acid domain, either in vivo or in vitro.

In a particular embodiment, a fusion protein according to the present invention comprises an immunoglobulin, i.e. the INSL3 polypeptide or variant disclosed hereabove is fused to all or a portion of an immunoglobulin, particularly the Fc portion of a human immunoglobulin. Methods for making immunoglobulin fusion proteins are well known in the art, such as the ones described in WO 01/03737, for example. The person skilled in the art will appreciate that the resulting fusion protein of the invention substantially retains the biological activity of the INSL3 polypeptides. More preferably said biological activity is at least one of the following: binding to the LGR8 receptor, modulation of the cAMP levels of LGR8 expressing cells, induction of the descent of the gonads in a mammal in particular in human, maturation and/or descent of the testes in a mammal in particular in human, enhancement of the survival of sperm cells in vitro and/or in vivo in a mammal in particular in human, male and/or female germ-cell maturation in a mammal in particular in human, development of ovarian follicles and/or induction of the maturation of the oocyte in vitro and/or in vivo in a mammal in particular in human, regulation of relaxin activity in the heart in a mammal in particular in human, stimulation of the proliferation of cancer cells (preferably of cells originating from the thyroid gland or prostate), action on kidney and/or thyroid function, central action on the brain, modulation of muscle function, effect on bone and/or blood cell function, reproductive action on the uterus. These biological activities can be verified using several biological assays that are known per se in the art (see for example the biological assays described at example 4). The fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length. Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu- Val-Leu-Gly-Gly-Gln-Phe-Met introduced between the INSL3 polypeptide sequence and the immunoglobulin sequence. The amino acid sequence derived from the immunoglobulin may be linked to the C-terminus or to the N-terminus of the INSL3 polypeptide of the present invention, preferably to the C-terminus. The resulting fusion protein has improved properties, such as an extended residence time in body fluids (half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.

In a preferred embodiment, the INSL3 polypeptide or variant of the present invention is fused to the constant region of an Ig molecule, e.g. an Fc portion of an Immunoglobulin. Preferably, it is fused to heavy chain regions, like the CH2 and CH3 domains, optionally with the hinge region of human IgGl, for example. The Fc part may e.g. be mutated in order to prevent unwanted activities, such as complement binding, binding to Fc receptors, or the like. Other iso forms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG2 or IgG4, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomeric or multimeric, hetero- or homomultimeric.

Further fusion proteins of the INSL3 polypeptides or variants of the present invention may be prepared by fusing domains isolated from other proteins allowing the formation or dimers, trimers, etc. Examples for protein sequences allowing the multimerization of the polypeptides of the Invention are domains isolated from proteins such as hCG (WO 97/30161), collagen X (WO 04/33486), C4BP (WO 04/20639), Erb proteins (WO 98/02540), or coiled coil peptides (WO 01/00814).

The present invention also relates to polypeptides as disclosed herein containing a signal peptide, along with the corresponding DNA sequence encoding such polypeptides. The signal peptide may be the naturally occurring signal peptide as disclosed herein, or may be a heterologus or synthetic signal peptide.

The present invention also relates to the above-disclosed INSL3 polypeptides and variants comprising an additional N-terminal amino acid residue, preferably a methionine. Indeed, depending on the expression system and conditions, polypeptides of the invention may be expressed in a recombinant host cell with a starting Methionine. This additional amino acid may then be either maintained in the resulting recombinant protein, or eliminated by means of an exopeptidase, such as Methionine Aminopeptidase, according to methods disclosed in the literature (Van Valkenburgh HA and Kahn RA, 2002; Ben-Bassat A, 1991).

3. Preparation of Polypeptides and fusion proteins of the present invention:

A. Nucleic acid encoding the polypeptides, proteins and fusion proteins of the present invention and vectors:

A further object of the present invention is an isolated nucleic acid molecule encoding the polypeptides, proteins and fusion proteins defined here above. In this regard, the term "nucleic acid molecule" encompasses all different types of nucleic acids, including without limitation deoxyribonucleic acids (e.g., DNA, cDNA, gDNA, synthetic DNA, etc.), ribonucleic acids (e.g., RNA, mRNA, etc.) and peptide nucleic acids (PNA). In a preferred embodiment, the nucleic acid molecule is a DNA molecule, such as a double-stranded DNA molecule or a cDNA molecule. The term "isolated" means nucleic acid molecules that have been identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the specific nucleic acid molecule as it exists in natural cells.

A particular object of this invention resides more specifically in an isolated nucleic acid molecule that comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 3 and SEQ ID NO: 4, or a complementary strand or degenerate sequence thereof, or a nucleic acid coding for the polypeptides of SEQ ID NO: 1 or 2, or a complementary strand. A degenerate sequence designates any nucleotide sequence encoding the same amino acid sequence as a reference nucleotide sequence, but comprising a distinct nucleotide sequence as a result of the genetic code degeneracy. In a preferred embodiment, the nucleic acid molecule is a DNA molecule, such as a double-stranded DNA molecule or a cDNA molecule. A further object of this invention is a vector comprising DNA encoding any of the above or below described polypeptides. The vector may be any cloning or expression vector, integrative or autonomously replicating, functional in any prokaryotic or eukaryotic cell. In particular, the vector may be a plasmid, cosmid, virus, phage, episome, artificial chromosome, and the like. The vector may comprise regulatory elements, such as a promoter, terminator, enhancer, selection marker, origin of replication, etc. Specific examples of such vectors include prokaryotic plasmids, such as pBR, pUC or pcDNA plasmids ; viral vectors, including retroviral, adenoviral or AAV vectors ; bacteriophages ; baculoviruses ; BAC or YAC, etc., as will be discussed below.

The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

B. Host Cells

A further aspect of the present invention is a recombinant host cell, wherein said cell comprises a nucleic acid molecule or a vector as defined above. The host cell may be a prokaryotic or eukaryotic cell. Examples of prokaryotic cells include bacteria, such as E.coli. Examples of eucaryotic cells are yeast cells, plant cells, mammalian cells and insect cells including any primary cell culture or established cell line (e.g., 3T3, Vero,

HEK293, TN5, etc.). Suitable host cells for the expression of glycosylated proteins are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CVl line transformed by SV40 (COS-7,

ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol, 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl, Acad. Sci. USA,

77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). Particularly preferred mammalian cells of the present invention are CHO cells.

C. Production of the Polypeptides and fusion proteins of the present invention:

Polypeptides and fusion proteins of this invention may be produced by any technique known per se in the art, such as by recombinant technologies, chemical synthesis, cloning, ligations, or combinations thereof. In a particular embodiment, the polypeptides or fusion proteins are produced by recombinant technologies, e.g., by expression of a corresponding nucleic acid in a suitable host cell. Another object of this invention is therefore a method of producing an INSL3 polypeptide or variant as described herein, the method comprising culturing a recombinant host cell of the invention under conditions allowing expression of the nucleic acid molecule, and recovering the polypeptide produced. The polypeptide produced may be glycosylated or not, or may contain other post-translational modifications depending on the host cell type used. Many books and reviews provide teachings on how to clone and produce recombinant proteins using vectors and prokaryotic or eukaryotic host cells, such as some titles in the series "A Practical Approach" published by Oxford University Press ("DNA Cloning 2: Expression Systems", 1995; "DNA Cloning 4: Mammalian Systems", 1996; "Protein Expression", 1999; "Protein Purification Techniques", 2001).

The vectors to be used in the method of producing a polypeptide according to the present invention can be episomal or non-/homologously integrating vectors, which can be introduced into the appropriate host cells by any suitable means (transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate- precipitation, direct microinjection, etc.). Factors of importance in selecting a particular plasmid, viral or retroviral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. The vectors should allow the expression of the polypeptide or fusion proteins of the invention in prokaryotic or eukaryotic host cells, under the control of appropriate transcriptional initiation / termination regulatory sequences, which are chosen to be constitutively active or inducible in said cell. A cell line substantially enriched in such cells can be then isolated to provide a stable cell line. Host cells are transfected or transformed with expression or cloning vectors described herein for protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al, supra.

For eukaryotic host cells (e.g. yeasts, insect or mammalian cells), different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived form viral sources, such as adenovirus, papilloma virus, Simian virus or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples are the TK promoter of the Herpes virus, the SV40 early promoter, the yeast gal4 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for repression and activation, so that expression of the genes can be modulated. The cells which have been stably transformed by the introduced DNA can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may also provide for phototrophy to an auxotrophic host, biocide resistance, e.g. antibiotics, or heavy metals such as copper, or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed (e.g., on the same vector), or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins of the invention.

Particularly suitable prokaryotic cells include bacteria (such as Bacillus subtilis or E. colϊ) transformed with a recombinant bacteriophage, plasmid or cosmid DNA expression vector. Such cells typically produce proteins comprising a N-terminal Methionine residue, such proteins representing particular objects of this invention. Preferred cells to be used in the present invention are eukaryotic host cells, e.g. mammalian cells, such as human, monkey, mouse, and Chinese Hamster Ovary (CHO) cells, because they provide post-translational modifications to protein molecules, including correct folding or glycosylation at correct sites. Alternative eukaryotic host cells are yeast cells (e.g., Saccharomyces, Kluyveromyces, etc.) transformed with yeast expression vectors. Also yeast cells can carry out post-trans lational peptide modifications including glycosylation. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids that can be utilized for production of the desired proteins in yeast. Yeast cells recognize leader sequences in cloned mammalian gene products and secrete polypeptides bearing leader sequences (i.e., pre-peptides).

For long-term, high-yield production of a recombinant polypeptide, stable expression is preferred. For example, cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. A cell line substantially enriched in such cells can be then isolated to provide a stable cell line.

A particularly preferred method of high-yield production of a recombinant polypeptide of the present invention is through the use of dihydro folate reductase

(DHFR) amplification in DHFR-deficient CHO cells, by the use of successively increasing levels of methotrexate as described in US 4,889,803. The polypeptide obtained may be in a glycosylated form.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines. In the baculo virus system, the materials for baculo virus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen. In addition to recombinant DNA technologies, the polypeptides or fusion proteins of this invention may be prepared by chemical synthesis technologies. Examples of chemical synthesis technologies are solid phase synthesis and liquid phase synthesis. As a solid phase synthesis, for example, the amino acid corresponding to the carboxy- terminus of the polypeptide to be synthesised is bound to a support which is insoluble in organic solvents and, by alternate repetition of reactions (e.g., by sequential condensation of amino acids with their amino groups and side chain functional groups protected with appropriate protective groups), the polypeptide chain is extended. Solid phase synthesis methods are largely classified by the tBoc method and the Fmoc method, depending on the type of protective group used. Totally synthetic proteins of size comparable to that of INSL3 are disclosed in the literature (Brown A et al., 1996).

The polypeptides of the present invention can be produced, formulated, administered, or generically used in other alternative forms that can be preferred according to the desired method of use and/or production. The proteins of the invention can be post-translationally modified, for example by glycosylation. The polypeptides or proteins of the invention can be provided in isolated (or purified) biologically active form, or as precursors, derivatives and/or salts thereof. As indicated above, the term "biologically active" means that such polypeptides have at least one of the biological activity described here above. These biological activities can be verified using several biological assays that are known per se in the art (non- limiting examples of such assays are described at example 4). "Precursors" are compounds which can be converted into the polypeptides of present invention by metabolic and/or enzymatic processing prior to or after administration thereof to cells or an organism. The term "salts" herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the polypeptides of the present invention. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid. Any of such salts should have substantially similar activity to the polypeptides of the invention. The term "derivatives" as used herein refers to derivatives that can be prepared from the functional groups present on the lateral chains of the amino acid moieties or on the amino- / or carboxy- terminal groups according to methods known per se in the art. Such derivatives include for example esters or aliphatic amides of the carboxyl-groups and N- acyl derivatives of free amino groups or O-acyl derivatives of free hydroxyl-groups and are formed with acyl-groups as for example alcanoyl- or aroyl-groups. Purification of the polypeptides of the invention can be carried out by a variety of methods known per se in the art, such as, without limitation, any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like. A particular purification procedure is affinity chromatography, using (monoclonal) antibodies or affinity groups which selectively bind the polypeptide and which are typically immobilized on a gel matrix contained within a column. Purified preparations of the proteins of the invention, as used herein, refers to preparations which contain less than 15% of contaminants, more preferably which comprise at least 90, 95 or 97% of the polypeptide.

4. Active conjugates or complex:

The polypeptides or fusion proteins of the invention can be in the form of active conjugates or complex with a heterologous moiety, which may be selected from cytotoxic agents, labels (e.g. biotin, fluorescent labels), drugs or other therapeutic agents, covalently bound or not, either directly or through the use of coupling agents or linkers. Useful conjugates or complexes can be generated using molecules and methods known per se in the art, for example for allowing the detection of the interaction with the LGR8 receptor (radioactive or fluorescent labels, biotin), the detection of LGR8 receptor expressing cells in a sample (radioactive or fluorescent labels, biotin), therapeutic efficacy (cytotoxic agents, drugs or other therapeutic agents). Cytotoxic agents include chemotherapeutic agents, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated proteins. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Useful conjugates or complexes can also be generated for improving the agents in terms of drug delivery efficacy. For this purpose, the polypeptides or fusion proteins of the invention can be in the form of active conjugates or complex with molecules such as polyethylene glycol and other natural or synthetic polymers (Harris JM and Chess RB, 2003; Greenwald RB et al, 2003; Pillai O and Panchagnula R, 2001). In this regard, the present invention contemplates chemically modified polypeptides and proteins as disclosed herein, in which the polypeptide or the protein is linked with a polymer. Typically, the polymer is water soluble so that the conjugate does not precipitate in an aqueous environment, such as a physiological environment. An example of a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation. In this way, the degree of polymerization can be controlled. An example of a reactive aldehyde is polyethylene glycol propionaldehyde, ormono- (Cl-ClO) alkoxy, or aryloxy derivatives thereof (see, for example, Harris, et al., U. S. Patent No. 5,252, 714). The polymer may be branched or unbranched. Moreover, a mixture of polymers can be used to produce the conjugates. The conjugates used for therapy can comprise pharmaceutically acceptable water- soluble polymer moieties. Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono- (Cl-ClO) alkoxy-PEG, aryloxy- PEG, poly- (N- vinyl pyrrolidone) PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bis- succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropyleneoxide/ethylene oxide co-polymer, polyoxyethylated polyols(e. g., glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers. Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000. A conjugate can also comprise a mixture of such water-soluble polymers.

One example of a conjugate comprises the INSL3 polypeptide variant of SEQ ID NO: 2 or amino acids 21 to 75 of SEQ ID NO: 2 and a polyallcyl oxide moiety attached to the N-terminus of the INSL3 polypeptide moiety. PEG is one suitable polyalkyl oxide. As an illustration, the INSL3 polypeptide or variant can be modified with PEG, a process known as "PEGylation." PEGylation can be carried out by any of the PEGylation reactions known in the art (see, for example, EP 0 154 316, Delgado et al, Critical Reviews in Therapeutic Drug Carrier Systems 9: 249 (1992), Duncan and Spreafico, Clin.Pharmacokinet. 27: 290 (1994), and Francis et al., Int J Hematol 68: 1 (1998)). For example, PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule. In an alternative approach, conjugates are formed by condensing activated PEG, in which a terminal hydroxy or amino group of PEG has been replaced by an activated linker (see, for example, Karasiewicz etal, U. S. Patent No. 5,382, 657).

5. Anti-INSL3 variant Polypeptide Antibodies:

Some drug candidates for use in the compositions and methods of the present invention are antibodies, antibody fragments or derivative thereof, which selectively bind to any of the above or below described polypeptides. In a more specific embodiment, the antibody, fragment or derivative thereof selectively binds to polypeptides of SEQ ID NO: 1, 2, or amino acids 21 to 75 of SEQ ID NO: 2 , or a variant of said polypeptides as described here above.

Within the context of this invention, the term "selective" binding indicates that the antibodies preferentially bind the target polypeptide or epitope, i.e., with a higher affinity than any binding to any other antigen or epitope. In other words, binding to the target polypeptide can be discriminated from non-specific binding to other antigens. It is preferred that the antibodies according to the present invention exhibit binding affinity (Ka) to the target polypeptide or epitope of 10⁶ M^"1 or greater, preferably 10⁷ M^"1 or greater, more preferably 10^s M^"1 or greater and most preferably 10⁹ M^"1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard G., Ann NY Acad. Sci. 51: 660-672, 1949). Antibodies of this invention may be monoclonal or polyclonal antibodies, or fragments or derivative thereof having substantially the same antigen specificity. A. Polyclonal Antibodies: Methods of preparing polyclonal antibodies from various species, including rodents, primates and horses, have been described for instance in Vaitukaitis et al. (J Clin Endocrinol Metab. 33 (1971) p. 988). Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the polypeptide of SEQ ID NO 1, 2, or amino acids 21 to 75 of SEQ ID NO: 2, or a variant as described hereabove or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). Repeated injections may be performed. Blood samples are collected and immunoglobulins or serum are separared. B. Monoclonal Antibodies: The antibodies may, alternatively, be monoclonal antibodies. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

Methods of producing monoclonal antibodies may be found, for instance, in Harlow et al (Antibodies: A laboratory Manual, CSH Press, 1988) or in Kohler et al (Nature 256 (1975) 495), incorporated therein by reference.

In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent (the immunizing agent will typically include the polypeptide of SEQ ID NO: 1, 2, or amino acids 21 to 75 of SEQ ID NO: 2 , or a variant as described hereabove or a fusion protein thereof) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT- deficient cells. Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

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

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purificationproceduressuch as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then trans fected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.

The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 [1991] and Marks et al., J. MoI. Biol, 222:581-597 (1991), for example.

The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in Ward et al (Nature 341 (1989) 544). C. Human and Humanized Antibodies:

The antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues.

Methods for humanizing non-human antibodies are well known in the art. Humanization can be essentially performed following the method of Winter and co- workers (Jones et al, Nature, 321 :522-525 (1986); Riechmann et al, Nature, 332:323- 327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. MoI. Biol, 227:381 (1991); Marks et al., J. MoI. Biol, 222:581 (1991)). The techniques of Cole et al., and Boerner et al., are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol, 147(l):86-95 (1991)). Similarly, human antibodies can be made by the introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 ( 1994); Morrison, Nature, 368: 812-13 (1994); Fishwild et al, Nature Biotechnology, 14:845-51 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol, 13 :65-93 (1995).

D. Immunoconjugates: The invention also pertains to immunoconjugates comprising an antibody conjugated to heterologous moieties, such as cytotoxic agents, labels, drugs or other therapeutic agents, covalently bound or not, either directly or through the use of coupling agents or linkers. Cytotoxic agent include chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Moreover, antibodies or antibody fragments of the present invention can be PEGylated using methods in the art and described herein. The antibodies disclosed herein may also be formulated as immuno liposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. ScL USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

E. Antibody fragments: The invention also pertains to "Antibody fragments" which comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al, Protein Eng., 8(10): 1057-1062 [1995]); single-chain antibody molecules; monobodies; and multispecific antibodies formed from antibody fragments.

"Fv" is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. "Single-chain antibody molecules" are fragments of an antibody comprising the

VH and VL domains of said antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the single-chain antibody molecule to form the desired structure for antigen binding. For a review of single-chain antibody molecules, see, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994). The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The term "monobodies" as used herein, refers to antigen binding molecules with a heavy chain variable domain and no light chain variable domain. A monobody can bind to an antigen in the absence of light chain and typically has three CDR regions designated CDRHl, CDRH2 and CDRH3. Monobodies include "camelid monobodies" obtained from a source animal of the camelid family, including animals with feet with two toes and leathery soles. Animals in the camelid family include camels, llamas, and alpacas. It has been reported that camels (Camelus dromedaries and Camelus bactrianus) often lack variable light chain domains when material from their serum is analyzed, suggesting that sufficient antibody specificity and affinity can be derived form VH domains (three CDR loops) alone. Monobodies also include modified VH from various animal sources, in particular mammals (for example mouse, rat, rabbit, horse, donkey, bovine or human), which can bind to an antigen in the absence of VL. Preferably, the VH is modified in positions at the VL interface to provide for binding of the VH to antigen in absence of the VL. Davies and Riechmann have for example demonstrated that "camelized monobodies" with high affinity (binding affinity (Ka) to the target polypeptide of 10⁷ M^"1 or greater) and high specificity can be generated (Davies & Riechmann, 1995, Biotechnology (N Y), 13(5):475-9). Non-specific binding of the VH through its interface for the light chain variable domain (VL) was prevented through three mutations (G44E, L45R and W47G) in this interface. These mutations were introduced to mimic camelid antibody heavy chains naturally devoid of light chain partners. F. Use of the Antibodies and antibodies fragment of the present invention:

Antibodies and antibodies fragment of the present invention have various utilities. For example, the antibodies may be used for detecting, dosing, purifying or neutralizing any INSL3 polypeptide or variant described here above. In a particular aspect, the invention thus resides in a method of detecting or dosing a INSL3 polypeptide or variant as defined above in a sample, comprising contacting such a sample with an antibody, fragment or derivative thereof as disclosed above, and determining the formation or dosing the (relative) quantity of an immune complex. The sample may be for instance any biological fluid, such as blood, plasma, serum, etc., optionally diluted and/or treated. The antibody, fragment or derivative thereof may be in suspension or immobilized on a support. The presence or amount of immune complexes may be determined by any technique known per se in the art, e.g., by ELISA, RIA, etc., e.g., using reporter antibodies, labelled antibodies, etc. In a more specific embodiment, the antibody, fragment or derivative thereof selectively binds to polypeptides of SEQ ID NO: 1 or a variant of said polypeptides as described here above.

Antibodies and antibodies fragment of the present invention are also useful for the affinity purification of INSL3 polypeptides or variants as described here above from recombinant cell culture or natural sources. In this process, the antibodies against the INSL3 polypeptide or variant are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the INSL3 polypeptide or variant to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the INSL3 polypeptide or variant, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the INSL3 polypeptide or variant from the antibody. In a more specific embodiment, the antibody, fragment or derivative thereof selectively binds to polypeptides of SEQ ID NO: 1 or a variant of said polypeptides as described here above.

Antibodies and antibodies fragment of the present invention can be used to block, inhibit, reduce, antagonize or neutralize the activity of the INSL3 polypeptides and variants of the present invention in particular in the treatment of specific human diseases. Antibodies of the present invention are therefore particularly useful as therapeutic agents. INSL3 is involved in various physiological actions, including descent of the gonads, maturation and descent of the testes, as well as the survival of sperm cells, development of ovarian follicles and maturation of the oocyte, regulation of relaxin activity in the heart. Furthermore, INSL3 has been associated to hyperplastic and neoplastic disorders of the thyroid gland and of the prostate suggesting a role for this relaxin superfamily member in the etiology of these pathologies.

A further object of this invention is therefore a pharmaceutical composition comprising an antibody or an antibody fragment as defined above, and a pharmaceutically acceptable carrier, excipient, or stabilizer. The present invention also relates to the use of an antibody or an antibody fragment as defined above for the manufacture of a medicament, preferably for treating a human subject. The present invention also pertains to methods of treating, preventing or ameliorating the symptoms of a disorder in a patient, the disorder involving upregulation of INSL3 expression or activity, the method comprising administering to the patient a pharmaceutical composition comprising an antibody or an antibody fragment as defined above. Within another aspect, the invention provides a method of treating, preventing or ameliorating the symptoms of a cardiovascular disease or a cancer in a subject, preferably a human subject, comprising administering an effective amount of the antibody as disclosed herein, thereby treating said pathological condition. The invention also pertains to the use of the antibody as disclosed herein in the manufacture of a medicament for the treatment of a cardiovascular disease or a cancer. Cardiovascular diseases according to the present invention include angina, myocardial infarctions such as acute myocardial infarctions, cardiac hypertrophy, and heart failure such as congestive heart failure, cardiomyopathy, valvular regurgitation, and intracardiac shunt and also include arterial diseases, such as atherosclerosis, hypertension, inflammatory vasculitides, Raynaud's disease and Raynaud's phenomenon, aneurysms, aortic stenosis and arterial restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease. Preferably the cardiovascular disease treated is chosen in the group consisting of angina, myocardial infarctions such as acute myocardial infarctions, cardiac hypertrophy, and heart failure such as congestive heart failure. Examples of cancer include but are not limited to, carcinoma including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms'tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer. The preferred cancer for treatment herein is adenocarcinoma, more preferably adenocarcinoma of the prostate, testis, ovary, breast, endometria, kidney, vulva, pancreas, salivary gland, or thyroid gland, even more preferably adenocarcinoma of the thyroid gland or prostate.

Within another aspect, the present invention provides a method of controlling the fertility level in a subject comprising administering an effective amount of the antibody as disclosed herein. The antibodies and antibodies fragment of the present invention may be used as a contraceptive. As used herein, "contraceptive" means an agent that when administered deliberately prevents conception or pregnancy, either directly or indirectly. Preferably the contraceptive is a female or male contraceptive, more preferably a human female or male contraceptive, even more preferably a human male contraceptive.

6. Pharmaceutical uses of the polypeptide or protein, nucleic acid molecules, vector or cells as defined above:

INSL3 has been involved in various physiological actions, including descent of the gonads. In particular, INSL3 has been shown to be involved in maturation and descent of the testes (Ivell & Hartnung, MoI Hum Reprod 2003, 9(4): 175 181), as well as the survival of sperm cells, development of ovarian follicles and maturation of the oocyte (Kawamura et al, 2004, PNAS 101, 7323-7328). INSL3 has also been implicated in regulation of relaxin activity in the heart (Tan et al., Eur J Pharmacol. 2002 Dec 20;457(2-3): 153-60), and may thus play an important regulatory role in cardiovascular disease. Furthermore, INSL3 has been associated to hyperplastic and neoplastic disorders of the thyroid gland (Hombach-Klonisch et al., Int J Oncol 2003; 22(5): 993-1001) and of the prostate (Klonisch T et al., Int J Oncol. 2005; 27(2):307- 15), suggesting a role for this relaxin superfamily member in the etiology of these pathologies.

The LGR8 receptor is expressed in the gubernaculum, ovary and testis, where in the latter organ it mediates INSL3's actions on testes descent (Bathgate et al., Trends in Endocrinology and Metabolism 2003, 14: 207- 213). In the ovary, LGR8 mediates follicular development and in both the testes and ovaries, it is implicated in male and female germ-cell maturation (Kawamura et al., 2004, PNAS 101, 7323-7328). LGR8 is also expressed in the kidney, bone, peripheral-blood leucocytes, muscle, brain, thyroid, and uterus (Hsu et al., Science, 295: 671-674). Hence, INSL3 has potential actions on kidney and thyroid function, central actions on the brain, modulation of muscle function, effects on bone and blood cell function and reproductive actions on the uterus.

Mutations and polymorphisms have been described in the human gene coding for

INSL3 and have been implicated in cryptorchidism (Bogatcheva NV, Agoulnik AI. Reprod Biomed Online. 2005;10(l):49-54). The vast majority of the mutations described so far lead to a single amino acid substitution in the pre-propeptide structure; the only exception is represented by the mutation R73X resulting in the termination of the translation. The most variable part of INSL3 is represented by the C-peptide (mutations or polymorphisms describe in this part of INSL3 include A60T, P93L, R102C and Rl 02H). Other mutations or polymorphisms described include: A24G, V43L, P49S and N110K.

A further object of this invention is therefore a pharmaceutical composition comprising a polypeptide or protein, nucleic acid, vector, recombinant cell as defined above, and a pharmaceutically acceptable carrier, excipient, or stabilizer. Preferably, the pharmaceutical composition of the present invention comprises an INSL3 polypeptide or variant as defined above. Even more preferably, the pharmaceutical composition of the present invention comprises a polypeptide consisting of the sequence of amino acids 21 to 75 of SEQ ID NO: 2 or a variant comprising one, two, three or four mutations chosen in the group consisting of: A24G, A60T, V43L and P49S, or a fusion protein as defined here above comprising such polypeptide.

Another aspect of the present invention relates to the use of a polypeptide or protein, nucleic acid molecule, vector or cell as disclosed above, for the manufacture of a medicament, preferably for treating a human subject.

The present invention also pertains to methods of treating, preventing or ameliorating the symptoms of a disorder in a patient, preferably a human subject, the disorder involving disregulation of INSL3 expression or activity, the method comprising administering to the patient a pharmaceutical composition as defined above. Preferably, the method comprises administering to the patient an effective amount of a polypeptide according to the invention; even more preferably, this polypeptide is consisting of the sequence of amino acids 21 to 75 of SEQ ID NO: 2 or a variant comprising one, two, three or four mutations chosen in the group consisting of: A24G, A60T, V43L and P49S, or a fusion protein as defined here above comprising such polypeptide.

In another aspect, the invention provides a method of treating, preventing or ameliorating the symptoms of a INSL3 -mediated disorder in a patient, preferably a human subject, wherein the disorder is selected from the group consisting of cancers, scleroderma, uncontrolled or abnormal collagen or fibronectin formation or breakdown, neurological disorders, angiogenic disorders, cardiovascular disorders, female reproductive disorders, conditions associated with pregnancy, renal disease, inflammatory bowel disease, cryptorchidism, disregulation of spermatogenesis and reproductive development including descent of the gonads, female or male infertility, the method comprising administering to the patient an effective amount of a polypeptide according to the invention. In a further aspect, the invention contemplates the use of a polypeptide or a pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of a INSL3 -mediated disorder in a patient, preferably a human subject, the disorder being selected from the group consisting of : cancers, scleroderma, uncontrolled or abnormal collagen or fibronectin formation or breakdown, neurological disorders, angiogenic disorders, cardiovascular disorders, female reproductive disorders, conditions associated with pregnancy, renal disease, inflammatory bowel disease, cryptorchidism, disregulation of spermatogenesis and reproductive development including descent of the gonads, female or male infertility.

The preferred disorders for treatment are cancers, cardiovascular disorders, female reproductive disorders, conditions associated with pregnancy, cryptorchidism, disregulation of spermatogenesis and reproductive development including descent of the gonads, female or male infertility, more preferably female reproductive disorders, cryptorchidism, disregulation of spermatogenesis and reproductive development including descent of the gonads or female or male infertility, even more preferably cryptorchidism or male infertility. Male infertility includes patients suffering spermatogenesis deficiences with oligospermia or azoospermia. Female infertility includes patients with follicle-genesis deficiency. Cardiovascular disorders according to the present invention include angina, myocardial infarctions such as acute myocardial infarctions, cardiac hypertrophy, and heart failure such as congestive heart failure, cardiomyopathy, valvular regurgitation, and intracardiac shunt and also include arterial diseases, such as atherosclerosis, hypertension, inflammatory vasculitides, Raynaud's disease and Raynaud's phenomenon, aneurysms, aortic stenosis and arterial restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease. Preferably the cardiovascular disease treated is chosen in the group consisting of angina, myocardial infarctions such as acute myocardial infarctions, cardiac hypertrophy, and heart failure such as congestive heart failure. Examples of cancer include but are not limited to, carcinoma including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non- Hodgkin's lymphoma, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms'tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer. The preferred cancer for treatment herein is adenocarcinoma, more preferably adenocarcinoma of the prostate, testis, ovary, breast, endometria, kidney, vulva, pancreas, salivary gland, or thyroid gland, even more preferably adenocarcinoma of the thyroid gland or prostate.

The pharmaceutical compositions of the present invention may contain, in combination with the polypeptides or proteins of the invention as active ingredient, suitable pharmaceutically acceptable diluents, carriers, biologically compatible vehicles and additives which are suitable for administration to an animal (for example, physiological saline solution) and optionally comprising auxiliaries (like excipients, stabilizers, or adjuvants) which facilitate the processing of the active compounds into preparations which can be used pharmaceutically. The pharmaceutical compositions may be formulated in any acceptable way to meet the needs of the mode of administration. For example, the use of biomaterials and other polymers for drug delivery, as well the different techniques and models to validate a specific mode of administration, are disclosed in literature (Luo B and Prestwich GD, 2001; CIe land JL et al., 2001).

"Pharmaceutically acceptable" is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which is administered. For example, for parenteral administration, the above active ingredients may be formulated in unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution. Carriers can be selected also from starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the various oils, including those of petroleum, animal, vegetable or synthetic origin (peanut oil, soybean oil, mineral oil, sesame oil).

Any accepted mode of administration can be used and determined by those skilled in the art to establish the desired blood levels of the active ingredients. For example, administration may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, rectal, oral, or buccal routes. The pharmaceutical compositions of the present invention can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, and the like, for the prolonged administration of the polypeptide at a predetermined rate, preferably in unit dosage forms suitable for single administration of precise dosages.

Parenteral administration can be by bolus injection or by gradual perfusion over time. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients known in the art, and can be prepared according to routine methods. In addition, suspension of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions that may contain substances increasing the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Pharmaceutical compositions include suitable solutions for administration by injection, and contain from about 0.01 to 99.99 percent, preferably from about 20 to 75 percent of active compound together with the excipient.

It is understood that the dosage administered will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art. The total dose required for each treatment may be administered by multiple doses or in a single dose. The pharmaceutical composition of the present invention may be administered alone or in conjunction with other therapeutics directed to the condition, or directed to other symptoms of the condition. Usually a daily dosage of active ingredient is comprised between 0.01 to 100 milligrams per kilogram of body weight per day. Ordinarily 1 to 40 milligrams per kilogram per day given in divided doses or in sustained release form is effective to obtain the desired results. Second or subsequent administrations can be performed at a dosage, which is the same, less than, or greater than the initial or previous dose administered to the individual.

7. Detection of nucleic acids coding for a transcriptional variant of INSL3 polypeptide of the present invention: A further aspect of the present invention relates to compositions and methods for detecting or dosing a nucleic acid, preferably RNA or cDNA, coding for a transcriptional variant of INSL3 polypeptide of the present invention in a sample. Such compositions include, for instance, any specific nucleic acid probes or primers which specifically recognise a nucleic acid encoding the transcriptional variants hereabove described.

A particular embodiment is directed to fragments of a nucleic acid sequence coding an INSL3 polypeptide or variant according to the present invention that may find use as hybridization probes. Such nucleic acid fragments are from 20 through 80 nucleotides in length, preferably from 20 through 60 nucleotides in length, more preferably 20 through 50 nucleotides in length, and most preferably from 20 through 38 nucleotides in length. In a preferred embodiment, the hybridization probes is derived from at least partially a sequence coding for a polypeptide of SEQ ID NO: 1, 2 or a variant of said polypeptide as defined hereabove or a complementary strand thereof. In a further preferred embodiment, the hybridization probe is a nucleic acid from 20 through 38 nucleotides in length coding for a polypeptide of SEQ ID NO: 1, 2 or a complementary strand thereof, and even more preferably a nucleic acid from 20 through 38 nucleotides of SEQ ID NO: 3, 4 or a complementary strand thereof. In this regard, the term "nucleic acid molecule" encompasses all different types of nucleic acids, including without limitation deoxyribonucleic acids (e.g., DNA, cDNA, gDNA, synthetic DNA, etc.), ribonucleic acids (e.g., RNA, mRNA, etc.) and peptide nucleic acids (PNA). In a preferred embodiment, the nucleic acid molecule is a DNA molecule, such as a double-stranded DNA molecule or a cDNA molecule. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as ³²P, ³³P or ³⁵S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the cDNA of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are well known in the art.

Other useful fragments of a nucleic acid sequence coding an INSL3 polypeptide or variant according to the present invention, and preferably fragments of the nucleic acid sequence coding for a polypeptide of SEQ ID NO: 1, 2 or a variant of said polypeptide as defined hereabove or a complementary strand thereof, or of a nucleic acid of SEQ ID NO: 3, 4 or a complementary strand thereof, include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of INSL3 DNA. Such a fragment generally comprises at least 14 nucleotides, preferably from 14 to 38 nucleotides, even more preferably from 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques 6:958, 1988). Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of INSL3 proteins and more particulary to block expression of a specific transcriptional variant.

A further use of the fragments of the nucleic acid sequence as defined here above is their use as primers as to amplify at least a distinctive fragment of a nucleic acid molecule encoding an INSL3 polypeptide or variant as defined above. A "primer" denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase. Typical primers of the present invention are single-stranded nucleic acid molecules of 6 to 50 nucleotides in length, more preferably of 8 to 40 nucleotides in length, even more preferably of 12 to 30 nucleotides in length. The sequence of the primer can be derived directly from the sequence of the target nucleic acid molecule. Perfect complementarity between the primer sequence and the target gene is preferred, to ensure high specificity. However, certain mismatch may be tolerated. In a preferred embodiment the primer is of 12 to 30 nucleotides, more preferably is of 15 to 20 nucleotides in length and is a fragment of SEQ ID NO: 3 or its complementary strand. Such primers include INSL3v-APl (SEQ ID NO: 5), INSL3v-AP2 (SEQ ID NO: 6), INSL3v-AP3 (SEQ ID NO: 7), INSL3v-AP4 (SEQ ID NO: 8) as described there after. These primers are particularly suitable as RT- PCR primers to specifically amplify a transcriptional variant encoded at least in part by exon IA, when associated to another primer chosen upstream or downstream by methods known in the art.

The present invention also relates to the RNA interference (RNAi) technology. RNA interference (RNAi) refers to a mechanism of post-transcriptional gene silencing (PTGS) in which double- stranded RNA (dsRNA) corresponding to a gene or mRNA of interest is introduced into an organism resulting in the degradation of the corresponding mRNA. In the RNAi reaction, both the sense and anti-sense strands of a dsRNA molecule are processed into small RNA fragments or segments ranging in length from 19 to 25 nucleotides (nt), preferably 21 to 23 nt, and having 2-nucleotide 3' tails. These dsRNAs are known as "guide RNAs" or "short interfering RNAs" (siRNAs). siRNAs can also include short hairpin RNAs (shRNAs) in which both strands of an siRNA duplex are included within a single RNA molecule. Alternatively, synthetic dsRNAs, which are 19 to 25 nt in length, preferably 21 to 23 nt, and have 2-nucleotide 3' tails, can be synthesized, purified and used in the reaction. The siRNA duplexes then bind to a nuclease complex composed of proteins that target and destroy endogenous mRNAs having homology to the siRNA within the complex. In this manner, specific mRNAs can be targeted and degraded, thereby resulting in a loss of protein expression from the targeted mRNA. The specific requirements and modifications of dsRNA are described in PCT Publication No. WOO 1/75164 (incorporated herein by reference). While dsRNA molecules can vary in length, it is preferable to use siRNA molecules which are 19- to 25-nt in length, most preferably 21- to 23-nucleotides in length, and which have characteristic 2- to 3- nucleotide 3' overhanging ends typically either (2'-deoxy) thymidine or uracil. The siRNAs typically comprise a 3' hydroxyl group. Single stranded siRNA as well as blunt ended forms of dsRNA can also be used. In order to further enhance the stability of the RNA, the 3' overhangs can be stabilized against degradation. In one such embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine. Alternatively, substitution of pyrimidine nucleotides by modified analogs, e.g., substitution of uridine 2-nucleotide overhangs by (2'-deoxy)thymide is tolerated and does not affect the efficiency of RNAi. The absence of a 2' hydroxyl group significantly enhances the nuclease resistance of the overhang in tissue culture medium. siRNA can be prepared using any of the methods known in the art including those set forth in PCT Publication No. WOO 1/75164 or using standard procedures for in vitro transcription of RNA and dsRNA annealing procedures as described in Elbashir et al. (Genes & Dev., 15:188-200, 2001). In the present invention, the dsRNA, or siRNA, is substantially complementary to at least a part of the mRNA sequence of an INSL3 variant mRNA as described herein and can reduce or inhibit the expression or biological activity of the INSL3 variant described herein. Desirably, the siRNA is 100% complementary to 18 to 25 consecutive nucleotides of the INSL3 variant described herein. Preferably, the decrease in the INSL3 variant described herein biological activity is at least 5% relative to cells treated with a control dsRNA, shRNA, or siRNA, more preferably at least 10%, 20%, or 25%, and most preferably at least 50%. Methods for assaying levels of protein expression are also well known in the art and include western blotting, immunoprecipitation, and ELISA. Methods for assaying the INSL3 polypeptides and variants biological activity include assays described herein.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Further aspects and advantages of the present invention will be disclosed in the following examples, which should be considered as illustrative only, and do not limit the scope of this application.

EXAMPLES

Example 1: Cloning of a human INSL3 variant lacking the connecting C-peptide and the A-chain.

An INSL3 variant lacking the connecting C-peptide and the A-chain and named

INSL3v was predicted in the Human gene of INSL3. Our prediction leads to a INSL3v protein encoded in 75 amino acids (225 bp) spanning 2 exons (the new exon identified has been named exon IA). The prediction contained an initiating methionine, a signal sequence and a stop codon (Figure 1). In order to generate the INSL3v protein both exons were amplified from genomic DNA by PCR (Figure 2). The amplified exons were mixed and a new PCR reaction was performed to amplify the re-assembled DNA. The full length PCR product corresponding to the INSL3v coding sequence (Figure 3) was subcloned into pCR-Bluntll-TOPO cloning vector to generate pCR-Bluntll-TOPO- INSL3v.

1.1 PCR amplification of exons encoding INSL3 V from genomic DNA.

PCR primers were designed to amplify exons 1 and IA of INSL3v (INSL3v-APl/ INSL3v-AP2 and INSL3v-AP3/ INSL3v-AP4, see Table 1). The reverse primer for exon 1 (INSL3v-AP2) had an overlap of 18 bp with exon IA of INSL3 at its 5' end. The forward primer for exon IA (INSL3v-AP3) had a overlap of 17 bp with exon 1 of INSL3 at its 5' end. The forward and reverse primers for exon IA actually overlapped by 11 bp themselves.

To generate exon 1 of INSL3v, the PCR reaction was performed in a final volume of 50 μl containing 1 μl of genomic DNA (0.1 μg/μl, Novagen Inc.), 1.5 μl of 10 mM dNTPs, 1 μl of 50 mM MgSO₄ (Invitrogen), 1.5 μl of INSL3v-APl (10 μM), 1.5 μl of INSL3v-AP2 (10 μM), 10 μl of 1OX Pfx buffer, 0.4 μl of Platinum^® Pfx DNA polymerase (2.5 U/μl, Invitrogen), and either OX, IX or 2X PCR_x Enhancer solution (Invitrogen). Exon IA of INSL3 was produced by the same method using PCR primers INSL3v-AP3 and INSL3v-AP4. Cycling was performed using an MJ Research DNA Engine, programmed as follows: 94 ⁰C, 5 min; 25 cycles of 94 ⁰C, 15 sec, 68 ⁰C, 30 sec; followed by 1 cycle at 68 ⁰C for 5 min and a holding cycle at 4 ⁰C. The PCR products were used as templates to generate the INSL3v ORF without purification.

1.2 Assembly of exons 1 and IA to generate the INSL3v ORF

Exons 1 and IA were assembled in a 50 μl PCR reaction containing 1 μl of exon

1 PCR product, 1 μl of exon 2 PCR product, 1.5 μl of 10 mM dNTPs, 1 μl of 50 mM MgSO₄ (Invitrogen), 1.5 μl of INSL3v-APl (10 μM), 1.5 μl of INSL3v-AP4 (10 μM),

10 μl of 1OX Pfx buffer, 0.4 μl of Platinum^® Pfx DNA polymerase (2.5 U/μl, Invitrogen), and 2X PCR_x Enhancer solution (Invitrogen). Cycling was performed using an MJ Research DNA Engine, programmed as follows: 94 ⁰C, 5 min; 25 cycles of 94 ⁰C, 15 sec, 68 ⁰C, 30 sec; followed by 1 cycle at 68 ⁰C for 5 min and a holding cycle at 4 ⁰C. Reaction products were visualised on a 2 % agarose gel (IX TAE). PCR products of the correct molecular weight (225 bp) were purified from the gel using the Qiagen MinElute DNA Purification System (Qiagen), eluted in 10 μl of EB buffer (1OmM Tris.Cl, pH 8.5) and subcloned directly.

1.3 Subcloning of PCR Products

The PCR product was subcloned into the topoisomerase I modified cloning vector (pCR-Bluntll-TOPO) purchased from the Invitrogen Corporation using the conditions specified by the manufacturer. Briefly, 4 μl of gel purified PCR product was incubated for 15 min at room temperature with 1 μl of TOPO vector and 1 μl salt solution. The reaction mixture was then transformed into E. coli strain TOPlO (Invitrogen) as follows: a 50 μl aliquot of One Shot TOPlO cells was thawed on ice and

2 μl of TOPO reaction was added. The mixture was incubated for 15 min on ice and then heat shocked by incubation at 42 ⁰C for exactly 30 s. Samples were returned to ice and 250 μl of warm (room temperature) SOC media was added. Samples were incubated with shaking (220 rpm) for 1 h at 37 ⁰C. The transformation mixture was then plated on L-broth (LB) plates containing kanamycin (40 μg/ml) and incubated overnight at 37 ⁰C.

1.4 Colony PCR

Colonies were inoculated into 50 μl sterile water using a sterile toothpick. A lO μl aliquot of the inoculum was then subjected to PCR in a total reaction volume of 20 μl containing IX AmpliTaq^® buffer, 200 μM dNTPs, 20 pmoles of T7 primer, 20 pmoles of SP6 primer, 1 unit of AmpliTaq^® DNA polymerase using an MJ Research DNA Engine. The cycling conditions were as follows: 94 ⁰C, 2 min; 30 cycles of 94 ⁰C, 30 sec, 48 ⁰C, 30 sec and 72 ⁰C for 30 sec. Samples were maintained at 4 ⁰C (holding cycle) before further analysis.

PCR reaction products were analyzed on 1 % agarose gels in 1 X TAE buffer. Colonies which gave the expected PCR product size (225 bp cDNA + 186 bp due to the multiple cloning site or MCS) were grown up overnight at 37 ⁰C in 5 ml L-Broth (LB) containing kanamycin (40 μg /ml), with shaking at 220 rpm.

1.5 Plasmid DNA preparation and sequencing

Miniprep plasmid DNA was prepared from the 5 ml culture using a Biorobot 8000 robotic system (Qiagen) or Wizard Plus SV Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 80 μl of sterile water. The DNA concentration was measured using a Spectramax 190 photometer (Molecular Devices). Plasmid DNA (200-500 ng) was subjected to DNA sequencing with the T7 primer using the BigDye Terminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. The primer sequence is shown in Table 1. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.

Sequence analysis identified a clone containing 100% match to the predicted INSL3v sequence, except that it was missing the 3 '-most nucleotide of the cds. This nucleotide (G) was added during subcloning into the pDONR221 Gateway entry vector (see below). The sequence of the cloned cDNA fragment is shown in Figure 3. The plasmid containing the cloned PCR product has been named pCR-Bluntll-TOPO-

INSL3v.

1.6 Construction of mammalian cell expression vectors for INSL3v

Plasmid pCR-BluntII-TOPO-INSL3v was used as PCR template to generate pEAK12d and pDEST12.2 expression clones containing the INSL3v ORF sequence with a 3 ' sequence encoding a 6HIS tag using the Gateway™ cloning methodology (Invitrogen).

1.6.1 Generation of Gateway compatible INSL3v ORF fused to an in frame 6HIS tag sequence.

The first stage of the Gateway cloning process involves a two step PCR reaction which generates the ORF of INSL3v flanked at the 5' end by an attBl recombination site and Kozak sequence, and flanked at the 3' end by a sequence encoding an in- frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA). The missing nucleotide (G) at the 3' end of the coding sequence was added in the Gateway reverse primer INSL3v-EX2.

The first PCR reaction (in a final volume of 50 μl) contains: 1 μl (30 ng) of plasmid pCR-BluntII-TOPO-INSL3v, 1.5 μl dNTPs (10 mM), 10 μl of 1OX Pfx polymerase buffer, 1 μl MgSO4 (50 mM), 0.5 μl each of gene specific primer (100 μM) (INSL3v-EXl and INSL3v-EX2), and 0.5 μl Platinum Pfx DNA polymerase (Invitrogen). The PCR reaction was performed using an initial denaturing step of 95 ⁰C for 2 min, followed by 12 cycles of 94 ⁰C for 15 s; 55 ⁰C for 30 s and 68 ⁰C for 2 min; and a holding cycle of 4 ⁰C. The amplification product was directly purified using the Wizard PCR Preps DNA Purification System (Promega) and recovered in 50 μl sterile water according to the manufacturer's instructions.

The second PCR reaction (in a final volume of 50 μl) contained 10 μl purified PCRl product, 1.5 μl dNTPs (10 mM), 5 μl of 1OX Pfx polymerase buffer, 1 μl MgSO₄ (50 mM), 0.5 μl of each Gateway conversion primer (100 μM) (GCP forward and GCP reverse) and 0.5 μl of Platinum Pfx DNA polymerase. The conditions for the 2nd PCR reaction were: 95 ⁰C for 1 min; 4 cycles of 94 ⁰C, 15 sec; 50 ⁰C, 30 sec and 68 ⁰C for 2 min; 25 cycles of 94 ⁰C, 15 sec; 55 ⁰C , 30 sec and 68 ⁰C, 2 min; followed by a holding cycle of 4 ⁰C. A 10 μl aliquot was visualized on 0.8 % agarose gel in 1 X TAE buffer (Invitrogen) in order to verify that the product was of the expected molecular weight (225 + 70 = 295 bp). The remaining 40 μl were loaded on 0.8 % agarose gel in 1 X TAE buffer gel and the band was purified using the Wizard PCR Preps DNA Purification System (Promega) and recovered in 50 μl sterile water according to the manufacturer's instructions.

1.6.2 Subcloning of Gateway compatible INSL3v ORF into Gateway entry vector pDONR221 and expression vectors pEAK12d and pDEST12.2

The second stage of the Gateway cloning process involves subcloning of the Gateway modified PCR product into the Gateway entry vector pDONR221 (Invitrogen) as follows: 5 μl of purified product from PCR2 were incubated with 1.5 μl pDONR221 vector (0.1 μg/μl), 2 μl BP buffer and 1.5 μl of BP clonase enzyme mix (Invitrogen) in a final volume of 10 μl at RT for 1 h. The reaction was stopped by addition of 1 μl proteinase K (2 μg/μl) and incubated at 37 ⁰C for a further 10 min. An aliquot of this reaction (1 μl) was used to transform E. coli DHlOB cells by electroporation as follows: a 25 μl aliquot of DHlOB electrocompetent cells (Invitrogen) was thawed on ice and 1 μl of the BP reaction mix was added. The mixture was transferred to a chilled 0.1 cm electroporation cuvette and the cells electroporated using a BioRad Gene- Pulser™ according to the manufacturer's recommended protocol. SOC media (0.5 ml) which had been pre-warmed to room temperature was added immediately after electroporation. The mixture was transferred to a 15 ml snap-cap tube and incubated, with shaking (220 rpm) for 1 h at 37 ⁰C. Aliquots of the transformation mixture (10 μl and 50 μl) were then plated on L-broth (LB) plates containing kanamycin (40 μg/ml) and incubated overnight at 37 ⁰C.

Plasmid miniprep DNA was prepared from 5 ml culture from 8 of the resultant colonies using a Qiaprep BioRobot 8000 system (Qiagen). Plasmid DNA (150-200 ng) was subjected to DNA sequencing with 21M13 and M13Rev primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4336919) according to the manufacturer's instructions. The primer sequences are shown in Table 1. Sequencing reactions were purified using Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.

Plasmid eluate (2 μl or approx. 150 ng) from one of the clones which contained the correct sequence (pENTR_INSL3v-6HIS, plasmid ID 16504) was then used in a recombination reaction containing 1.5 μl of either pEAK12d vector or pDEST12.2 vector (0.1 μg / μl), 2 μl LR buffer and 1.5 μl of LR clonase (Invitrogen) in a final volume of 10 μl. The mixture was incubated at RT for 1 h, stopped by addition of 1 μl proteinase K (2 μg/μl) and incubated at 37 ⁰C for a further 10 min. An aliquot of this reaction (1 μl) was used to transform E. coli DHlOB cells by electroporation as follows: a 25 μl aliquot of DHlOB electrocompetent cells (Invitrogen) was thawed on ice and 1 μl of the LR reaction mix was added. The mixture was transferred to a chilled 0.1 cm electroporation cuvette and the cells electroporated using a BioRad Gene-Pulser™ according to the manufacturer's recommended protocol. SOC media (0.5 ml) which had been pre-warmed to room temperature was added immediately after electroporation. The mixture was transferred to a 15 ml snap-cap tube and incubated, with shaking (220 rpm) for 1 h at 37 ⁰C. Aliquots of the transformation mixture (10 μl and 50 μl) were then plated on L-broth (LB) plates containing ampicillin (100 μg/ml) and incubated overnight at 37 ⁰C.

Colony PCR was performed from 4 of the resultant colonies resuspended in 20μl water. Colony PCR reaction (in a final volume of 20 μl) contained 5 μl of resuspended colony, 0.5 μl dNTPs (10 mM), 2 μl of 1OX PCR buffer containing 15 mM MgCl₂, 1 μl of each vector specific primers (10 μM) (pEAK12F and pEAK12R or M13Rev and 21M13) and 0.2 μl of AmpliTaq DNA polymerase (AppliedBio systems). The conditions for the PCR reaction were: 96 ⁰C for 2 min; 35 cycles of 94 ⁰C, 30 sec; 55 ⁰C, 30 sec and 72 ⁰C for 45 sec; followed by a holding cycle of 4 ⁰C. PCR reactions were visualized on 0.8 % agarose gel in 1 X TAE buffer (Invitrogen) in order to verify the presence of insert. The expected molecular weight of the PCR product amplified was 543 bp in pEAK12d vector and 464 bp in pDEST12.2 vector.

CsCl gradient purified maxi-prep DNA was prepared from a 500 ml culture of a positive clone containing the expected insert (pEAK12d_INSL3v-6HIS, plasmid ID 16505) using the method described by Sambrook J. et al, 1989 (in Molecular Cloning, a Laboratory Manual, 2^nd edition, Cold Spring Harbor Laboratory Press), Plasmid DNA was resuspended at a concentration of 1 μg/μl in sterile water (or 10 mM Tris-HCl pH 8.5). DNA was sequence verified and stored at -20 ⁰C.

Endotoxin- free maxi-prep DNA was also prepared from a 500 ml culture of a positive clone containing the expected insert (pDEST12.2_INSPINSL3v-6HIS, plasmid ID 16506) using the EndoFree Plasmid Mega kit (Qiagen) according to the manufacturer's instructions. Purified plasmid DNA was resuspended in endotoxin free

TE buffer at a final concentration of at least 3 μg/μl, sequence verified, and stored at -

20 ⁰C.

Example 2: Expression of a human INSL3 variant lacking the connecting C- peptide and the A-chain.

2.1 Functional genomics expression in mammalian cells of the cloned, His-tagged plasmid #16505

Human Embryonic Kidney 293 cells expressing the Epstein-Barr virus Nuclear Antigen (HEK293-EBNA, Invitrogenj were maintained in suspension in Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH). Cells are inoculated at IxIO⁶ cells/ml in 250ml FEME (DMEM/Ham's F-12 1:1 19 mM HEPES, 5g/L Glucose, 7.5 mM L-Glutamine, 4ml/L ITS-X) (all Invitrogen-Life Technologies) medium supplemented with 1% FCS. For the transfection-mix 500μg DNA (#16505) plus 10 μg reporter-gene DNA is diluted in 50ml FEME 1% FCS. Then ImI PEI (lmg/1 Polysciences, USA) is added. This mix is incubated for 10 minutes at room temperature. After 10 minutes the transfection mix is added to the cells and the culture is incubated at 37°C in the incubator for 90 min. Finally the volume is topped up with the remaining 200ml FEME 1%FCS containing 2.5ml Pen-Strep to prevent contamination due to non- sterility of DNA. Confirmation of positive transfection was done by qualitative fluorescence examination at day 6 (Axiovert 10 Zeiss). On day 6 (harvest day), supernatant (500ml) was centrifuged (4°C, 40Og) and placed into a pot bearing a unique identifier.

One aliquot (500μl) was kept for QC of the 6His-tagged protein (internal bioprocessing QC). 2.2 Purification of the cloned, His-tagged plasmid #16505

The 500 ml culture medium sample containing the recombinant protein with a C- terminal 6His tag was diluted with one volume cold buffer A (50 mM NaH₂PO₄; 600 mM NaCl; 8.7 % (w/v) glycerol, pH 7.5) to a final volume of 1000 ml. The sample was filtered through a 0.22 μm sterile filter (Millipore, 500 ml filter unit) and kept at 4⁰C in a 1 liter sterile square media bottle (Nalgene).

The purification was performed at 4⁰C on a VISION workstation (Applied Biosystems) connected to an automatic sample loader (Labomatic). The purification procedure was composed of two sequential steps, metal affinity chromatography on a Poros 20 MC (Applied Biosystems) column charged with Ni ions (10 x 50 mm, 3.93 ml), followed by buffer exchange on a Sephadex G-25 medium (Amersham Pharmacia) gel filtration column (l,0 x 15 cm). For the first chromatography step the metal affinity column was regenerated with 30 column volumes of EDTA solution (100 mM EDTA; 1 M NaCl; pH 8.0), recharged with Ni ions through washing with 15 column volumes of a 100 mM NiSO₄ solution, washed with 10 column volumes of buffer A, followed by 7 column volumes of buffer B (50 mM NaH₂PO₄; 600 mM NaCl; 8.7 % (w/v) glycerol, 400 mM; imidazole, pH 7.5), and finally equilibrated with 15 column volumes of buffer A containing 15 mM imidazole. The sample was transferred, by the Labomatic sample loader, into a 200 ml sample loop and subsequently charged onto the Ni metal affinity column at a flow rate of 20 ml/min. The charging procedure was repeated 5 times in order to transfer the entire sample (1000 ml) onto the Ni column. Subsequently the column was washed with 12 column volumes of buffer A, followed by 28 column volumes of buffer A containing 20 mM imidazole. During the 20 mM imidazole wash loosely attached contaminating proteins were eluted of the column. The recombinant His-tagged protein was finally eluted with 10 column volumes of buffer B at a flow rate of 2 ml/min, and the eluted protein was collected in a 2.7 ml fraction. For the second chromatography step, the Sephadex G-25 gel-filtration column was regenerated with 2 ml of buffer D (1.137 M NaCl; 2.7 mM KCl; 1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; pH 7.2), and subsequently equilibrated with 4 column volumes of buffer C (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; 20 % (w/v) glycerol; pH 7.4). The peak fraction eluted from the Ni-column was automatically, through the integrated sample loader on the VISION, loaded onto the Sephadex G-25 column and the protein was eluted with buffer C at a flow rate of 2 ml/min. The desalted sample was recovered in a 2.7 ml fraction. The fraction was filtered through a 0.22 μm sterile centrifugation filter (Millipore), aliquoted, frozen and stored at -80° C. An aliquot of the sample was analyzed on SDS-PAGE (4-12 % NuPAGE gel; Novex) by Coomassie blue staining and Western blot with anti-His antibodies.

Coomassie Blue staining: The NuPAGE gel was stained in a 0.1 % coomassie blue R250 staining solution (30 % methanol, 10 % acetic acid) at room temperature for 1 h and subsequently destained in 20 % methanol, 7.5 % acetic acid until the background was clear and the protein bands clearly visible.

Western blot: Following the electrophoresis the proteins were electro transferred from the gel to a nitrocellulose membrane at 290 mA for 1 hour at 4⁰C. The membrane was blocked with 5 % milk powder in buffer E (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; 0.1 % Tween 20, pH 7.4) for 1 h at room temperature, and subsequently incubated with a mixture of 2 rabbit polyclonal anti-His antibodies (G- 18 and H-15, 0.2ug/ml each; Santa Cruz) in 2.5 % milk powder in buffer E overnight at 4°C. After further 1 hour incubation at room temperature, the membrane was washed with buffer E (3 x 10 min), and then incubated with a secondary HRP-conjugated anti- rabbit antibody (DAKO, HRP 0399) diluted 1/3000 in buffer E containing 2.5 % milk powder for 2 hours at room temperature. After washing with buffer E (3 x 10 minutes), the membrane was developed with the ECL kit (Amersham) for 1 min. The membrane was subsequently exposed to a Hyperfilm (Amersham), the film developed and the Western blot image visually analyzed.

Example 3: Tissue Distribution of the human INSL3 variant lacking the connecting C-peptide and the A-chain.

The expression pattern of the predicted INSL3v mRNA may be determined using RT- PCR analysis. cDNA templates of various tissues may be amplified using variant- specific primers, to determine tissue expression of the variants. Example 4: Biological activity of the polypeptides of the present invention.

The biological activity of the polypeptides of this invention can be verified using several biological assays that are known per se in the art.

4.1 Binding of the polypeptides of the present invention to the LGR8 receptor in an in vitro cellular test:

Different tests are described in the art to test the binding of a ligand to its receptor and these techniques are known to those skilled in the art. For example, the binding of the polypeptides of the present invention to the LGR8 receptor can be tested by the test disclosed by Bullesbach EE and Schwabe C (J Biol Chem. 2005. 280(15):14586-90). Cells (293T/17) are stably transfected with a LGR8 expressing vector, using for example TransFast transfection reagent (Promega) according to the manufacturer's protocol. Clones expressing the LGR8 receptor are selected and are cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and the corresponding selective marker. For the binding assay, transfected 293T/17 cells expressing LGR8 are grown to 80% confluence and then dislodged with 0.5 M EDTA (pH 7.5, 1 ml/10 ml of conditioned medium) for 10 min at 37 ⁰C. Cells are collected by centrifugation at 2000 X g for 10 min, suspended two times in 1 ml of ice-cold binding buffer (20 mM Hepes, pH 7.5, 1% bovine serum albumin, 0.1 mg/ml lysine, 1.5 mM CaC12, 50 mM NaCl, 0.01% NaN3), and recentrifuged at 4 ⁰C for 10 min at 2000 X g. The pellet is reconstituted to 25x10⁶ cells/ml of binding buffer. For binding assays ¹²⁵I- labeled polypeptide to be tested, or wild-type human ¹²⁵I-INSL3 as a control, in 20 μl of binding buffer is placed into a 1.5-ml Eppendorf vial together with 40 μl of binding buffer with or without different concentration of unlabeled polypeptide or wild-type human INSL3 and 40 μl (10⁶ cells) of the cell suspension and incubated at room temperature for 60 min. Thereafter the cells are diluted with 1 ml of ice-cold binding buffer and collected by centrifugation (10 min at 2000 x g at 4 ⁰C); the pellet is washed with 1 ml of the same buffer and centrifuged for 10 min at 5000 x g at 4 ⁰C. The tips of the vials containing the pellets are placed in counting tubes and transferred to a γ counter for analysis. The total binding is determined in the absence and nonspecific binding in the presence of unlabeled polypeptide. Specific binding at a given ¹²⁵I- polypeptide concentration is defined as the difference in bound radioactivity between samples incubated in the absence of unlabeled polypeptide or in the presence of an excess of unlabeled polypeptide. Scatchard plot analyses of the binding data are performed.

4.2 Modulation of the cAMP levels ofLGR8 expressing cells by the polypeptides of the present invention:

Assay 1:

INSL3 activity may be assessed by measuring cAMP level following activation of the LGR8 receptor. An example of this assay is described by Gorlov IP, et al. (Hum MoI Genet., 2002, l l(19):2309-18 and the subject matter relating to the cloning and expression of the GREAT gene in mammalian cells and the measurement of the cAMP level is hereby incorporated into the present application by reference.

Assay 2:

Human 293 T cells stably trans fected with human LGR8 are preincubated in the presence of 0.25mM 3-isobutyl-l-methyl xanthine (IBMX, Sigma) before treatment with the polypeptide to be tested or with wild-type human INSL3 as a positive control and without such polypeptide as a negative control. At the end of the incubation, measurement of cAMP levels can be made using any known techniques, such as the well-characterized cAMP ELISA (Satoko et al, J. Biol Chem, 278 : 7855- 7862). AU experiments are repeated at least three times.

4.3 In vitro induction of oocyte maturation by the polypeptides of the present invention:

Assay 1:

For evaluating oocyte maturation, cumulus-enclosed oocytes (CEOs) are obtained from pregnant mare serum gonadotropin (PMSG)-treated rats and cultured as described (Vaknin, K. M., Lazar, S., Popliker, M. & Tsafriri, A. (2001) Biol. Reprod.

64, 299-309). CEOs are collected in the L15 medium containing 0.25 mM of MIX within 20 min of ovary retrieval, washed twice in L15 medium without MIX, and transferred to the test medium without MIX. The CEOs are cultured with or without different doses of the polypeptide to be tested or other hormones. For controls, the CEOs are cultured in L15 medium containing MIX. At the end of culture, the occurrence of germinal vesicle breakdown (GVBD) in the oocytes is examined after removing cumulus cells surrounding the CEOs by using a small-bore pipette under Hoffman modulation contrast microscopy (Nikon, Tokyo).

Kawamura K, et al, Proc Natl Acad Sci U S A. 2004 May l l;101(19):7323-8 have shown that INSL3 induces oocyte maturation in vitro by using such a model. Assay 2: Induction of maturation of preovulatory follicles by the polypeptides of the present invention. For preovulatory follicle cultures, follicles are excised from the ovary after PMSG treatment for 48 h. The follicles, 20-30 per vial, are cultured with or without different doses of the polypeptide to be tested or luteinizing hormone (LH) (5 mg/ml) in the L15 medium. Some follicles are also treated with Pertussis toxin (1 mg/ml) to suppress Gi activity. The vials are flushed at the start of the culture with O2/N2 (1/1) and cultured at 37⁰C with gentle shaking. After culture, individual CEOs are dissected from each follicle to examine the occurrence of GVBD. Kawamura K, et al., Proc Natl Acad Sci U S A. 2004 May l l;101(19):7323-8 have shown that INSL3 induces oocyte maturation in vitro by using such a model.

4.4 In vivo induction of oocyte maturation by the polypeptides of the present invention:

To test the effect of the polypeptides of the present invention on oocyte maturation in vivo, intrabursal injection of the polypeptide to be tested is performed as described (Vaknin, K. M., Lazar, S., Popliker, M. & Tsafriri, A. (2001) Biol. Reprod. 64, 299-309). Immature rats are primed with PMSG (15 IU) and, at 48 h later, lightly anesthetized, and one of the ovaries is exteriorized through a small lumbosacral incision. The polypeptide to be tested or PBS as a negative control are injected through a 30-gauge needle threaded into the ovarian bursa via the adjoining fat pad. After injection, the ovary is returned to the abdominal cavity and skin is clipped. For positive control, hCG (10 units) is administrated by i.p. injection. At different intervals after treatment, CEOs are collected by puncturing the largest ovarian follicles to examine the occurrence of oocyte GVBD. Kawamura K, et al, Proc Natl Acad Sci U S A. 2004 May l l;101(19):7323-8 have shown that INSL3 induces oocyte maturation in vivo by using such a model.

4.5 In vivo enhancement of the survival of sperm cells by the polypeptides of the present invention:

In order to test the in vivo activity of the polypeptides of the present invention on the testis, male Sprague-Dawley rats at 28 days of age are treated s.c. with the GnRH antagonist ganirelix acetate (250 mg/kg/day) to suppress pituitary gonadotropin secretion. Some animals are treated with the GnRH antagonist together with s.c. injections of hCG (75 units/day) or the polypeptides to test. Animals are killed 5 days later, and organ weight is determined. Testes are stored at -7O⁰C before RNA or DNA extraction, or fixed in Bouin's solution for in situ labeling of DNA ends. DNA from whole testis is isolated and quantitated with spectrophotometry at 260 nm. Aliquots of DNA (500 ng) from each sample are labeled at 3' ends with ³²P-dideoxy-ATP (3,000 Ci/mmol; Amersham Biosciences) by using terminal transferase (25 units per sample; Boehringer-Mannheim) as described by Billig, H., Furuta, L, Rivier, C, Tapanainen, J., Parvinen, M. & Hsueh, A. J. (1995) Endocrinology 136, 5-12.

Labeled samples are fractionated through 2% agarose gels. After electrophoresis, gels are dried for 2 h in a slab-gel dryer without heat and exposed to Kodak X-Omat AR film at -70° C. DNA fragmentation is visualized by autoradiography and quantitated by β- counting of low molecular weight (<15 kb) DNA fragments. For in situ DNA 3 '-end labeling, fixed testicular tissues are embedded in paraffin and cut into 3-m m sections. To detect apoptotic DNA fragmentation, the Fluorescein in situ cell death detection kit (Roche Diagnostics) is used according to manufacturer's instructions, and sections are counterstained with propidium iodide (0.01 mg/ml, Sigma).

Kawamura K, et al., Proc Natl Acad Sci U S A. 2004 May l l;101(19):7323-8 have shown that rat INSL3 is a survival factor for male germ cells in such a model.

4.6 In vivo induction of the descent of the gonads by the polypeptides of the present invention:

Insl3 -deficient mice have been described (Nef S, Parada LF. Nat Genet. 1999 Jul;22(3):295-9). These mice can be use as a model to test the ability of the polypeptides of the present invention to induce the descent of the gonads and/or the maturation and/or descent of the testes. The polypeptide to be tested is injected to the deficient mice at different stage of development and at different dosage and/or different modes of administration are tested. The biological effect of such a treatment is followed, in particular the effect on the gonads.

4.7 Stimulation of the proliferation of cancer cells by the polypeptides of the present invention:

In vitro and in vivo models to test the ability of polypeptides to stimulate the proliferation of cancer cells are well known in the art. For example the proliferation of one or more cell line(s) derived from the cancer to be tested (in particular cells originating from the thyroid gland or prostate) can be measured in vitro by using different techniques well known in the art. Such techniques include for example incorporation of [³H] -thymidine, MTT cell proliferation assay, counting of cell numbers... and generally consist in comparing the level of proliferation with or without the polypeptide to be tested. In vivo models including for example human tumors cells xenografted onto athymic mice can also be used to test the ability of the polypeptides of the present invention to induce proliferation of cancer cells in vivo. These models can also be used to test the ability of the antibodies of the present invention to inhibit the proliferation of cancer cells.

Table 1

Underlined sequence = Kozak sequence Bold = Stop codon

Italic sequence = His tag Highlighted sequence = overlap with adjacent exon

Claims

1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising the sequence set forth at SEQ ID NO: 1; b) a polypeptide comprising a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1.

2. An polypeptide according to claim 1 selected from the group consisting of: a) a polypeptide comprising the sequence set forth at SEQ ID NO: 2; b) a polypeptide comprising the sequence set forth at amino acids 21 to 75 of SEQ ID NO: 2; c) a polypeptide comprising a polypeptide differing solely from a) or b) by one, two, three or four mutations chosen in the group consisting of: A24G, A60T, V43L and P49S; d) a polypeptide comprising a sequence having at least 80% amino acid sequence identity with a) or b). 3. A polypeptide according to claim 1 selected from the group consisting of: a) a polypeptide consisting of SEQ ID NO: 1; b) a polypeptide consisting of a polypeptide having at least 80% amino acid sequence identity with SEQ ID NO: 1.

4. A polypeptide according to claim 2 selected from the group consisting of: a) a polypeptide consisting of SEQ ID NO: 2; b) a polypeptide consisting of the sequence set forth at amino acids 21 to 75 of SEQ ID NO: 2; c) a polypeptide consisting of a polypeptide differing form a) or b) by one, two, three or four mutations chosen in the group consisting of: A24G, A60T, V43L and P49S; d) a polypeptide consisting of a polypeptide having at least 80% amino acid sequence identity with a) or b).

5. An isolated polypeptide of any one of the preceding claims, which binds the human LGR8 receptor.

6. A fusion protein comprising a polypeptide of any one of claims 1 to 5 operably linked to an additional amino acid domain.

7. The fusion protein of claim 6, wherein the polypeptide is operably linked to the GST sequence, a His tag sequence, a multimerication domain, the constant region of an immunoglobulin molecule or a heterodimeric protein hormone such as human chorionic gonadotropin (hCG).

8. An isolated nucleic acid molecule encoding a polypeptide of any one of claims 1 to 7.

9. An isolated nucleic acid molecule of claim 8, which is a cDNA molecule.

10. An isolated nucleic acid molecule of claim 9, which comprises or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 3 and SEQ ID

NO: 4, or a complementary strand or degenerate sequence thereof, or a nucleic acid coding for the polypeptides of SEQ ID NO: 1 or 2, or a complementary strand.

11. A vector comprising a nucleic acid molecule of any one of claims 8 to 10.

12. A recombinant host cell, wherein said cell comprises a nucleic acid molecule of any one of claims 8 to 10 or a vector of claim 11.

13. The host cell of claim 12, which is a prokaryotic or eukaryotic cell.

14. A method of producing a polypeptide of any one of claims 1 to 7, the method comprising culturing a recombinant host cell of claim 12 or 13 under conditions allowing expression of the nucleic acid molecule, and recovering the polypeptide produced.

16. The polypeptide of any one of claims 1 to 7 in the form of active conjugates or complex.

17. The polypeptide according to claim 16 which is pegylated.

18. An antibody, or a fragment or derivative thereof, which selectively binds to a polypeptide of any one of claims 1 to 7.

19. An antibody of claim 18, which is a monoclonal antibody or a fragment or derivative thereof.

20. An antibody of claim 18 or 19, which is a human or a humanized antibody or a fragment or derivative thereof.

21. An immunoconjugate comprising an antibody according to any one of claims 18 to 20 conjugated to a heterologous moiety.

22. The polypeptide according to any one of claims 1 to 7, or 16 to 17 for use as a medicament.

23. A pharmaceutical composition comprising a polypeptide of any one of claims 1 to 7 or 16 to 17, a nucleic acid molecule of any one of claims 8 to 10, a vector of claim 11 or a cell of claim 12 or 13, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

24. A method of treating, preventing or ameliorating the symptoms of a disorder in a patient, the disorder involving disregulation of INSL3 expression or activity, the method comprising administering to the patient a pharmaceutical composition of claim 23.

25. A method of treating, preventing or ameliorating the symptoms of a INSL3- mediated disorder in a patient, wherein the disorder is selected from the group consisting of cancers, scleroderma, uncontrolled or abnormal collagen or fibronectin formation or breakdown, neurological disorders, angiogenic disorders, cardiovascular disorders, female reproductive disorders, conditions associated with pregnancy, renal disease, inflammatory bowel disease, cryptorchidism, disregulation of spermatogenesis and reproductive development including descent of the gonads, female or male infertility, the method comprising administering to the patient an effective amount of a polypeptide according to any one of claims 1 to 7, or 16 to 17 or of a pharmaceutical composition according to claim 23.

26. Use of a polypeptide according to any one of claims 1 to 7, or 16 to 17 or of a pharmaceutical composition according to claim 23 in the manufacture of a medicament for the treatment of a INSL3 -mediated disorder in a patient, the disorder being selected from the group consisting of : cancers, scleroderma, uncontrolled or abnormal collagen or fibronectin formation or breakdown, neurological disorders, angiogenic disorders, cardiovascular disorders, female reproductive disorders, conditions associated with pregnancy, renal disease, inflammatory bowel disease, cryptorchidism, disregulation of spermatogenesis and reproductive development including descent of the gonads, female or male infertility.

27. The method or use according to claim 25 or 26 wherein the disorder is selected from the group consisting of cancers, cardiovascular disorders, female reproductive disorders, conditions associated with pregnancy, cryptorchidism, disregulation of spermatogenesis and reproductive development including descent of the gonads, female or male infertility, more preferably female reproductive disorders, cryptorchidism, disregulation of spermatogenesis and reproductive development including descent of the gonads or female or male infertility, even more preferably cryptorchidism or male infertility. 28. The method or use according to claim 27 wherein the disorder is a cardiovascular disorder selected from the group consisting of: angina, myocardial infarctions such as acute myocardial infarctions, cardiac hypertrophy, and heart failure such as congestive heart failure, or a cancer selected from the group consisting of adenocarcinoma of the prostate, testis, ovary, breast, endometria, kidney, vulva, pancreas, salivary gland, or thyroid gland, more preferably adenocarcinoma of the thyroid gland or prostate.

29. The antibody, or fragment or derivative thereof according to any one of claims 18 to 20 for use as a medicament.

30. A pharmaceutical composition comprising an antibody, or a fragment or a derivative thereof of any one of claims 18 to 20, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

31. A method of treating, preventing or ameliorating the symptoms of a cardiovascular disease or a cancer in a subject, the method comprising administering to the patient an effective amount of an antibody, or a fragment or a derivative thereof of any one of claims 18 to 20. 32. Use of an antibody, or a fragment or a derivative thereof of any one of claims 18 to 20 in the manufacture of a medicament for the treatment of a cardiovascular disease or a cancer.

33. A nucleic acid probe, wherein said probe selectively hybridizes to a nucleic acid as defined in any one of claims 8 to 10 or the complementary strand thereof.

34. A nucleic acid primer that can be used to amplify at least a distinctive fragment of a nucleic acid molecule encoding an INSL3 polypeptide according to any one of claims 1 to 7.