AU9037898A - Insulin-like polypeptide and uses therefor - Google Patents

Description

WO 99/15664 PCT/US98/17888 INSULIN-LIKE POLYPEPTIDE AND USES THEREFOR FIELD OF THE INVENTION The present invention relates to a novel insulin-like polypeptide (ILP), nucleic acid encoding ILP, vectors and host cells comprising ILP encoding nucleic acid, and methods of producing ILP. 5 BACKGROUND OF THE INVENTION Insulin is a well-studied member of a family of homologous proteins including insulin-like growth factors (IGF-I, and -II,), relaxin, placentin. and other like proteins. Human insulin is a small protein with a molecular weight of 5.8 kDa. It is composed of two amino acid chains (A and B) connected to each other by disulfide linkages. A third amino acid chain (C-peptide) is cleaved from proinsulin to produce mature insulin. 10 An important effect of mature insulin is its ability to increase the rate of glucose transport through the membranes of most cells in the body. In the complete absence of insulin, the overall rate of glucose transport into cells becomes only about one-fourth the normal value. On the other hand, when great excesses of insulin are secreted and when an excess of glucose is available to be transported, the rate of glucose transport into cells may be as great as five times normal. Thus, the rate of glucose transport for many tissues 15 can be altered as much as 20-fold. Insulin promotes glucose transport into cells by stimulating a process of facilitated diffusion in which insulin combines with a membrane receptor molecule. Enhanced transport of glucose through the cell membrane by insulin is particularly effective in skeletal muscle and adipose tissue. In addition, insulin enhances glucose transport into the heart and certain smooth muscle organs, such as the uterus. When there 20 is an excess of both insulin and glucose in the blood, glycogen stores in skeletal muscle increase markedly, and there is a moderate enhancement of glycogen in the skin, glands, and other tissues. In adipose tissue, the excess glucose transported into the fat cells is largely converted into fat and stored in this form. In liver cells, after a large portion of the excess glucose has been stored as glycogen and the glycogen content has reached its limit in these cells, most of the remaining excess glucose is converted into fat. Thus, a rapid and potent 25 effect of insulin is to promote fat storage in the adipose tissue. Insulin also affects protein metabolism by increasing active transport of amino acids into cells. accelerating translation of mRNA to protein, and increasing transcription of DNA to form the mRNA for subsequent translation. The metabolic effects of insulin make it nearly as important to growth as growth hormone. A lack 30 of insulin causes extreme wasting of body proteins, with consequent release of amino acids into the circulating body fluids and elevated plasma amino acid levels. Protein wasting is one of the most serious of all the effects of severe diabetes mellitus, leading to extreme weakness and abnormal organ function. A related molecule, IGF-I, is a peptide present in blood plasma, cerebral spinal fluid, and other body fluids. It comprises 70 amino acids, including three disulfide bonds. IGF-I can stimulate growth of a wide 35 range of cell types and can mediate the effects of growth hormone on skeletal growth. Most tissues and especially the liver produce IGF-1 together with specific IGF-binding proteins. These molecules are under the control of growth hormone (GH). Like GH, IGF-I is a potent anabolic protein (see, for example, Tanner et al. (1977) Acta Endocrinol. 84:681-696; and Uthne et al. (1974) J. Clin. Endocrinol. Metab. 39:548-554).

-I

SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 IGF-I has been isolated from human serum and produced recombinantly (see, for example, EP 123,228 and EP 128,733). Insulin-like growth factors, including IGF-1 and IGF-II, are chemically related to human proinsulin in that they contain A and B domains connected by a C domain region, and have high homology to proinsulin. 5 The IGFs further contain a D domain at the C-terminus that is not found in proinsulin. The IGFs are functionally homologous to insulin as well by stimulating phosphorylation of specific tyrosine residues within the cytoplasmic domain of the receptors to which they bind. Proteins with extensive homologies to human IGF-I are present in samples of IGF-I purified from plasma of other species. IGF-I has both systemic and local effects and appears to be associated with different 10 specific binding proteins, several of which have been sequenced and are termed IGFBP- 1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, Mac 25 (IGFBP-7), and prostacyclin-stimulating factor (PSF) or endothelial cell-specific molecule (ESM-1). Mac 25 is described, for example, in Oh et al., J. Biol. Chem., 271: 30322 30325 (1996). PSF is described in Yamauchi et al., Biochemical Journal, 303: 591-598 (1994). ESM-l is described in Lassalle et al., J. Biol. Chem., 271: 20458-20464 (1996). For other identified IGFBPs, see, e.g., 15 EP 375.438 published 27 June 1990: EP 369,943 published 23 May 1990; WO 89/09268 published 5 October 1989; Wood et al.. Molecular Endocrinology, 2:1176-1185 (1988); Brinkman et al., The EMBO J., 7: 2417 2423 (1988); Lee et al., Mol. Endocrinol., 2: 404-411 (1988); Brewer et al., BBRC, 152: 1289-1297 (1988); EP 294,021 published 7 December 1988; Baxter et al., BBRC, 147: 408-415 (1987); Leung et al., Nature, 330: 537-543 (1987); Martin et al., J. Biol. Chem., 261: 8754-8760 (1986); Baxter et al., Comp. Biochem. Physiol., 20 91B: 229-235 (1988); WO 89/08667 published 21 September 1989; WO 89/09792 published 19 October 1989; and Binkert etal., EMBO J., 8: 2497-2502 (1989). These binding proteins appear to modulate the biological functions and availability of IGF-I in both positive and negative manners. Analogues with changed affinities for the binding proteins have been produced and changes of biological activities related to sequence variation have been found. IGF-I appears to act mainly by interactions with the IGF-type 1 receptor exposed on the 25 other surface of plasma membranes in many different cell types. Binding to IGF type 2- and insulin receptors also seems to be of importance. The availability of recombinant human IGF-I (rhIGF-I) has provided a means to evaluate the scope of the hormone's affects on body fuel metabolism (see, for example Boulware, S.D. et al. (1992) Am. J. Physiol. 262:E130-EI33 and references cited therein). In normal fasted rats, rhIGF-I has been shown to 30 produce hypoglycemia, even when infused simultaneously with anti-insulin serum. While both insulin and rhlGF-I have been shown to stimulate peripheral glucose uptake, rhIGF-I had little or no suppressive effect on production of glucose by the liver (Jacob, R. et al. (1989) J. Clin. Invest. 83:1717-1723). rhIGF-I also had no detectable effect on free fatty acid (FFA) levels in rats, a feature distinguished from that of insulin. Researchers have found that an intravenous bolus injection of IGF-I lowers blood glucose levels in 35 humans (Guler et al. (1987) N. Engl. J. Med. 317:137-140). Additionally, IGF-I promotes growth in several metabolic conditions characterized by low IGF-I levels, such as hypophysectomized rats (Guler et al., Endocrinology, 118:Supp 129 abstract, Skottner et al., (1987) J. Endocrinol. 112:123-132, Guler et al., (1988) PNAS USA 85:4889-4893; Froesch et al., in Endocrinology Intl. Congress Series 655, Labrie and Proulx, eds., -2 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Amsterdam: Excerpta Medica, 1984). diabetic rats (Scheiwiller et al. (1986) Nature 323:169-171 (1986)), and dwarf rats (Skottner et al. (1989) Endocrinology 124:2519-2526). The kidney weight of hypophysectomized rats increases substantially upon prolonged infusions of IGF-I subcutaneously (Guler et al.. Proceedings of the 1 st European Congress of Endocrinology 103:abstract 12-390 (Copenhagen, 1987)). An additional use 5 for IGF-I is its administration to improve glomerular filtration and renal plasma flow in human patients (see, for example, EP 327,503; and Guler et al. (1989) PNAS USA 86:2868-2872). In human subjects, it was found that a continuous infusion of rhIGF-I produced marked changes in glucose, lipid, and amino acid metabolism, which may have resulted from a possible combination of direct actions of the hormone as well as its ability to modulate other glucoregulatory hormones (Boulware, et al. (1992) supra). Similar observed metabolic 10 response to rhlGF-I and insulin suggested that IGF-I and insulin activate a similar cascade of cellular events or that they bind the same receptor (Boulware, et al. (1992) supra). IGFs have been found in both the developing and adult eye in the aqueous (Tripathi et al. (1991) Dev. Drug Res. 22:1-23) and vitreous humor (Grant et al. (1991) Diabetes 35:416-420) and have been suggested to promote the survival of retinal neuronal cells (WO 93/08826). 15 Relaxin was originally determined to be a protein produced in, and acting upon, the tissues of the mammalian reproductive tract to facilitate parturition (Sherwood, O.D. in "The Physiology of Reproduction," E. Knobil and J.D. Neill, eds, p. 861. Raven, New York (1994); and Wade, J.D. and Tregear, G.W. in Methods in Enzymlogy 289:637-646 (1997)). The principal actions of relaxin were considered to be a lengthening of the pubic ligaments, widening of the pelvis, dampening of uterine contractions, and softening and dilating of 20 the cervix (Wade, J.D. and Tregear, G.W. (1997) supra). However, later studies indicated that relaxin had a wider physiological role as capable of causing changes in fluid balance via the relationship between plasma osmolality and arginine vasopressin (Weisenger, R.S. et al. (1995) J. Endocrinology 137:505-510); changes in heart rate (chronotropic activity) and heart muscle contractility (ionotropic activity) (Kakouris, H. et al. (1995) Lancet 339:1076-1978); and is present in male seminal plasma (Winslow, J.W. et al. (1992) 25 Endocrinology (1992) 130:2660-2668). Relaxin receptors have been observed in three tissues by autoradiography using 32P-labeled synthetic relaxin, which tissues include uterus, brain, and heart (Osheroff, P. and Ho, W.-H. (1993) J. Biol. Chem. 268:15193-15199). Relaxin binding regions were observed in brain in regions associated control of cardiovascular functions such as blood pressure and fluid and electrolyte homeostasis. 30 Relaxin has been shown to be more potent than angiotensin II or endothelin, suggesting that it may play a role in cardiovascular disorders (Summers, R.J. et al. in "Recent Progress in Relaxin Research," A.H. MacLennan et al. eds., p. 487, Global, Singapore (1995)). It has also been shown to inhibit collagen deposition, leading to its potential use in skin disorders such as scleroderma. The insulin C-peptide was recently shown to have biological activity. Injection of human C-peptide 35 prevented or attenuated vascular and neural (electrophysiological)dysfunction and impaired Na-- - and K+ dependent adenosine triphosphate activity in tissues of diabetic rats (Ido. Y. et al. (1997) Science 277:563 566). -3-SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 SUMMARY OF THE INVENTION An aspect of the subject invention provides a nucleic acid sequence (SEQ ID NO: 1) of a gene, pro-ilp, which uniquely encodes a novel human insulin-like polypeptide expressed in the colon and uterus, as well as in liver, placenta, lung, and eye. The new gene encodes an insulin-like polypeptide, pro-ILP (SEQ ID NO:2), 5 which is a member of the insulin/IGF family. The pro-ILP may be processed to form two amino acid chains: an A chain (SEQ ID NO:9), and a B chain (SEQ ID NO:10), which amino acid chains are covalently linked by disulfide bonds. A third amino acid chain (SEQ ID NO:21) is the C-peptide of ILP generated by the processing of pro-ILP (SEQ ID NO:2) to mature ILP, comprising covalently bonded amino acid chains A (SEQ ID NO:9) and B (SEQ ID NO:10). The nucleic acid sequences (SEQ ID NOS: 18, 19, 20) encoding the 10 A, B and C amino acid chains, respectively, are also provided by the invention. As described herein, the term "pro-ilp gene" will be used interchangeably with "ilp gene" referring to SEQ ID NO: I (Fig. 6). ILP refers to the mature polypeptide comprising the A and B chains covalently linked by disulfide bonds. The ILP C peptide (SEQ ID NO:21) is expected to exist as a separate peptide following processing of the pro ILP (SEQ ID:2). The invention further embodies the ILP polypeptides or fragments thereof described, supra, as well 15 as antibodies (including monoclonal antibodies) to the polypeptides. Further embodiments include a chimeric molecule comprising an ILP polypeptide fused to a heterologous amino acid sequence, in which the heterologous amino acid sequence includes, but is not limited to an epitope tag sequence or a Fc region of an immunoglobulin. Another aspect of the invention includes a method for determining expression of ilp in a cell. 20 Preferably, the diagnostic test comprises providing a cell extract or a tissue sample containing cells suspected of expressing ilp and determining the presence of mRNA encoding ILP by hybridization of the mRNA to a detectable probe complementary to the sequence complementary to SEQ ID NO: I (Fig. 6) or a fragment thereof. Another aspect of the invention includes a method of diagnosing a physiologic or pathologic 25 condition of the uterus, colon or other ILP-expressing cell or tissue, which method includes the steps of hybridizing a detectable probe to expressed mRNA encoding the ILP present in a tissue sample, a cell extract or other sample thereof and comparing the amount of hybridized detectable probe on the test sample to a control sample from healthy tissue. The detectable probe is complementary to the nucleic acid of SEQ ID NOS:1, 18, 19, 20 or a fragment thereof. 30 An aspect of the invention includes the antisense nucleic acids of the pro-ILP gene or a fragment thereof; cloning or expression vectors containing the pro-ILP gene or the A and/or B and/or C chains; host cells or organisms transformed with expression vectors containing the pro ilp or nucleic acid encoding mature A. B, and/or C chains: a method for the production and recovery of purified ILP from host cells; and purified ILP and/or C-peptide. 35 In a further aspect, the invention encompasses a transgenic animal comprising an altered ilp in which the polypeptide encoded by the altered gene is not biologically active (non-functional), deleted, or has no more than 70% wild type activity, preferably no more that 50% activity and more preferably has no more than 25% activity of the native ILP polypeptide (a "knockout" animal). In addition, a transgenic animal of the invention -4 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 includes a transgenic animal comprising and expressing a native ILP, or a fragment or variant thereof. Such transgenic animals are useful for the screening of potential ILP agonists and antagonists. Aspects of the invention further concern pharmaceutical compositions comprising an ILP covalently linked (A and B chains) and/or an ILP C-peptide as defined herein in admixture with a pharmaceutically 5 acceptable carrier. Dosages and administration of ILP or ILP C-peptide in a pharmaceutical composition may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics (see, for example, Mordenti, J. and Rescigno, A. (1992) Pharmaceutical Research 9:17-25; Morenti, J. et al. (1991) Pharmaceutical Research 8:1351-1359; and Mordenti, J. and Chappell, W. (1989) "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development, Yacobi et al. (eds), Pergamon Press, 10 NY, pp. 42-96, each of which references are herein incorporated by reference in its entirety). In an aspect of the invention, the isolated nucleic acid encoding the ILP or ILP C-peptide of the invention, or fragment thereof, may also be used for in vivo or ex vivo gene therapy. Preferably, the nucleic acid is incorporated into an expression cassette comprised within a retroviral vector for delivery of the nucleic acid sequence to a cell of an animal. 15 In another aspect of the invention, a nucleic acid sequence encoding an ILP, or ILP C-peptide or fragment or variant thereof, as part of an expression cassette, is introduced into a cell of an animal such that the ILP-encoding nucleic acid or ILP C-peptide nucleic acid sequence is expressed in the cell. Preferably, the ILP-encoding or ILP C-peptide-encoding nucleic acid sequence comprises sequences (such as a promotor sequence) for the control of ILP expression within the cell. Embodiments of the invention include the 20 expression cassette, vectors encoding the expression cassette and host cells comprising the expression cassette. Preferred host cells include, but are not limited to, bacteria (such as E. coli), yeast such as S. cerevisiae), and mammalian cells (such as CHO cells). In yet another aspect of the invention, a method of producing an ILP is disclosed In a further aspect of the invention, a host cell expressing an ILP or ILP agonist or an ILP C-peptide 25 or ILP C-peptide agonist is introduced into an animal, preferably a human, such that ILP, ILP agonist, ILP C peptide. or ILP C-peptide agonist produced by the host cell is effective in treating a disorder responsive to increased local or systemic ILP administration. Cells genetically engineered to express an ILP, fragment or variant thereof, can be implanted in the host to provide effective levels of factor or factors. The cells can be prepared, encapsulated, and implanted as provided in U.S. Patents 4,892,538, and 5,011,472, WO 92/19195, 30 WO 95/05452, or Aeischer et al. (1996) Nature 2:696-699, for example, which references are herein incorporated by reference in their entirety. It is another embodiment of the invention that the insulin-like peptide of the invention is useful in the treatment of disorders related to neurophysiological function affecting fluid homeostasis, electrolyte homeostasis, cardiovascular function, blood pressure, somatic or cardiac ionotropic activity, cardiac 35 chronotropic activity, and collagen deposition. These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the structure, synthesis, and usage as more fully set -5 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 forth below. Each reference cited herein is herein incorporated by reference in its entirety with particular attention to the description of subject matter associated with the context of the citation. DESCRIPTION OF THE DRAWINGS Fig. 1 depicts the nucleotide sequence for the ilp (SEQ ID NO:I) and the predicted amino acid 5 sequence of ILP (SEQ ID NO:2), the colon- and uterine-expressed insulin-like polypeptide. The signal sequence, A chain (SEQ ID NO:9), B chain (SEQ ID NO: 10), and C-Chain (SEQ ID NO:21) are also indicated by overlining. The cysteine residues that are predicted to participate in disulfide bond linkages between the A and B chains are indicated by the encircled numbers I through 6 below the cysteine residues within the sequence, where the linkages are between I and 4; 2 and 6; and 3 and 5. Primer oligonucleotides 10 IN2328985.f, IN2328985.p, and IN2328985.r, designed based on homology to relaxin and used to isolate the full length ilp, are indicated by overlining and underlining. Fig. 2 shows the amino acid sequence alignment of ILP (SEQ ID NO:2) with other polypeptide of the insulin/IGF family: h-Insulin (SEQ ID NO:3); h-IGF-1 (SEQ ID NO:4); h-IGF2 (SEQ ID NO:5); h preRelaxin (SEQ ID NO:6); h-Placentin (SEQ ID NO:7); h-Leydig insulin-like peptide precursor (SEQ ID 15 NO:8): and h-ILP (SEQ ID NO:2). Alignments shown were produced using the multisequence alignment program "ALIGN" (Genentech, Inc.). Fig. 3 displays an analysis of pro-ILP hydrophobicity based on the predicted amino acid sequence and composition. The plot indicates that ILP contains a hydrophobic region at the N-terminus characteristic of a signal sequence. 20 Fig. 4 shows a relatedness tree of some human insulin/IGF polypeptide family members (including GenBank accession numbers) h-preIGFI (P70277), h-preIGF2 (P01344), h-prepro insulin (P10042), h-ILP (SEQ ID NO:2), h-prepro-relaxin (P94621), h-Leydig insulin-like peptide precursor (P51460), h-placentin (R89134) The phylogenetic tree was generated by the "ALIGN" program. Fig. 5 shows the nucleic acid sequences of Genentech DNA 26648 (SEQ ID NO:14: Incyte EST 25 INC2328985), and Incyte EST INC778319 (SEQ ID NO:15). Fig. 6 shows the nucleic acid sequence of Genentech DNA 27865 within which the coding nucleic acid sequence of pro-ILP is indicated as SEQ ID NO: I. The deduced amino acid sequence of pro-ILP is indicated as SEQ ID NO:2. Before the present polypeptide, nucleic acids, vectors, and host cells and processes for making such 30 are described, it is to be understood that this invention is not limited to the particular compositions of matter and processes described, as such compounds and methods may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims. DESCRIPTION OF THE EMBODIMENTS 35 Definitions As used herein, "uterine-expressed insulin-like polypeptide" or "insulin-like polypeptide" or "ILP", refers to a naturally occurring ILP or active fragments thereof, having the amino acid sequence shown in SEQ -6 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCTIUS98/17888 ID NO:2. The ILP polypeptide may be encoded by ilp having the nucleic acid sequence shown in SEQ ID NO: 1 (Fig. 6) encoded within SEQ ID NO:22 shown in Fig. I (SEQ ID NO: 1; within Genentech DNA 27865 (Fig. 6): within the plasmid DNA27865-1091 having the ATCC designation: 209296). ILP may also be defined as the polypeptide encoded by an mRNA transcribed from the nucleic acid sequence of SEQ ID NO: 1. 5 The ILP polypeptide of the invention encompasses a polypeptide comprised of an A chain (SEQ ID NO:9) and a B chain (SEQ ID NO:10) linked by disulfide bonds, which A and B chain amino acid chains are within the deduced amino acid sequence of the ILP (SEQ ID NO:2). The C-peptide of pro-ILP encoded by SEQ ID NO:20 and SEQ ID NO:21 is also encompassed by the invention. It is understood that due to the degeneracy of the genetic code, the nucleic acid encoding the ILP may be substituted such that the amino acid sequence 10 of SEQ ID NO:2 is encoded by the substituted nucleic acid sequence. This definition encompasses not only the polypeptide isolated from a native ILP source, such as, but not limited to, uterus, colon, liver, placenta, lung and eye, from human or other mammalian species, but also the polypeptide prepared by recombinant or synthetic methods. It also includes variant forms including functional derivatives, allelic variants, naturally occurring isoforms and analogues thereof. The ILP can be "native ILP" which refers to endogenous ILP 15 polypeptide which has been isolated from a mammal. The ILP can also be "native sequence ILP" insofar as it has the same amino acid sequence as a native ILP. "Mature ILP" is soluble or secreted ILP released from the cell (i.e. lacking an N-terminal hydrophobic sequence) and further encompasses ILP comprised of two amino acid chains, and A chain (SEQ ID NO:9) and a B chain (SEQ ID NO:10) linked by interchain and intrachain disulfide bonds as indicated in Fig. 1. 20 The ILP of the invention encompasses the naturally occurring, recombinant, or synthetic forms of the ILP, with or without the initiating methionine, whether purified from native source, synthesized, produced by recombinant DNA technology or by any combination of these and/or other methods. The novel ILPs of the invention specifically include the human pro-ILP, the amino acid sequence of which is shown in Fig. I (SEQ ID NO:2): mature human ILP (SEQ ID NOS: 9 and 10) and ILP C-peptide (SEQ ID NO: 21). The novel 25 native human ILPs of the present invention are about 114 amino acids in length, but may be longer or shorter while maintaining the biological activity of the native ILP, which biological activities include, but are not limited to, receptor binding, activation of physiological processes within a cell expressing a receptor for ILP, or binding to an antibody raised to the ILP of SEQ ID NO:2 or mature ILP (SEQ ID NOS 9 and 10) or ILP C-peptide (SEQ ID NO:21). The novel native human ILP further includes a polypeptide comprised of two 30 amino chains, an A chain (SEQ ID NO:9) and a B chain (SEQ ID NO:10) linked by disulfide bonds. Optionally, ILP is associated with native glycosylation, or other post-translational derivatization. By "naturally occurring ILP" is meant ILP produced by human cells that have not been genetically engineered and specifically contemplates various ILP forms arising from post-translational modifications of the polypeptide including but not limited to disulfide bond formation, acetylation, carboxylation, 35 glycosylation, phosphorylation, lipidation and acylation. A "functional derivative" of a polypeptide is a compound having a qualitative biological activity in common with the native polypeptide. Thus, a functional derivative of a native novel ILP of the present invention is a compound that has a qualitative biological activity in common with such native ILP. -7 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 "Functional derivatives" include, but are not limited to. fragments of native polypeptide from any animal species (including humans), derivatives of native polypeptide (human and non-human) and their fragments. and peptide and non-peptide analogs of native polypeptide, provided that they have a biological activity in common with a respective native polypeptide. Derivative further refers to polypeptide derived from naturally 5 occurring ILP by chemical modifications such as ubiquitination, labeling (e.g., with radionuclides, various enzymes, etc.), pegylation (derivatization with polyethylene glycol) or by insertion or substitution by chemical synthesis using an amino acid, such as ornithine, that is not normally or naturally found in human proteins. The term "derivative" is also used to define amino acid sequence and glycosylation variants, and covalent modifications of a native polypeptide. 10 By "fragments" is meant regions within the sequence of a mature native polypeptide. Preferably ILP fragments will have a consecutive sequence of at least 10, and more preferably at least 20, amino acid residues of the ILP. The preferred fragments have about 10-100 amino acid residues which are identical to a portion of the sequence of ILP in SEQ ID NO:2. To have activity, the ILP fragment of the invention has sufficient length to display biologic and/or immunologic activity. Fragment can also include a portion of each of the 15 A and B chains (SEQ ID Nos:9 and 10) linked by one or more disulfide bonds. Similarly, with regard to nucleic acids, fragment may mean a region within the sequence of a nucleic acid encoding ILP. Preferably a nucleic acid fragment comprising a portion of the ILP gene will have a consecutive sequence of at least 20, preferably at least 50 nucleic acid residues. Preferably the nucleic acid fragment will comprise a sufficient number of nucleic acid residues to be long enough for use in polymerase chain reaction (PCR) or various 20 hybridization procedures, such as amplification or identification of portions ofmRNA or DNA molecules. "Oligonucleotides" or "nucleic acid probes" are prepared based on the cDNA sequence which encodes ILP provided by the present invention. Oligonucleotides comprise portions of the DNA sequence having at least about 15 nucleotides, usually at least about 20 nucleotides. Nucleic acid probes comprise portions of the sequence having fewer nucleotides than about 6 kb. usually fewer than about I kb. After appropriate 25 testing to eliminate false positives, these probes may be used to determine whether mRNA encoding ILP is present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh, P.S. et al. (1992) PCR Methods App. 1:241-250. Probes may be derived from naturally occurring or recombinant single- or double-stranded nucleic acids or be chemically synthesized. They may be labeled by nick translation, Klenow fill-in reaction, PCR or other methods well known in the art (see, for example, 30 Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; or Ausubel. F.M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, NYC; each reference herein incorporated by reference in its entirety). "Non-peptide analogs" are organic compounds that display substantially the same surface as peptide analogs of the native polypeptide. Thus, the non-peptide analogs of the native novel ILPs of the present 35 invention are organic compounds that display substantially the same surface as peptide analogs of the native ILPs. Such compounds interact with other molecules in a similar fashion as the peptide analogs, and mimic a biological activity of a native ILP of the present invention. Preferably, amino acid sequence variants of the present invention have at least about 60% amino acid sequence identity, more preferably at least about 75 % -8 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 amino acid sequence identity, and most preferably at least about 90% amino acid sequence identity with a native ILP of the present invention. Preferably, the sequence variants show the highest percentage amino acid conservation at amino acid residues conserved between the novel ILP of the present invention and other members of the ILP family (see Fig. 2). 5 The terms "isolated" or "substantially pure" refer to a polypeptide or nucleic acid which is free of other polypeptide or nucleic acids as well as lipids, carbohydrates or other materials with which it is naturally associated. An exception is made for glycosylation wherein sugar moieties are covalently attached to amino acids of the ILP polypeptide of the invention. One of ordinary skill in the art can purify an ILP polypeptide or nucleic acid encoding the polypeptide using standard techniques appropriate for each type of molecule. 10 The term "percent amino acid sequence identity" with respect to the ILP sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the ILP sequence having the deduced amino acid sequence described in Fig. 1 (SEQ ID NO:2) or the A, B or C peptides (SEQ ID NOS:9, 10 and 21), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part 15 of the sequence identity. N-terminal, C-terminal, or internal extensions, deletions, or insertions into the ILP sequence shall be construed as affecting sequence identity or homology. Another type of ILP variant is "chimeric ILP", which term encompasses a polypeptide comprising full-length ILP or a fragment thereof fused or bonded to a heterologous polypeptide. The chimera will normally share at least one biological property with ILP. Examples of chimeric ILPs include immunoadhesins 20 and epitope tagged ILP. In another embodiment, the heterologous polypeptide is thioredoxin, a salvage receptor binding epitope, cytotoxic polypeptide or enzyme (e.g., one which converts a prodrug to an active drug). The terms "covalent modification" and "covalent derivatives" are used interchangeably and include, but are not limited to, modifications of a native polypeptide or a fragment thereof with an organic 25 proteinaceous or non-proteinaceous derivatizing agent, fusions to heterologous polypeptide sequences, and post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected sides or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. Certain post-translational modifications are the result of the action of recombinant host 30 cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, tyrosyl or threonyl residues, methylation of the ac-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular 35 Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)). Covalent derivatives/modifications specifically include fusion proteins comprising native ILP sequences of the present invention and their amino acid sequence variants, such as immunoadhesins, and N-terminal fusions to heterologous signal sequences. -9 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 The term "biological activity" in the context of the present invention is defined as the possession of at least one adhesive, regulatory or effector function qualitatively in common with a native polypeptide. Preferred functional derivatives within the scope of the present invention are unified by retaining binding characteristics of a native ILP of the present invention. 5 The phrase "activating an ILP receptor" refers to the act of causing an ILP receptor to mediate physiological changes within a cell expressing the receptor on its surface. Generally, this will involve binding of ILP to an ILP receptor. "Identity" or "homology" with respect to a native polypeptide and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues 10 of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art. For example, the sequences disclosed herein were analyzed using "ALIGN", Genentech, Inc. 15 The term "agonist" is used to refer to peptide and non-peptide analogs of the native ILPs (where native ILP refers to pro-ILP, mature ILP or ILP C-peptide) of the present invention and to antibodies specifically binding such native ILPs provided that they retain at least one biological activity of a native ILP. Preferably, the agonists of the present invention retain the qualitative binding recognition properties and receptor activation properties of the native ILP polypeptide. 20 The term "antagonist" is used to refer to a molecule inhibiting a biological activity of a native ILP of the present invention wherein native ILP refers to pro-ILP, mature ILP or ILP C-peptide. Preferably, the antagonists herein inhibit the binding of a native ILP of the present invention. Preferred antagonists essentially completely block the binding of a native ILP to an ILP receptor to which it otherwise binds. An ILP "antagonist" is a molecule which prevents, or interferes with, an ILP effector function (e.g. a molecule which 25 prevents or interferes with binding and/or activation of an ILP receptor by ILP). Such molecules can be screened for their ability to competitively inhibit ILP receptor activation by monitoring binding of native ILP in the presence and absence of the test antagonist molecule, for example. Examples of ILP antagonists include neutralizing antibodies against ILP. An antagonist of the invention also encompasses an antisense polynucleotide against the ILP gene, which antisense polynucleotide blocks transcription or translation of the 30 ILP gene, thereby inhibiting its expression and biological activity. Ordinarily, the terms "amino acid" and "amino acids" refer to all naturally occurring L-a-amino acids. In some embodiments, however, D-amino acids may be present in the polypeptide or peptides of the present invention in order to facilitate conformational restriction. For example, in order to facilitate disulfide bond formation and stability, a D amino acid cysteine may be provided at one or both termini of a peptide functional 35 derivative or peptide antagonist of the native ILPs of the present invention. The amino acids are identified by either the single-letter or three-letter designations: Asp D aspartic acid lie I isoleucine Thr T threonine Leu L leucine -10 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Ser S serine Tyr Y tvrosine Glu E glutamic acid Phe F phenylalanine Pro P proline His H histidine Gly G glycine Lys K lysine 5 Ala A alanine Arg R arginine Cys C cysteine Trp W tryptophan Val V valine Gin Q glutamine Met M methionine Asn N asparagine The term "amino acid sequence variant" refers to molecules with some differences in their amino acid 10 sequences as compared to a native amino acid sequence. Substitutional variants are those that have at least one amino acid residue in a native sequence removed and a different amino acid inserted in its place at the same position. Insertional variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native sequence. Immediately adjacent to an amino acid means connected to 15 either the a-carboxy or a-amino functional group of the amino acid. Deletional variants are those with one or more amino acids in the native amino acid sequence removed. "Antibodies (Abs)" and "immunoglobulins (Igs)" are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include 20 both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptide of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between 25 the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one and (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino 30 acid residues are believed to form an interface between the light and heavy chain variable domains (Clothia et al., J. Mol. Biol. 186, 651-663 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82, 4592-4596 (1985)). The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda (X). based on the amino acid sequences of their constant 35 domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, lgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG-1, IgG -11 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains that correspond to the-different classes of immunoglobulins are called a, delta, epsilon, y, and i, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The term "antibody" is used in the broadest sense and specifically covers single monoclonal 5 antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments (e.g., Fab, F(ab') 2 , and Fv), so long as they exhibit the desired biological activity. 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 10 except for possible naturally occurring mutations that may be present in minor amounts. 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. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1975), or may be made 15 by recombinant DNA methods (see, e.g. U.S. Patent No. 4,816,567 (Cabilly et al.) and Mage and Lamoyi, in Monoclonal Antibody Production Techniques and Applications, pp. 79-97, Marcel Dekker, Inc., New York (1987)). The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554 (1990), for example. "Humanized" forms of non-human (e.g. murine) antibodies are specific chimeric immunoglobulins, 20 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. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient antibody are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, 25 affinity and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human FR residues. Furthermore, the humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or FR sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or 30 substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see: Jones et al., Nature 321, 522-525 (1986); Reichmann et al., Nature 332, 323-329 (1988); EP-B-239 400 published 30 September 1987; Presta, Curr. Op. Struct. 35 Biol. 2 593-596 (1992); and EP-B-451 216 published 24 January 1996), which references are herein incorporated by reference in their entirety. -12 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 By "neutralizing antibody" is meant an antibody molecule as herein defined which is ableto block or significantly reduce an effector function of native sequence ILP. For example, a neutralizing antibody may inhibit or reduce the ability of ILP to activate an ILP receptor. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in 5 which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567, Cabilly et al.; 10 Morrison et al., Proc. Natl. Acad. Sci. USA 81. 6851-6855 (1984)). In the context of the present invention the expressions "cell", "cell line", and "cell culture" and "host cell" are used interchangeably, and all such designations include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological property, as screened for in the originally transformed cell, 15 are included in the invention. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. The terms "replicable expression vector", "expression vector" and "vector" refer to a piece of DNA, usually double-stranded, which may have inserted into it a piece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell, or not naturally found in the host 20 cell in the context of an expression vector. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA may be generated. In addition, the vector contains the necessary elements that permit translating the foreign DNA into a polypeptide. Many molecules of the polypeptide encoded by the foreign DNA can thus be rapidly synthesized. 25 The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other sequences. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancer. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic 30 acid sequence. For example, DNA for a presequence or a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of 35 a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice. -13 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 "Oligonucleotides" are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods, such as phosphotriester, phosphite, or phosphoramidite chemistry, using solid phase techniques such as those described in EP 266,032, published 4 May 1988. or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al., Nucl. Acids Res. 14, 5399 5 (1986). They are then purified on polyacrylamide gels. By "solid phase" is meant a non-aqueous matrix to which a reagent of interest (e.g., ILP or an antibody thereto) can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can 10 comprise the well of an assay plate: in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149, herein incorporated by reference in its entirety. The terms "transformation" and "transfection" are used interchangeably herein and refer to the process of introducing DNA into a cell. Following transformation or transfection, the ILP DNA may integrate 15 into the host cell genome, or may exist as an extrachromosomal element. If prokaryotic cells or cells that contain substantial cell wall constructions are used as hosts, the preferred methods of transfection of the cells with DNA is the calcium treatment method described by Cohen et al., Proc. Natl. Acad Sci. US.A., 69:2110 2114 (1972) or the polyethylene glycol method of Chung et al., Nuc. Acids. Res. 16:3580 (1988). If yeast are used as the host, transfection is generally accomplished using polyethylene glycol, as taught by Hinnen, Proc. 20 Natl. Acad Sci. U.S.A., 75:1929-1933 (1978). If mammalian cells are used as host cells, transfection generally is carried out by the calcium phosphate precipitation method, Graham et al., Virology 52:546 (1978), Gorman et al., DNA and Protein Eng. Tech. 2:3-10 (1990). However, other known methods for introducing DNA into prokaryotic and eukaryotic cells, such as nuclear injection, electroporation, or protoplast fusion also are suitable for use in this invention. 25 Particularly useful in this invention are expression vectors that provide for the transient expression in mammalian cells of DNA encoding ILP. In general, transient expression involves the use of an expression vector that is able to efficiently replicate in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Transient expression systems, comprising a suitable expression vector and a host cell, allow for the 30 convenient positive identification of polypeptide encoded by cloned DNAs, as well as for the rapid screening of such polypeptide for desired biological or physiological properties. It is further envisioned that the ILP of this invention may be produced by homologous recombination, as provided for in WO 91/06667, published 16 May 1991. Briefly, with respect to ILP, this method involves transforming a cell containing an endogenous ILP gene with a homologous DNA, which homologous DNA 35 comprises (a) an amplifiable gene (e.g. a gene encoding dihydrofolate reductase (DHFR)), and (b) at least one flanking sequence, having a length of at least about 150 base pairs, which is homologous with a nucleotide sequence in the cell genome that is within or in proximity to the gene encoding ILP. The transformation is carried out under conditions such that the homologous DNA integrates into the cell genome by recombination. -14 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Cells having integrated the homologous DNA are then subjected to conditions which select for amplification of the amplifiable gene, whereby the ILP gene is amplified concomitantly. The resulting cells are then screened for production of desired amounts of ILP. Flanking sequences that are in proximity to a gene encoding ILP are readily identified, for example, by the method of genomic walking, using as a starting point 5 the nucleotide sequence of human ILP (SEQ ID NO:]; within Genentech DNA27865; Fig. 6) within SEQ ID NO:22 of Fig. 1. "Isolated nucleic acid encoding ILP" is RNA or DNA free from at least one contaminating source nucleic acid with which it is normally associated in the natural source and preferably substantially free of any other mammalian RNA or DNA. The phrase "free from at least one contaminating source nucleic acid with 10 which it is normally associated" includes the case where the nucleic acid is present in the source or natural cell but is in a different chromosomal location or is otherwise flanked by nucleic acid sequences not normally found in the source cell or not normally found adjacent to the ILP encoding nucleic acid in the source cell. An example of isolated ILP encoding nucleic acid is RNA or DNA that encodes a biologically active ILP sharing at least 75%, more preferably at least 80%, still more preferably at least 85%, even more preferably 15 90%. and most preferably 95% sequence identity with the human pro-ILP (SEQ ID NO:1), human mature ILP (SEQ ID NOS: 18 and 19) where the A and B chains are covalently linked by disulfide bonds; or LIP C peptide (SEQ ID NO:20). Hybridization is preferably performed under "stringent conditions" which means (1) employing low ionic strength and high temperature for washing, for example, 0.015 sodium chloride/0.0015 M sodium 20 citrate/0.1% sodium dodecyl sulfate at 50 0 C, or (2) employing during hybridization a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42oC. Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate). 50 mM sodium phosphate (pH 6/8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated 25 salmon sperm DNA (50 .tg/ml), 0.1% SDS, and 10% dextran sulfate at 42 0 C. with washes at 42'C in 0.2 x SSC and 0.1% SDS. Yet another example is hybridization using a buffer of 10% dextran sulfate, 2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55 °C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55 'C. Where desired, a "signal or leader sequence" can direct the polypeptide through the membrane of a 30 cell. Such a sequence may be naturally present on the polypeptide of the present invention or provided from heterologous protein sources by recombinant DNA techniques. A polypeptide "fragment," "portion," or "segment" is a stretch of amino acid residues of at least about 5 amino acids, often at least about 7 amino acids, typically at least about 9 to 13 amino acids, and, in various embodiments, at least about 17 or more amino acids. To be active, ILP polypeptide must have sufficient length 35 to display biologic and/or immunologic activity. "Immunoadhesins" or "ILP - immunoglobulin chimeras" are chimeric antibody-like molecules that combine the functional domain(s) of a binding protein (usually a receptor, a cell-adhesion molecule or a ligand) with the an immunoglobulin sequence. The most common example of this type of fusion protein -15 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 combines the hinge and Fc regions of an immunoglobulin (Ig) with domains of a cell-surface receptor that recognizes a specific ligand. This type of molecule is called an "immunoadhesin", because it combines "immune" and "adhesion" functions; other frequently used names are "Ig-chimera", "Ig-" or "Fc-fusion protein", or "receptor-globulin." 5 "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those prone to have the disorder of those in which the disorder is to be prevented. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans. domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, and the 10 like. Preferably, the mammal herein is a human. "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including 15 ascorbic acid: low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins: hydrophilic polymers such as polyvinylpyrrolidone: amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TweenTM, polyethylene 20 glycol (PEG), and PluronicsTM. General Procedures for the Production of an ILP by Recombinant DNA Technologyev A. Identification and isolation of nucleic acid encoding a novel insulin-like polvpeptide, ILP. The native ILPs of the present invention may be isolated from cDNA or genomic libraries. For example, a suitable cDNA library can be constructed by obtaining polyadenylated mRNA from cells known 25 to express the desired ILP, and using the mRNA as a template to synthesize double stranded cDNA. Suitable sources of the mRNA are embryonic and adult mammalian tissues. mRNA encoding native ILPs of the present invention is expressed, for example, in adult mammalian (preferably human) colon, uterus, liver, placenta, lung and eye. The gene encoding the novel ILPs of the present invention can also be obtained from a genomic library, such as a human genomic cosmid library, or a mouse-derived embryonic stem cell (ES) 30 genomic library. Libraries, either cDNA or genomic, are screened with probes designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries, suitable probes include monoclonal and polyclonal antibodies that recognize and specifically bind to an ILP of the invention. For cDNA libraries. suitable probes include carefully selected oligonucleotide probes (usually of about 20-80 bases in length) that 35 encode known or suspected portions of an ILP polypeptide from the same or different species, and/or complementary or homologous cDNAs or fragments thereof that encode the same or a similar gene. Appropriate probes for screening genomic DNA libraries include, without limitation, oligonucleotides, cDNAs. or fragments thereof that encode the same or a similar gene, and/or homologous genomic DNAs or -16 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 fragments thereof. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures as described in Chapters 10-12 of Sambrook et al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989, herein incorporated by reference in its entirety. 5 If DNA encoding an ILP of the present invention is isolated by using carefully selected oligonucleotide sequences to screen cDNA libraries from various tissues, the oligonucleotide sequences selected as probes should be sufficient in length and sufficiently unambiguous that false positive selections are minimized. The actual nucleotide sequence(s) is/are usually designed based on regions that have the least codon redundance. The oligonucleotides may be degenerate at one or more positions. The use of degenerate 10 oligonucleotides is of particular importance where a library is screened from a species in which preferential codon usage is not known. The oligonucleotide must be labeled such that it can be detected upon hybridization to DNA in the library being screened. The preferred method of labeling is to use ATP (e.g., y 32 P) and polynucleotide kinase to radiolabel the 5' end of the oligonucleotide. However, other methods may be used to label the 15 oligonucleotide, including, but not limited to, biotinylation or enzyme labeling. cDNAs encoding the novel ILPs can also be identified and isolated by other known techniques of recombinant DNA technology, such as by direct expression cloning, or by using the polymerase chain reaction (PCR) as described in U.S. Patent No. 4,683,195, issued 28 July 1987, in section 14 of Sambrook et al., supra, or in Chapter 15 of Current Protocols in Molecular Biology, Ausubel et al., supra (1989), which references 20 are herein incorporated by reference in their entirety. Once cDNA encoding a new native ILP from one species has been isolated, cDNAs from other species can also be obtained by cross-species hybridization. According to this approach, human or other mammalian cDNA or genomic libraries are probed by labeled oligonucleotide sequences selected from known ILP sequences (such as murine or human sequences) in accord with known criteria. Preferably, the probe 25 sequence should be sufficient in length and sufficiently unambiguous that false positives are minimized. Typically, a 32P-labeled oligonucleotide having about 30 to 50 bases is sufficient, particularly if the oligonucleotide contains one or more codons for methionine or tryptophan. Isolated nucleic acid will be DNA that is identified and separated from contaminant nucleic acid encoding other polypeptide from the source of nucleic acid. Hybridization is preferably performed under "stringent conditions", as defined herein. 30 Once the sequence is known, the gene encoding a particular ILP can also be obtained by chemical synthesis, following one of the methods described in Engels and Uhlmann, Agnew. Chem. Int. Ed. Engl. 28, 716 (1989), herein incorporated by reference in its entirety. These methods include triester, phosphite, phosphoramidite and H-phosphonate methods, PCR and other autoprimer methods, and oligonucleotide syntheses on solid supports. 35 B. Cloning and expression of nucleic acid encoding a novel ILP. Once the nucleic acid encoding a novel ILP is available, it is generally ligated into a replicable expression vector for further cloning (amplification of the DNA), or for expression. -17 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Expression and cloning vectors are well known in the art and contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. The selection of the appropriate vector will depend on 1) whether it is to be used for DNA amplification or for DNA expression, 2) the size of the DNA to be inserted into the vector, and 3) the host cell to be transformed with the vector. Each vector contains 5 various components depending on its function (amplification of DNA of expression of DNA) and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: 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 the above listed components, the desired coding and control sequences, employs standard ligation 10 techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. For analysis to confirm correct sequences in constructed plasmids, the ligation mixtures are commonly used to transform E. coli cells, e.g. E. coli K12 strain 294 (ATCC 31,446) and successful transformants selected by ampicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced by the 15 method of Messing et al., Nucleic Acids Res. 9. 309 (1981) or by the method of Maxam et al., Methods in Enzymology 65, 499 (1980). The polypeptide of the present invention may be expressed in a variety of prokaryotic and eukaryotic host cells. Suitable prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. A preferred cloning host is E. coli 294 (ATCC 31,446) although other gram negative or gram positive 20 prokaryotes such as E. coli B, E. coli X 1776 (ATCC 31,537), E. coli W3110 (ATCC 27,325), Pseudomonas species, or Serratia Marcesans are suitable. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for vectors herein. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species and strains are commonly 25 available and useful herein, such as S. pombe (Beach and Nurse, Nature 290:140 (1981)), Kluvveromyces lactis (de Louvencourt. L. et al., J. Bacteriol. 154:737-742 (1983)); yarrowia (EP 402,226); Pichia pastoris (EP 183,070), Trichoderma reesia (EP 244,234), Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA 76:5259-5263 (1979)); and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun. 112:284-289 (1983); Tilburn et al., Gene 26:205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. 30 USA 81:1470-1474 (1984)) and A. niger (Kelly and Hynes, EMBO J. 4:475-479 (1985)). Suitable host cells may also derive from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture, although cells from mammals such as humans are preferred. Examples of invertebrate cells include plants and insect cells. Numerous baculoviral strains and variants and 35 corresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito). Drosophila melangaster (fruitfly), and Bombyx mori host cells have been identified. See, e.g. Luckow et al., Bio/Technology 6:47-55 (1988); Miller et al., in Genetic Engineering, Setlow, J.K. et al., eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature -18 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 315:592-594 (1985). A variety of such viral strains are publicly available, e.g. the L-1 variant of Autographa californica NPV. and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodopterafrugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as 5 hosts. Typically, plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens, which has been previously manipulated to contain the ILP DNA. During incubation of the plant cell culture with A. tumefaciens, the DNA encoding an ILP is transferred to the plant cell host such that it is transfected, and will, under appropriate conditions, express the ILP DNA. In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and 10 polyadenylation signal sequences. Depicker et al., J. Mol. Appl. Gen. 1:561 (1982). In addition, DNA segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in recombinant DNA-containing plant tissue. See EP 321,196 published 21 June 1989. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture 15 (tissue culture) is well known (see for example, Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)). Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO; Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216 20 (1980)); mouse sertolli cells (TM4. Mather, Biol. Reprod. 23:243-251 (1980)): monkey kidney cells (CVI ATCC CCL 70): African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2. HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals N.Y. Acad. 25 Sci. 383:44068 (1982)); MRC 5 cells: FS4 cells: and a human hepatoma cell line (Hep G2). Preferred host cells are human embryonic kidney 293 and Chinese hamster ovary cells. Particularly useful in the practice of this invention are expression vectors that provide for the expression in mammalian cells of DNA encoding a novel ILP herein. Where transient expression is preferred, expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that 30 the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Transient systems, comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptide encoded by cloned DNAs, as well as for the rapid screening of such polypeptide for desired biological or physiological properties. Thus, transient expression systems are particularly useful in the invention for purposes of identifying analogs and 35 variants of a native ILP of the invention. Other methods, vectors, and host cells suitable for adaptation to the synthesis of the ILPs in recombinant vertebrate cell culture are described for example, in Getting et al., Nature 293. 620-625 (1981); Mantel et al., Nature 281, 40-46 (1979): Levinson et al.; EP 117,060 and EP 117,058. Particularly useful -19 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 plasmids for mammalian cell culture expression of the ILP polypeptide are pRK5 (EP 307,247) pRK5B (Holmes et al.. Science, 253: 1278-1280 (1991)), or pSVI6B (PCT Publication No. WO 91/08291). Other cloning and expression vectors suitable for the expression of the ILPs of the present invention in a variety of host cells are, for example, described in EP 457,758 published 27 November 1991. A large 5 variety of expression vectors is now commercially available. An exemplary commercial yeast expression vector is pPIC.9 (Invitrogen), while an commercially available expression vector suitable for transformation ofE. coli cells is PETI5b (Novagen). C. Culturing the Host Cells. Prokarvote cells used to produced the ILPs of this invention are cultured in suitable media as describe 10 generally in Sambrook et al., supra. Mammalian cells can be cultured in a variety of media. Commercially available media such as Ham's FIO (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham and Wallace, Meth. Enzymol. 58, 44 (1979); Barnes and Sato, Anal. Biochem. 102, 255 15 (1980). US 4.767,704: 4.657.866: 4.927,762; or 4.560,655; WO 90/03430; WO 87/00195 or US Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin T M drug) trace elements (defined as inorganic 20 compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH and the like, suitably are those previously used with the host cell selected for cloning or expression, as the case may be, and will be apparent to the ordinary artisan. 25 The host cells referred to in this disclosure encompass cells in in vitro cell culture as well as cells that are within a host animal or plant. It is further envisioned that the ILPs of this invention may be produced by homologous recombination, or with recombinant production methods utilizing control elements introduced into cells already containing DNA encoding the particular ILP. 30 D. Detecting Gene Amplification and/or Expression. Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA 77, 5201-5205 (1980)), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe. based on the sequences provided herein. Various labels may be employed, most 35 commonly radioisotopes, particularly 32P. However, other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a polynucleotide. The biotin then serves as a site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies may be employed that can recognize specific duplexes, -20 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to the surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected. 5 Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. A particularly sensitive staining technique suitable for use in the present invention is described by Hse et al., Am. J. Clin. Pharm. 75, 734-738 (1980). Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either 10 monoclonal or polyclonal, and may be prepared in any animal. Conveniently, the antibodies may be prepared against a native ILP polypeptide, or against a synthetic peptide based on the DNA sequence disclosed herein. E. Amino Acid Sequence Variants of a Native ILP. Amino acid sequence variants of native ILPs are prepared by methods known in the art by introducing appropriate nucleotide changes into a native ILP DNA, or by in vitro synthesis of the desired polypeptide. 15 There are two principal variables in the construction of amino acid sequence variants: the location of the mutation site and the nature of the mutation. With the exception of naturally-occurring alleles, which do not require the manipulation of the DNA sequence encoding the native ILP, the amino acid sequence variants of ILPs are preferably constructed by mutating the DNA, either to arrive at an allele or an amino acid sequence variant that does not occur in nature. 20 Amino acid alterations can be made at sites that differ in novel ILPs from various species, or in highly conserved regions, depending on the goal to be achieved. Sites at such locations will typically be modified in series, e.g. by (1) substituting first with conservative choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue or residues, or (3) inserting residues of the same or different class adjacent to the located site, or combinations of options 1-3. One helpful technique for 25 such modifications is called "alanine scanning" (Cunningham and Wells, Science 244, 1081-1085 (1989)). Naturally-occurring amino acids are divided into groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophobic: cys, ser, thr; (3) acidic: asp, glu; 30 (4) basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe. Conservative substitutions involve exchanging a member within one group for another member within the same group, whereas non-conservative substitutions will entail exchanging a member of one of 35 these classes for another. Substantial changes in function or immunological identity are made by amino acid substitutions that are less conservative, i.e. differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side -21 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 chain. The substitutions which in general are expected to produce the greatest changes in the properties of the novel native ILPs of the present invention will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl. is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side 5 chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl: or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine. Such substitutions are expected to have their most significant effect when made at those amino acids concerned between ILP and other members of the insulin family (see, for example, Fig.2). In particular, substitutions that affect the processing of the ILP of the ILP are expected to have 10 significant effects. Such amino acids are those within approximately 10 amino acids on each side of the A, B, and C chain cleavage sites of pro-ILP (SEQ ID NO:2). Substitutional variants of the novel ILPs of the present invention also include variants where functionally homologous (having at least about 40%-50% homology) domains of other proteins are substituted by routine methods for one or more of the domains within the novel ILP structure. 15 Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptide containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions (i.e. insertions within the novel ILP amino acid sequence) may range generally from about 1 to 10 residues, more preferably 1 to 5 residues, more preferably I to 3 residues. An example of a terminal insertion includes fusion of a heterologous N-terminal signal 20 sequence to the N-terminus of the ILP molecule to facilitate the secretion of the mature ILP or a fragment thereof from recombinant host cells. Such signal sequences will generally be obtained from, and thus be homologous to, a signal sequence of the intended host cell species. Suitable sequences include STII or Ipp for E. coli, alpha factor for yeast, and viral signals such as herpes gD for mammalian cells. Other insertional variants of the native ILP molecules include the fusion of the N- or C-terminus of 25 the ILP molecule to immunogenic polypeptide, e.g. bacterial polypeptide such as beta-lactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as immunoglobulin regions (preferably immunoglobulin constant regions), albumin, or ferritin, as described in WO 89/02922 published on 6 April 1989. Further insertional variants are immunologically active derivatives of the novel ILPs, containing an 30 epitope of an immunologically competent extraneous polypeptide, i.e. a polypeptide which is capable of eliciting an immune response in the animal to which the fusion is to be administered or which is capable of being bound by an antibody raised against an extraneous polypeptide. Typical examples of such immunologically competent polypeptide are allergens, autoimmune epitopes, or other potent immunogens or antigens recognized by pre-existing antibodies in the fusion recipient, including bacterial polypeptide such 35 as trpLE, 3-glactosidase, viral polypeptide such as herpes gD protein, and the like. Immunogenic fusions are produced by cross-linking in vitro or by culture of cells transformed with recombinant DNA encoding an immunogenic polypeptide. It is preferable that the immunogenic fusion be one in which the immunogenic sequence is joined to or inserted into a novel ILP molecule or fragment thereof -22 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 by one or more peptide bonds. These products therefore consist of a linear polypeptide chain containing the ILP epitope and at least one epitope foreign to the ILP. It will be understood that it is within the scope of this invention to introduce the epitopes anywhere within an ILP molecule of the present invention or a fragment thereof. These immunogenic insertions are particularly useful when formulated into a pharmacologically 5 acceptable carrier and administered to a subject in order to raise antibodies against the ILP molecule, which antibodies in turn are useful as diagnostics, in tissue-typing, or in purification of the novel ILPs by standard immunoaffinity techniques. Alternatively, in the purification of the ILPs of the present invention, binding partners for the fused extraneous polypeptide, e.g. antibodies, receptors or ligands, are used to adsorb the fusion from impure admixtures, after which the fusion is eluted and, if desired, the novel ILP is recovered from 10 the fusion, e.g. by enzymatic cleavage. Since it is often difficult to predict in advance the characteristics of a variant ILP, it will be appreciated that some screening will be needed to select the optimum variant. Such screening includes, but is not limited to, arrays of receptor binding. After identifying the desired mutation(s), the gene encoding an ILP variant can, for example, be 15 obtained by chemical synthesis as described herein. More preferably, DNA encoding an ILP amino acid sequence variant is prepared by site-directed mutagenesis of DNA that encodes an earlier prepared variant or a nonvariant version of the ILP. Site-directed (site-specific) mutagenesis allows the production of ILP variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient 20 size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. In general, the techniques of site-specific mutagenesis are well known in the art, as exemplified by publications such as, Edelman et al.. DNA 2., 183 (1983). As will be appreciated, the site-specific mutagenesis technique typically employs a phage vector that exists in both 25 a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage, for example, as disclosed by Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam (1981). This and other phage vectors are commercially available and their use is well known to those skilled in the art. A versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA 30 fragments using M13-derived vectors was published by Zoller, M.J. and Smith, M., Nucleic Acids Res. 10, 6487-6500 (1982)). Also, plasmid vectors that contain a single-stranded phage origin of replication (Veira et al., Meth. Enzymol. 153, 3 (1987)) may be employed to obtain single-stranded DNA. Alternatively, nucleotide substitutions are introduced by synthesizing the appropriate DNA fragment in vitro, and amplifying it by PCR procedures known in the art. 35 The PCR amplification technique may also be used to create amino acid sequence variants of a novel ILP. In a specific example of PCR mutagenesis, template plasmid DNA (1 ltg) is linearized by digestion with a restriction endonuclease that has a unique recognition site in the plasmid DNA outside of the region to be amplified. Of this material, 100 ng is added to a PCR mixture containing PCR buffer, which contains the four -23 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 deoxynucleotide triphosphates and is included in the GeneAmp R kits (obtained from Perkin-Elmer Cetus, Norwalk, CT and Emeryville, CA). and 25 pmole of each oligonucleotide primer, to a final volume of 50 pl. The reaction mixture is overlayered with 35 pl mineral oil. The reaction is denatured for 5 minutes at 100oC, placed briefly on ice, and then 1 pl Thermus aquaticus (Taq) DNA polymerase (5 units/pl), purchased from 5 Perkin-Elmer Cetus, Norwalk, CT and Emeryville, CA) is added below the mineral oil layer. The reaction mixture is then inserted into a DNA Thermal Cycler (Perkin-Elmer Cetus) programmed as follows: (as an example) 2 min. 55oC, 30 sec. 72oC, then 19 cycles of the following: 10 30 sec. 94oC, 30 sec. 55oC, and 30 sec. 72oC. At the end of the program, the reaction vial is removed from the thermal cycler and the aqueous phase transferred to a new vial, extracted with phenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA 15 is recovered by standard procedures. This material is subsequently subjected to appropriate treatments for insertion into a vector. Cassette mutagenesis is another method useful for preparing variants and is based on the technique described by Wells et al. (Gene 34:315 (1985)). Additionally, the so-called phagemid display method may be useful in making amino acid sequence 20 variants of native or variant ILPs or their fragments. This method involves 1) constructing a replicable expression vector comprising a first gene encoding a receptor to be mutated, a second gene encoding at least a portion of a natural or wild-type phage coat protein wherein the first and second genes are heterologous, and a transcription regulatory element operably linked to the first and second genes. thereby forming a gene fusion encoding a fusion protein: 2) mutating the vector at one or more selected positions within the first gene thereby 25 forming a family of related plasmids: 3) transforming suitable host cells with the plasmids: 4) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein: 5) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on 30 the surface of the particle; 6) contacting the phagemid particles with a suitable antigen so that at least a portion of the phagemid particles bind to the antigen; and 7) separating the phagemid particles that bind from those that do not. Steps 4 through 7 can be repeated one or more times. Preferably in this method the plasmid is under tight control of the transcription regulatory element, and the culturing conditions are adjusted so that the amount or number of phagemid particles displaying more than one copy of the fusion protein on the 35 surface of the particle is less than about 1%. Also, preferably, the amount of phagemid particles displaying more than one copy of the fusion protein is less than 10% of the amount of phagemid particles displaying a single copy of the fusion protein. Most preferably, the amount is less than 20%. Typically in this method, the expression vector will further contain a secretory signal sequence fused to the DNA encoding each subunit -24 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 of the polypeptide and the transcription regulatory element will be a promoter system. Preferred promoter systems are selected from lac Z, APL, tac. T7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof. Also, normally the method will employ a helper phage selected from M13K07, M13R408, M13-VCS, and Phi X 174. The preferred helper phage is M I3K07. and the preferred coat protein 5 is the M13 Phage gene III coat protein. The preferred host is E. coli, and protease-deficient strains of E. coli. Further details of the foregoing and similar mutagenesis techniques are found in general textbooks, such as, for example, Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al. eds., supra. F. Covalent Modifications. 10 Covalent modifications of the novel ILPs of the present invention are included within the scope of the invention. Such modifications are traditionally introduced by reacting targeted amino acid residues of the ILPs with an organic derivatizing agent that is capable of reacting with selected amino acid side chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying 15 residues important for biological activity, for immunoassays of the ILP, or for the preparation of anti-lLP antibodies for immunoaffinity purification of the recombinant. For example, complete inactivation of the biological activity of the protein after reaction with ninhydrin would suggest that at least one arginyl or lysyl residue is critical for its activity, whereafter the individual residues which were modified under the conditions selected are identified by isolation ofa peptide fragment containing the modified amino acid residue. Such 20 modifications are within the ordinary skill in the art and are performed without undue experimentation. Derivatization with bifunctional agents is useful for preparing intramolecular aggregates of the ILPs with polypeptide as well as for cross-linking the ILP polypeptide to a water insoluble support matrix or surface for use in assays or affinity purification. In addition, a study of interchain cross-links will provide direct information on conformational structure. Commonly used cross-linking agents include 1,1-bis(diazoacetyl)-2 25 phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, homobifunctional imidoesters, and bifunctional maleimides. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates which are capable of forming cross-links in the presence of light. Alternatively, reactive water insoluble matrices such as cyanogen bromide activated carbohydrates and the systems reactive substrates described in U.S. Patent Nos. 3,959,642; 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; 30 4,055,635; and 4,330,440 are employed for protein immobilization and cross-linking. Certain post-translational modifications are the result of the action of post-translational deamidation of glutamine and asparagine to the corresponding glutamyl and aspartyl residues. For example, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention. 35 Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, threonyl or tyrosyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)). -25 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Further derivatives of the ILPs herein are the so called "immunoadhesins", which are chimeric antibody-like molecules combining the functional domain(s) of a binding protein (usually a receptor, a cell adhesion molecule or a ligand) with the an immunoglobulin sequence. The most common example of this type of fusion protein combines the hinge and Fc regions of an immunoglobulin (Ig) with domains of a cell-surface 5 receptor that recognizes a specific ligand. This type of molecule is called an "immunoadhesin", because it combines "immune" and "adhesion" functions; other frequently used names are "Ig-chimera", "Ig-" or "Fc fusion protein", or "receptor-globulin." Immunoadhesins reported in the literature include, for example, fusions of the T cell receptor (Gascoigne et al.. Proc. Natl. Acad. Sci. USA 84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 10 (1989): Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA 9:347-353 (1990); Byrn et al., Nature 344:667-670 (1990)); L-selLP (homing receptor) (Watson et al., J. Cell. Biol. 110:2221 2229 (1990)); Watson et al., Nature 349:164-167 (1991)); E-selLP (Mulligan et al., J. Immunol. 151:6410-17 (1993); Jacob et al., Biochemistry 34:1210-1217 (1995)); P-selLP (Mulligan et al., supra; Hollenbaugh et al., Biochemistry 34:5678-84 (1995)); ICAM- 1 (Stauton et al., J. Exp. Med. 176:1471-1476 (1992); Martin et al., 15 J. Virol. 67:3561-68 (1993); Roep et al., Lancet 343:1590-93 (1994)); ICAM-2 (Damle et al., J. Immunol. 148:665-71 (1992)); ICAM-3 (Holness et al., J. Biol. Chem. 270:877-84 (1995)); LFA-3 (Kanner et al., J. Immunol. 148:2023-2029 (1992)); LI glycoprotein (Doherty et al., Neuron 14:57-66 (1995)); TNF-RI (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-539 (1991); Lesslauer et al., Eur. J. Immunol. 21:2883-86 (1991); Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); TNF-R2 (Zack et al., Proc. Natl. 20 Acad. Sci. USA 90:2335-39 (1993); Wooley et al., J. Immunol. 151:6602-07 (1993)); CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley et al., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp. Med. 174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144 (1991)); NP receptors (Bennett et al., J. Biol. Chem. 266:23060-23067 (1991)); IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 abstr. 1448 (1991)); IFN-yR a- and P-chain (Marsters et al., Proc. Natl. Acad. Sci. USA 92:5401-05 25 (1995)): trk-A, -B. and -C (Shelton et al., J. Neurosci. 15:477-91 (1995)); IL-2 (Landolfi, J. Immunol. 146:915-19 (1991)); IL-10 (Zheng etal., J. Immunol. 154:5590-5600 (1995)). The simplest and most straightforward immunoadhesin design combines the binding region(s) of the 'adhesin' protein with the hinge and Fc regions of an immunoglobulin heavy chain. Ordinarily, when preparing the ILP-immunoglobulin chimeras of the present invention, nucleic acid encoding the desired ILP 30 polypeptide will be fused to at least one of the chains preferably at the C-terminus of the chain to the N terminus of nucleic acid encoding the C-terminus of an immunoglobulin constant domain sequence, however fusion to the N-terminus of the immunoglobulin is also possible. Typically, in such fusions the encoded chimeric polypeptide will retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a 35 constant domain, or immediately N-terminal to the CHI of the heavy chain or the corresponding region of the light chain. The precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion or binding characteristics of the ILP immunoglobulin chimeras. -26 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 In a preferred embodiment, the sequence of a native, mature ILP polypeptide is fused to the N terminus of the C-terminal portion of an antibody (in particular the Fc domain), containing the effector functions of an immunoglobulin, e.g. IgG- 1. It is possible to fuse the entire heavy chain constant region to the ILP sequence. However, more preferably, a sequence beginning in the hinge region just upstream of the 5 papain cleavage site (which defines IgG Fc chemically; residue 216, taking the first residue of heavy chain constant region to be 114, or analogous sites of other immunoglobulins) is used in the fusion. In a particularly preferred embodiment, an ILP polypeptide chain is fused to the hinge region and CH2 and CH3 or CHI1, hinge. CH2 and CH3 domains of an IgG-1, IgG-2, or lgG-3 heavy chain. The precise site at which the fusion is made is not critical, and the optimal site can be determined by routine experimentation. 10 In some embodiments, the ILP-immunoglobulin chimeras are assembled as multimers, and particularly as homodimers or homotetramers (WO 91/08298). Generally, these assembled immunoglobulins will have known unit structures. A basic four chain structural unit is the form in which IgG, lgD, and IgE exist. A four unit is repeated in the higher molecular weight immunoglobulins; IgM generally exists as a pentamer of basic four units held together by disulfide bonds. IgA globulin, and occasionally IgG globulin, 15 may also exist in multimeric form in serum. In the case of multimer, each four unit may be the same or different. Various exemplary assembled ILP-immunoglobulin chimeras within the scope of the invention are schematically diagrammed below: (a) ACL-ACL; 20 (b) ACH-[ACH, ACL-ACH, ACL-VHCH, or VLCL-ACH]; (c) ACL-ACH-[ACL-ACH, ACL-VHCH, VLCL-ACH, or VLCL-VHCH]; (d) ACL-VHCH-[ACH, or ACL-VHCH, or VLCL-ACH]; (e) VLCL-ACH-[ACL-VHCH, or VLCL-ACH]; and (f) [A-Y]n-[VLCL-VHCH] 2 , 25 wherein each A represents identical or different novel ILP polypeptide amino acid sequences; VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; 30 CH is an immunoglobulin heavy chain constant domain; n is an integer greater than 1; Y designates the residue of a covalent cross-linking agent. In the interest of brevity, the foregoing structures only show key features: they do not indicate joining (J) or other domains of the immunoglobulins, nor are disulfide bonds shown. However, where such domains 35 are required for binding activity, they shall be construed as being present in the ordinary locations which they occupy in the immunoglobulin molecules. Although the presence of an immunoglobulin light chain is not required in the immunoadhesins of the present invention, an immunoglobulin light chain might be present either covalently associated to an ILP -27 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 immunoglobulin heavy chain fusion polypeptide. or directly fused to the ILP polypeptide. In the former case. DNA encoding an immunoglobulin light chain is typically coexpressed with the DNA encoding the ILP immunoglobulin heavy chain fusion protein. Upon secretion, the hybrid heavy chain and the light chain will be covalently associated to provide an immunoglobulin-like structure comprising two disulfide-linked 5 immunoglobulin heavy chain-light chain pairs. Methods suitable for the preparation of such structures are, for example, disclosed in U.S. Patent No. 4,816,567 issued 28 March 1989. In a preferred embodiment, the immunoglobulin sequences used in the construction of the immunoadhesins of the present invention are from an IgG immunoglobulin heavy chain constant domain. For human immunoadhesins, the use of human IgG-1 and IgG-3 immunoglobulin sequences is preferred. A major 10 advantage of using IgG-1 is that IgG-1 immunoadhesins can be purified efficiently on immobilized protein A. In contrast, purification of IgG-3 requires protein G, a significantly less versatile medium. However, other structural and functional properties of immunoglobulins should be considered when choosing the Ig fusion partner for a particular immunoadhesin construction. For example, the IgG-3 hinge is longer and more flexible, so it can accommodate larger 'adhesin' domains that may not fold or function properly when fused 15 to IgG- 1. While lgG immunoadhesins are typically mono- or bivalent, other Ig subtypes like IgA and IgM may give rise to dimeric or pentameric structures, respectively, of the basic Ig homodimer unit. Multimeric immunoadhesins are advantageous in that they can bind their respective targets with greater avidity than their IgG-based counterparts. Reported examples of such structures are CD4-IgM (Traunecker et al., suora); ICAM-IgM (Martin et al., J. Virol. 67, 3561-68 (1993)); and CD2-IgM (Arulanandam et al., J. Exp. Med. 177, 20 1439-50 (1993)). For ILP-lg immunoadhesins, which are designed for in vivo application, the pharmacokinetic properties and the effector functions specified by the Fc region are important as well. Although IgG-1, IgG-2 and IgG-4 all have in vivo half-lives of 21 days, their relative potencies at activating the complement system are different. IgG-4 does not activate complement, and IgG-2 is significantly weaker at complement activation 25 than IgG-1. Moreover, unlike IgG-1, IgG-2 does not bind to Fc receptors on mononuclear cells or neutrophils. While IgG-3 is optimal for complement activation, its in vivo half-life is approximately one third of the other IgG isotypes. Another important consideration for immunoadhesins designed to be used as human therapeutics is the number of allotypic variants of the particular isotype. In general, IgG isotypes with fewer serologically-defined allotypes are preferred. For example, IgG-1 has only four serologically-defined allotypic 30 sites, two of which (Glm and 2) are located in the Fc region; and one of these sites Glml, is non immunogenic. In contrast, there are 12 serologically-defined allotypes in IgG-3, all of which are in the Fc region: only three of these sites (G3m5, 11 and 21) have one allotype which is nonimmunogenic. Thus, the potential immunogenicity of a y3 immunoadhesin is greater than that of a y l immunoadhesin. ILP-Ig immunoadhesins are most conveniently constructed by fusing the cDNA sequence encoding 35 the ILP portion in-frame to an Ig cDNA sequence. However, fusion to genomic Ig fragments can also be used (see, e.g. Gascoigne et al., Proc. Natl. Acad. Sci. USA 84:2936-2940 (1987); Aruffo et al., Cell 61:1303-1313 (1990); Stamenkovic etal., Cell 66:1133-1144 (1991)). The latter type of fusion requires the presence of Ig regulatory sequences for expression. cDNAs encoding IgG heavy-chain constant regions can be isolated based -28 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 on published sequence from cDNA libraries derived from spleen or peripheral blood lymphocytes, by hybridization or by polymerase chain reaction (PCR) techniques. Other derivatives of the novel ILPs of the present invention, which possess a longer half-life than the native molecules comprise the ILP or an ILP-immunoglobulin chimera, covalently bonded to a 5 nonproteinaceous polymer. The nonproteinaceous polymer ordinarily is a hydrophilic synthetic polymer, i.e., a polymer not otherwise found in nature. However, polymers which exist in nature and are produced by recombinant or in vitro methods are useful, as are polymers which are isolated from native sources. Hydrophilic polyvinyl polymers fall within the scope of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularly useful are polyalkylene ethers such as polyethylene glycol (PEG); 10 polyelkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; branched or unbranched polysaccharides which comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid including homopolysaccharides and 15 heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid: polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparon. The polymer prior to cross-linking need not be, but preferably is, water soluble, but the final conjugate must be water soluble. In addition, the polymer should not be highly immunogenic in the conjugate form, nor should it possess viscosity 20 that is incompatible with intravenous infusion or injection if it is intended to be administered by such routes. Preferably the polymer contains only a single group which is reactive. This helps to avoid cross linking of protein molecules. However, it is within the scope herein to optimize reaction conditions to reduce cross-linking, or to purify the reaction products through gel filtration or chromatographic sieves to recover substantially homogenous derivatives. 25 The molecular weight of the polymer may desirably range from about 100 to 500,000, and preferably is from about 1,000 to 20,000. The molecular weight chosen will depend upon the nature of the polymer and the degree of substitution. In general, the greater the hydrophilicity of the polymer and the greater the degree of substitution, the lower the molecular weight that can be employed. Optimal molecular weights will be determined by routine experimentation. 30 The polymer generally is covalently linked to the novel ILP or to the ILP-immunoglobulin chimeras through a multifunctional crosslinking agent which reacts with the polymer and one or more amino acid or sugar residues of the ILP or ILP-immunoglobulin chimera to be linked. However, it is within the scope of the invention to directly crosslink the polymer by reacting a derivatized polymer with the hybrid, or vice versa. The covalent crosslinking site on the ILP or ILP-Ig includes the N-terminal amino group and epsilon 35 amino groups found on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl or other hydrophilic groups. The polymer may be covalently bonded directly to the hybrid without the use of a multifunctional (ordinarily bifunctional) crosslinking agent. Covalent binding to amino groups is accomplished by known chemistries based upon cyanuric chloride, carbonyl diimidazole, aldehyde reactive -29 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 groups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde: PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde, succinimidyl active esters, activated dithiocarbonate PEG, 2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate activated PEG.) Carboxyl groups are derivatized by coupling PEG-amine using carbodiimide. 5 Polymers are conjugated to oligosaccharide groups by oxidation using chemicals, e.g. metaperiodate, or enzymes, e.g. glucose or galactose oxidase, (either of which produces the aldehyde derivative of the carbohydrate), followed by reaction with hydrazide or amino derivatized polymers, in the same fashion as is described by Heitzmann et al., P.N.A.S., 71:3537-41 (1974) or Bayer et al., Methods in Enzymology 62:310 (1979), for the labeling of oligosaccharides with biotin or avidin. Further, other chemical or enzymatic 10 methods which have been used heretofore to link oligosaccharides are particularly advantageous because, in general, there are fewer substitutions than amino acid sites for derivatization, and the oligosaccharide products thus will be more homogeneous. The oligosaccharide substituents also are optionally modified by enzyme digestion to remove sugars, e.g. by neuraminidase digestion, prior to polymer derivatization. The polymer will bear a group which is directly reactive with an amino acid side chain, or the N- or 15 C-terminus of the polypeptide linked, or which is reactive with the multifunctional cross-linking agent. In general, polymers bearing such reactive groups are known for the preparation of immobilized proteins. In order to use such chemistries here, one should employ a water soluble polymer otherwise derivatized in the same fashion as insoluble polymers heretofore employed for protein immobilization. Cyanogen bromide activation is a particularly useful procedure to employ in crosslinking polysaccharides. 20 "Water soluble" in reference to the starting polymer means that the polymer or its reactive intermediate used for conjugation is sufficiently water soluble to participate in a derivatization reaction. "Water soluble" in reference to the polymer conjugate means that the conjugate is soluble in physiological fluids such as blood. The degree of substitution with such a polymer will vary depending upon the number of reactive sites 25 on the protein, whether all or a fragment of the protein is used, whether the protein is a fusion with a heterologous protein (e.g. an ILP-immunoglobulin chimera), the molecular weight, hydrophilicity and other characteristics of the polymer, and the particular protein derivatization sites chosen. In general, the conjugate contains about from I to 10 polymer molecules, while any heterologous sequence may be substituted with an essentially unlimited number of polymer molecules so long as the desired activity is not significantly adversely 30 affected. The optimal degree of cross-linking is easily determined by an experimental matrix in which the time, temperature and other reaction conditions are varied to change the degree of substitution, after which the ability of the conjugates to function in the desired fashion is determined. The polymer, e.g. PEG, is cross-linked by a wide variety of methods known in the art for the covalent modification of proteins with nonproteinaceous polymers such as PEG. Certain of these methods, however, 35 are not preferred for the purposes herein. Cyanuronic chloride chemistry leads to many side reactions, including protein cross-linking. In addition, it may be particularly likely to lead to inactivation of proteins containing sulfhydryl groups. Carbonyl diimidazole chemistry (Beauchamp et al., Anal Biochem. 131:25-33 (1983)) requires high pH (>8.5), which can inactivate proteins. Moreover, since the "activated PEG" -30 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 intermediate can react with water, a very large molar excess of "activated PEG" over protein is required. The high concentrations of PEG required for the carbonyl diimidazole chemistry also led to problems in purification, as both gel filtration chromatography and hydrophilic interaction chromatography are adversely affected. In addition, the high concentrations of "activated PEG" may precipitate protein, a problem that has 5 been noted previously (Davis, U.S. Patent No. 4,179,337). On the other hand, aldehyde chemistry (Royer, U.S. Patent No. 4,002,531) is more efficient since it requires only a 40-fold molar excess of PEG and a 1-2 hr incubation. However, the manganese dioxide suggested by Royer for preparation of the PEG aldehyde is problematic "because of the pronounced tendency of PEG to form complexes with metal-based oxidizing agents" (Harris et al., J. Polym. Sci. Polym. Chem. Ed. 22, 341-52 (1984)). The use of a Moffatt oxidation, 10 utilizing DMSO and acetic anhydride, obviates this problem. In addition, the sodium borohydride suggested by Royer must be used at high pH and has a significant tendency to reduce disulfide bonds. In contrast, sodium cyanoborohydride, which is effective at neutral pH and has very little tendency to reduce disulfide bonds is a preferred reagent. The long half-life conjugates of this invention are separated from the unreacted starting materials by 15 gel filtration. Heterologous species of the conjugates are purified from one another in the same fashion. The polymer also may be water-insoluble, as a hydrophilic gel. The novel ILPs may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are 20 disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A., Ed. (1980). G. Antibody preparation. (i) Polyclonal antibodies Polyclonal antibodies to an ILP, or fragment of the ILP, of the present invention generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the ILP and an adjuvant. It may 25 be useful to conjugate the ILP or a fragment containing the target amino acid sequence to a protein that is immunogenic in the species to be immunized, e.g. keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC1 2 , or R 1 N=C=NR, where R and R 1 are 30 different alkyl groups. Animals are immunized against the immunogenic conjugates or derivatives by combining approximately I mg or I pg of conjugate (for rabbits or mice, respectively) with three volumes of Freud's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of conjugate in Freud's complete adjuvant by subcutaneous 35 injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for anti-ILP antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same ILP, but conjugated to a different protein and/or through a different cross-linking -31 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are used to enhance the immune response. (ii) Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., 5 the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. For example, the anti-ILP monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1975), or may be made by recombinant DNA 10 methods (Cabilly, et al., U.S. Pat. No. 4,816,567). DNA encoding the monoclonal antibodies of the invention is 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 15 transfected 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 DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison, et al., Proc. Nat. Acad. Sci. 81:6851 (1984), or by covalently joining to the immunoglobulin coding sequence 20 all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of an ILP monoclonal antibody of the invention. Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of 25 an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an ILP and another antigen-combining site having specificity for a different antigen. Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents 30 for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate. For diagnostic applications, the antibodies of the invention typically will be labeled with a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 3 H, 14 C, 3 2 p, 35S, or 1251, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or 35 luciferin: biotin, radioactive isotopic labels, such as, e.g., 1251, 3 2 p, 14 C, or 3 H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter, et al., Nature 144:945 (1962): David, et al., -32 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren. J. Histochem. and Cytochem. 30:407 (1982). The antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (see, for 5 example, Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987). (iii) Humanized antibodies Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an 10 "import" variable domain. 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 a rodent complementary domain region (CDR) or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (Cabilly, supra), wherein substantially less than an intact 15 human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. It is important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies 20 are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in 25 the functioning of the candidate immunoglobulin sequence. i.e. the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding (see, for example, WO 92/22653). 30 Alternatively, it is now possible to produce transgenic animals (e.g. mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such 35 germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g. Jakobovits et al., Proc. Natl. Acad. Sci. USA 90, 2551-255 (1993); Jakobovits et al., Nature 362, 255-258 (1993). (iv) Bispecific antibodies SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities may be for an ILP of the present invention, while the other one may for any other antigen. for example, another member of the insulin family. Such constructs can also be referred to as bispecific immunoadhesins. 5 Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305, 537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, 10 which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in PCT application publication No. WO 93/08829 (published 13 May 1993), and in Traunecker et al., EMBO 10:3655-3659 (1991). This problem may be overcome by selecting a common light chain for each arm of the bispecific antibody such that binding specificity of each antibody is maintained, as disclosed in US Application Serial No. 08/850,058, filed May 5, 1997. 15 According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, and second and third constant regions of an immunoglobulin heavy chain (CH2 and CH3). It is preferred to have the first heavy chain constant region (CH1) containing the site necessary for light chain 20 binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are cotransfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or 25 all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance. In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure 30 facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in PCT application WO 94/04690 published 3 March 1994. For further details of generating bispecific antibodies see, for example, Suresh et al, Methods in 35 Enzymology 121, 210 (1986). (v) Heteroconjugate antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been -34 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 proposed to target immune system cells to unwanted cells (U.S. Patent No. 4.676,980), and for treatment of HIV infection (PCT application publication Nos. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross 5 linking techniques. H. Diagnostic Kits & Articles of Manufacture. Since the invention provides a diagnostic assay (i.e. for detecting the presence of ILP in a sample using antibodies or DNA markers and for detecting expression of the ILP gene in a tissue sample) as a matter of convenience, the reagents for these assays can be provided in a kit, i.e., a packaged combination of reagents, 10 for combination with the sample to be tested. The components of the kit will normally be provided in predetermined ratios. Thus, a kit may comprise the antibody or ILP (DNA or polypeptide or fragment thereof) labeled directly or indirectly with a suitable label. Where the detectable label is an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g. a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives may be included such as stabilizers, buffers and the 15 like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration. The kit also suitably includes instructions for carrying out the bioassay. 20 In another embodiment of the invention, an article of manufacture is provided which contains materials useful for the treatment of disorders associated with ILP overexpression or decreased expression as described herein. The article of manufacture comprises a container and a label wherein the label provides instructions for the administration of the ILP or ILP agonist or antagonist for treatment of a mammal according to the invention. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The 25 containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is ILP or an agonist or antagonist thereof. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. The 30 article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. I. Peptide and non-peptide analogs. 35 Peptide analogs of the ILPs of the present invention are modeled based upon the three-dimensional structure of the native polypeptide. Peptides may be synthesized by well known techniques such as the solid phase synthetic techniques initially described in Merrifield, J. Am. Chem. Soc. 15:2149-2154 (1963). Other peptide synthesis techniques are, for examples, described in Bodanszky et al., Peotide Synthesis, John Wiley -35 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 & Sons, 2nd Ed.. 1976, as well as in other reference books readily available for those skilled in the-art. A summary of peptide synthesis techniques may be found in Stuart and Young, Solid Phase Peotide Svnthelia, Pierce Chemical Company, Rockford, IL (1984). Peptides may also be prepared by recombinant DNA technology, using a DNA sequence encoding the desired peptide. 5 In addition to peptide analogs, the present invention also contemplates non-peptide (e.g. organic) compounds which display substantially the same surface as the peptide analogs of the present invention, and therefore interact with other molecules in a similar fashion. J. Use of the ILPs. Amino acid sequence variants of the native ILPs of the present invention may be employed 10 therapeutically to compete with the normal binding of the native proteins to an ILP receptor. Thus, where the variant binds but does not activate a receptor, the ILP amino acid sequence variants are useful as competitive inhibitors of the biological activity of native ILP. Native ILP and its amino acid sequence variants are useful in the identification and purification of a native ILP receptor. The purification is preferably performed by immunoadhesins comprising an ILP amino 15 acid sequence retaining the qualitative ability of a native ILP of the present invention to recognize its native ILP receptor. The native ILPs of the present invention are further useful as molecular markers of the tissues in which an ILP receptor is expressed. Furthermore, the ILPs of the present invention provides valuable sequence motifs which can be 20 inserted or substituted into other native members of the insulin family of molecules. The alteration of these native proteins by the substitution or insertion of sequences from the novel ILP of the present invention can yield variant molecules with altered biological properties, such as receptor binding affinity or receptor specificity. For example, one or more ILP domains of another member of the insulin family may be entirely or partially replaced by ILP domain sequences derived from an ILP of the present invention. Similarly, 25 sequences from an ILP disclosed herein may be substituted or inserted into the amino acid sequences of other insulin family members. Additionally, anti-ILP antibodies of the invention are useful in kits for the diagnosis of disease related to ILP and for methods of detecting the presence or absence of ILP in a sample, such as a body fluid or tissue sample, as described herein. The present invention provides a nucleotide sequence uniquely identifying a 30 novel insulin-like polypeptide which is expressed, for example, in colon and uterus. As a result of expression in these organs, the nucleic acid, ilp, the polypeptide, ILP, and antibodies to ILP are useful in diagnostic assays based on ILP production in cases of disease affecting the colon or uterus. A test for excess expression of ILP can diagnose an abnormal condition of the organ from which the cell or tissue sample was obtained. Such abnormal conditions include, but are not limited to, colon cancer, uterine cancer, ovarian cancer, 35 adenocarcinoma, colitis inflammatory bowel disease, pelvic inflammatory disease, gastrointestinal bleeding, Crohn's disease, abnormal uterine contraction, constipation, irritable bowel syndrome, diabetes, and obesity. The nucleotide sequences encoding ILP (or their complement) have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as -36 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 hybridization probes. use as oligomers for PCR, use for chromosome and gene mapping, use- in the recombinant production of ILP, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding ILP disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art. Furthermore, the 5 nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g., the triplet genetic code, specific base pair interactions, etc. Although nucleotide sequences which encode ILP and/or ILP variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring ILP gene under stringent conditions, it may 10 be advantageous to produce nucleotide sequences encoding ILP or ILP derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding ILP and/or ILP derivatives without altering the encoded amino acid sequence include the production 15 of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence. It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of ILP-encoding nucleotide sequences, some bearing minimal homology to the nucleotide sequence of any known and naturally occurring gene may be produced. The invention has specifically 20 contemplated each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring ILP. and all such variations are to be considered as being specifically disclosed. Nucleotide sequences encoding ILP may be joined to a variety of other nucleotide sequences by 25 means of well established recombinant DNA techniques (cf Sambrook J et al. (1989) Molecular Cloninc: A Laboratory Manual. Cold Spring Harbor Laboratory, New York). Useful nucleotide sequences for joining to ilp include an assortment of cloning vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Vectors of interest include expression vectors, replication vectors, 30 probe generation vectors, sequencing vectors, and the like. In general, vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for the host cell. Another aspect of the invention is to provide for ilp-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding ILP. Such probes may also be used for 35 the detection of similar insulin-like peptide encoding sequences. The hybridization probes of the subject invention may be derived from the nucleotide sequences of SEQ ID NO: I or its complement or from genomic sequences including promoters, enhancer elements and introns of naturally occurring ilp. Hybridization probes -37 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 may be labeled by a variety of reporter groups. including radionuclides such as 3 2 P or 3 5 S. or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like. PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences which encode ILP. Such probes used in PCR may 5 be of recombinant origin, may be chemically synthesized, or a mixture of both and comprise a discrete nucleotide sequence for diagnostic use or a degenerate pool of possible sequences for identification of closely related genomic sequences. Other means of producing specific hybridization probes for ilp include the cloning of nucleic acid sequences encoding ILP and ILP derivatives into vectors for the production of mRNA probes. Such vectors 10 are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. It is now possible to produce a DNA sequence, or portions thereof, encoding ILP and ILP derivatives entirely by synthetic chemistry, after which the gene can be inserted into any of the many available DNA 15 vectors using reagents, vectors and cells that are known in the art at the time of the filing of this application. Moreover, synthetic chemistry may be used to introduce mutations into the ilp sequence or any portion thereof The nucleotide sequence can be used to construct an assay to detect disease associated with abnormal levels of expression of ilp. The nucleotide sequence can be labeled by methods known in the art and added to a fluid or tissue sample from a mammal under hybridizing conditions. After an incubation period, the 20 sample is washed with a compatible fluid which optionally contains a dye (or other label requiring a developer) if the nucleotide has been labeled with an enzyme. After the compatible fluid is rinsed off, the dye is quantitated and compared with a standard. If the amount of dye is significantly elevated, the nucleotide sequence has hybridized with the sample, and the assay indicates of ilp expression and the presence of disease. 25 The nucleotide sequence for i/p can be used to construct hybridization probes for mapping that gene. The nucleotide sequence provided herein may be mapped to a chromosome and specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries 30 or flow-sorted chromosomal preparations specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosome spreads has been described, for example, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, NYC. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of genetic map data can 35 be found in genome issue of Science (1994) 265:1981. Correlation between the location of ilp on a physical chromosomal map and a specific disease (or predisposition to a specific disease) can help delimit the region of DNA associated with that genetic disease. The nucleotide sequence of the subject invention may be used to detect differences in gene sequence between normal and carrier or affected individuals. -38 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Nucleotide sequences encoding ILP may be used to produce purified ILP using well known methods of recombinant DNA technology (see, for example. Sambrook, J. et al. (1989) supra: or Goeddel (1990) Gene Expression Technology, Methods and Enzymologyev, Vol 185, Academic Press. San Diego). ILP may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species 5 in which ilp nucleotide sequences are endogenous or from a different species. Advantages of producing ILP by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures. Cells transformed with DNA encoding ILP may be cultured under conditions suitable for the expression of the ILP and the recovery of the protein from the cell culture. ILP produced by a recombinant 10 cell may be secreted or may be contained intracellularly, depending on the particular genetic construction used. In general, it is more convenient to prepare recombinant proteins in secreted form. Purification steps vary with the production process and the particular protein produced. In addition to recombinant production, ILP fragments may be produced by direct peptide synthesis using solid-phase techniques (Stewart, et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San 15 Francisco; Merrifield, J. (1963) J Am Chem Soc 85:2149-2154. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.) in accordance with the instructions provided by the manufacturer. Various fragments of ILP may be chemically synthesized separately and combined using chemical methods to produce the full length molecule. 20 ILP used for antibody induction does not require biological activity; however, it must be immunogenic. Peptides used to induce specific antibodies may have an amino acid sequence of at least five amino acids, preferably at least 10 amino acids. They should mimic a portion of the amino acid sequence of the protein and may contain the entire amino acid sequence of ILP. Short stretches of ILP amino acid sequence may be fused with those of another protein such as keyhole limpet hemocyanin and the chimeric 25 molecule used for antibody production. Antibodies specific for ILP may be produced by inoculation of an appropriate animal with the polypeptide or an antigenic fragment. An antibody is specific for ILP if it is produced against an epitope of the polypeptide and binds to at least part of the natural or recombinant protein. Antibody production includes not only the stimulation of an immune response by injection into animals, but also analogous steps in the 30 production of synthetic antibodies or other specific-binding molecules such as the screening of recombinant immunoglobulin libraries (Orlandi, R. et al. (1989) PNAS 86:3833-3837; or Huse, W.D. et al. (1989) Science 256:1275-1281) or the in vitro stimulation of lymphocyte populations. Current technology provides for a number of highly specific binding reagents based on the principles 35 of antibody formation (Winter, G. and Milstein, C. (1991) Nature 349:293-299) . These techniques may be adapted to produce molecules that specifically bind ILP. An additional embodiment of the subject invention is the use of ILP-specific antibodies, inhibitors, receptors or their analogs as bioactive agents to promote the survival or growth of cells, treat disease of the -39 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 colon, uterus or other organs and tissues such as the eye, which diseases include, but are not limited to colon cancer, uterine cancer, ovarian cancer, adenocarcinoma, colitis inflammatory bowel disease, pelvic inflammatory disease, gastrointestinal bleeding. Crohn's disease, abnormal uterine contraction, constipation, irritable bowel syndrome, diabetes, and obesity; or other physiologic and pathologic problems which affect 5 the function of the indicated organs. Bioactive compositions comprising agonists, antagonists, receptors or inhibitors of ILP may be administered in a suitable therapeutic dose determined by any of several methodologies including clinical studies on mammalian species to determine maximal tolerable dose and on normal human subjects to determine safe dose. Additionally, the bioactive agent may be complexed with a variety of well established 10 compounds or compositions which enhance stability or pharmacological properties such as half-life. It is contemplated that the therapeutic, bioactive composition may be delivered by intravenous infusion into the bloodstream or any other effective means which could be used for treating problems of the colon, uterus, or related tissue. Dosages and administration of ILP, ILP agonist or ILP antagonist in a pharmaceutical composition 15 may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics (see, for example. Mordenti. J. and Rescigno, A. (1992) Pharmaceutical Research 9:17-25: Morenti, J. et al. (1991) Pharmaceutical Research 8:1351-1359; and Mordenti, J. and Chappell, W. (1989) "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drue Development, Yacobi et al. (eds), Pergamon Press, NY, pp. 42-96, each of which references are herein incorporated by reference in its entirety). An effective 20 amount of ILP or ILP agonist or antagonist to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the mammal. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 10 ng/kg to up to 100 mg/kg of the mammal's body weight or more per day, preferably about 1 pg/kg/day to 10 mg/kg/day. Typically, the 25 clinician will administer ILP or ILP agonist or antagonist until a dosage is reached that achieves the desired effect for treatment of the above mentioned disorders. ILP or an ILP agonist or ILP antagonist may be administered alone or in combination with another to achieve the desired pharmacological effect. ILP itself, or agonists, antibodies, inhibitors, receptors or antagonists of ILP can provide different effects when administered therapeutically. Such compounds for 30 treatment will be formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although the pH may vary according to the characteristics of the ILP, agonist, antibody, inhibitor, receptor or antagonist being formulated and the condition to be treated. Characteristics of the treatment compounds include solubility of the molecule, half-life and antigenicity/immunogenicity; these and other characteristics may aid in defining an effective carrier. 35 Native human proteins are preferred for treatment, but organic or synthetic molecules resulting from drug screens may be equally effective in particular situations. ILP or ILP agonists, or antibodies, inhibitors, receptors or antagonists may be delivered by known routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol, -40 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 transdermal patch and bandage; injectable, intravenous and lavage formulations: and orally administered liquids and pills, particularly formulated to resist stomach acid and enzymes. The particular formulation, exact dosage, and route of administration will be determined by the attending physician and will vary according to each specific situation. 5 Such determinations of administration are made by considering multiple variables such as the condition to be treated, the type of mammal to the treated, the compound to be administered, and the pharmacokinetic profile of the particular treatment compound. Additional factors which may be taken into account include disease state (e.g. severity) of the patient, age, weight, gender, diet, time of administration, drug combination, reaction sensitivities, and tolerance/response to therapy. Long acting treatment compound 10 formulations (such as liposomally encapsulated ILP or PEGylated ILP or ILP polymeric microspheres, such as polylactic acid-based microspheres) might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular treatment compound. Normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 pg/kg/day to 10 mg/kg/day, depending upon the route of administration. 15 Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. Nos. 4,657,760: 5.206.344; or 5,225,212. each of which patents is herein incorporated by reference in its entirety. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting the uterus or colon, for example, may necessitate delivery in a manner different from that to another organ or tissue. 20 Where sustained release administration of ILP is desired, microencapsulation of the protein or polypeptide is contemplated. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon-y (rhIFN-y), interleukin-2, and MN rgpl20. Johnson et al., Nat. Med., 2: 795-799 (1996); Yasuda, Biomed. Ther., 27: 1221-1223 (1993); Hora et al., Bio. Technol. 8: 755-758 (1990): Cleland, "Design and Production of Single Immunization Vaccines 25 Using Polylactide Polyglycolide Microsphere Systems." in Vaccine Design: The Subunit and Adiuvant Approach , Powell and Newman, eds, (Plenum Press, New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S Pat. No. 5,654,010. WO 96/07399 refers to several proteins, including IGF I. See also EP 257,368 on a microsphere composition with IGF-I or GH for slow release. The sustained release formulations of these proteins were developed using poly-lactic-coglycolic acid 30 (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body. Moreover, the degradability of this polymer can be adjusted from months to years depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide/glycolide polymer," in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drue Delivery Systems (Marcel Dekker: New York, 1990), 35 pp. 1-41. There is a need in the art for a sustained-release formulation of ILP with release characteristics suitable for the treatment of any disease or disorder requiring administration of ILP. For a formulation that can provide a dosing of approximately 80 pg/kg/day in mammals with a maximum body weight of 85 kg, the -41 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 largest dosing would be approximately 6.8 mg ILP per day. In order to achieve this dosing level, a sustained release formulation which contains a maximum possible protein loading (15-20% w/w ILP) with the lowest possible initial burst (<20%) is necessary. A continuous (zero-order) release of ILP from microparticles for 1-2 weeks is also desirable. In addition, the encapsulated protein to be released should maintain its integrity 5 and stability over the desired release period. It is contemplated that conditions or diseases of the uterus, colon, or other urogenital tissues may precipitate damage that is treatable with ILP or ILP agonist where ILP expression is reduced in the diseased state; or with antibodies to ILP, ILP receptors, or ILP antagonists where the expression of ILP is increased in the diseased state. These conditions or diseases may be specifically diagnosed by the tests discussed above 10 for physiologic and pathologic problems which affect the function of the organ. The instant invention is shown and described herein in what is considered to be the most practical, and the preferred embodiments. It is recognized, however, that departures may be made therefrom which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art upon reading this disclosure. 15 EXAMPLES The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make the compounds and compositions of the invention and how to practice the methods of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to insure accuracy with respect to numbers used (e.g. 20 amounts, temperature, etc.), but some experimental errors and deviation should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in degrees C, and pressure is at or near atmospheric. Example 1: Selection of Expressed Sequence Tags with Homolovgy to Insulin Family of Proteins The nucleic acid sequence of the relaxin molecule, a member of the insulin family of proteins, was 25 used to search for homologous sequences in a human colon cDNA library of expressed sequence tags (EST) from Incyte, Inc. Two ESTs were obtained, Incyte INC2328985 (Genentech DNA 26648: SEQ ID NO:14; Fig. 5) and INC778319 (SEQ ID NO:15; Fig. 5), each having approximately 40% homology to a region of the relaxin nucleic acid sequence, and represent sequences within a gene of an insulin-like polypeptide (ILP). The EST corresponding to SEQ ID NO: 15 was used to clone the full length ILP gene. 30 The full length ILP gene sequence was cloned using oligonucleotide primers, the design of which was based on the nucleic acid sequence of the EST corresponding to SEQ ID NO:15 (Incyte EST INC2328985; Genentech DNA 26648). The oligonucleotide primers were 5'CAC ATT CAG TCC TCA GCA AAA TGA A-3' (IN2328985.f; SEQ ID NO:11 I); 5'-GAG AAT AAA AAC AGA GTG AAA ATG GAG CCC TTC ATT TTG C-3' (IN2328985.p; SEQ ID NO: 12); and 5'-CTC AGC TTG CTG AGC TTG AGG GA-3' (IN2328985.r; 35 SEQ ID NO:13). Example 2: Construction of a full-length cDNA library. In general, the construction of a genomic DNA library typically includes the following steps: (1) isolation of genomic DNA, (2) partial or complete digestion of the DNA, and (3) size fractionation. The DNA -42 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 is then ligated to a vector, and introduced into a host cell, e.g. E coli (by transformation with a plasmid-vector or by in vitro packaging into bacteriophage particles and subsequent infection of E. coli). The latter steps are substantially the same for genomic and cDNA libraries. The size of a library of random genomic DNA fragments that is required to ensure representation of all sequences present in the genome will depend on the 5 size of the genome and the size of the cloned fragments (see, Clark and Carbon. Cell 92, 91-99 (1976)). There are a number of different procedures for the preparation of genomic DNA, all of which start with some form of cell lysis, followed by deproteinization and recovery of the DNA. Typical protocols for the preparation of genomic DNA from mammalian, plant tissues and bacteria are described, e.g. in Ausubel et al., supra, Units 2.2-2.4. Digestion of the genomic DNA is performed by restriction enzymes, following routine procedures 10 of partial or complete digestion. In order to avoid distortions, it is important to select an enzyme that cuts the DNA with high frequency but without any bias in selection of one site over another. A partial digestion method for the maximization of the randomness of DNA sequence in genomic libraries is described, for example, in Seed eta!., Gene 19, 201-209 (1982). Protocols for enzymatic manipulation of DNA are disclosed in Ausubel et al., supra, Unit 3. The completely or partially digested DNA must then be size fractionated to 15 remove small and large fragments, which would interfere with subsequent cloning. Methods for size fractionation are well known in the art and are typically based on sucrose gradient fractionation or preparative gel electrophoresis. The DNA is then ligated into a vector, which is introduced into a host cell, typically E. coli. General techniques for the construction of genomic DNA libraries are disclosed, for example, in Ausubel et al., supra, especially in Units 5.1.1-5.1.2; 5.3.2-5.3.6; 5.4.1-5.4.3; and 5.7.1-5.7.3. Introduction of the 20 library into E. coli can be performed by any standard transformation techniques, including CaCI 2 transfection, and electroporation. In a typical procedure of constructing recombinant cDNA libraries, poly(A) + mRNAs are isolated from cells, preferably a cell type in which the mRNA encoding the desired polypeptide is produced in large quantities. The mRNAs are then converted into double stranded cDNA (dscDNA) in vitro using the enzyme 25 reverse transcriptase to synthesize complementary cDNA strands from the mRNA template. In order to obtain double-stranded DNA suitable for ligation into a vector, the dscDNA copy of the mRNA is methylated and equipped with suitable (usually EcoRI) linkers. Methods for methylation of DNA are well known in the art, and involve the use of commercially available methylases which covalently join methyl groups to adenine or cytosine residues within specific target sequences. For example, EcoRI methylates an adenine residue within 30 the EcoRl recognition sequence. In the process of converting mRNA into double stranded cDNA in vitro, a first cDNA strand is synthesized by the reverse transcriptase and separated from the mRNA by treatment with alkali or using a nuclease such as the enzyme RNase H. Conveniently, this step can be achieved using a reverse transcriptase that also has RNase H activity. E. coli DNA polymerase then uses the first cDNA strand as a template for the synthesis of the second cDNA strand, thereby producing a population of dscDNA 35 molecules from the original poly(A) mRNA. After converting the 5' and 3' ends into blunt ends, the dscDNA can be ligated to linkers/adaptors and subsequently ligated into suitable vectors and transformed or packaged into a cell, thereby forming the library. For methods for preparing high-quality cDNA libraries see, for example, Gubler and Hoffman. Gene 25, 263-269 (1983); Okayama and Berg, Mol. Cell. Biol. 2, 161-170 -43 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 (1982): and Kato et al, Gene 150, 243-250 (1994). Typical protocols for making cDNA libraries are also described in Ausubel et aL., supra, especially in Units 5.2.1: 5.5.2-5.5.7; 5.6.1-5.6.8: and 5.8.1-5.8.11. An optional method for converting mRNA into dscDNA is disclosed in copending patent application Serial No. 08/872,861 filed 15 October 1996. herein incorporated by reference in its entirety. According to 5 this method, reverse transcriptase-producing cells are transformed with vectors in which the 5' end of a mRNA molecule having a 5' oligonucleotide cap is ligated to a single-stranded 5' overhang complementary to the oligonucleotide cap, and the 3' end of the mRNA molecule is ligated to a single-stranded 3' overhang complementary to the 3' end of the mRNA molecule, so that the reverse transcriptase produced by the cell converts the mRNAs into dscDNAs to form a cDNA library. 10 As an alternative, a cDNA library may be prepared such that the library is enriched in signal sequences. This library is enriched in amino terminal signal sequences which are within a cloning vector that possesses both a unique restriction site at the 5' end of the inserted cDNA clone and a DNA promotor 5' to the inserted cDNA. Details of the generation and use of such a library is disclosed in U.S. Application Serial No. 08/815,520 filed February 27, 1997, herein incorporated by reference in its entirety. 15 According to the method disclosed in U.S. Application Serial No. 08/815,520, mammalian signal sequences are detected based upon their ability to effect the secretion of a starch degrading enzyme (e.g. amylase) lacking a functional native signal sequence. The secretion of the enzyme is monitored by the ability of the transformed yeast cells, which cannot degrade starch naturally or have been rendered unable to do so, to degrade and assimilate soluble starch. 20 Briefly, the method involves transforming non-amylolytic yeast cells with exogenous DNA containing the coding sequence of a randomly selected, unidentified mammalian signal peptide ligated to DNA encoding an amylase, which amylase lacks a functional native signal peptide. Preferably, the exogenous DNA is from a mammalian cDNA library enriched in signal sequences and the mammalian coding sequence is inserted amino terminal to, and in-frame with the secretion defective amylase gene. It is also preferred that 25 the ATG start codon is eliminated or mutated at the N-terminus of the signal sequence as well as at the N terminus of the mature amylase gene, such that translation is initiated only from the start codon of the mammalian signal peptide to be identified. The transformed yeast cells are then screened, for their ability to degrade starch. Positive clones are isolated and the mammalian cDNA is purified. The recombinant cDNA library preferably is a mammalian cDNA library. The DNA identified preferably is a full-length cDNA 30 encoding a novel secreted or transmembrane polypeptide. According to a method disclosed in U.S. Application Serial No. 08/815,520, the cDNA library enriched in signal sequences is created using the following procedure. The vector used for preparing the cDNA library contains a first unique restriction site and a DNA promotor region 5' to the inserted cDNA. An mRNA transcript is transcribed from the insert cDNA. Next, random DNA oligonucleotide primers are used 35 for reverse transcription of the mRNA to create cDNA fragments of the full-length cDNA clone. The cDNA fragments corresponding to lengths between approximately 500 bp and 1000 bp are ligated to an adapter oligonucleotide coding for a second unique restriction site. The cDNA fragments are then digested with a restriction enzyme that cuts at the first unique restriction site. The cDNA fragments are then ligated into the -44 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 amylase expression vector described above which has been digested with enzymes compatible with the first and second restriction sites of the cDNA. Selected clones are used to isolate the full length cDNA from the original cDNA library. Isolation of mRNA: 5 A cDNA library was constructed from human uterus mRNA obtained from Clontech Laboratories, Inc. Palo Alto, CA USA, catalog no. 6537-1. The following protocol is described in "Instruction Manual: SUPERSCRIPT@ Lamda System for cDNA Synthesis and I cloning," cat. No. 19643-014, Life Technologies, Gaithersburg, MD, USA which is herein incorporated by reference. Unless otherwise noted, all reagents were also obtained from Life 10 Technologies. The overall procedure can be summarized into the following steps: (1) First strand synthesis; (2) Second strand synthesis; (3) Adaptor addition; (4) Enzymatic digestion; (5) Gel isolation of cDNA: (6) Ligation into vector; and (7) Transformation. First strand synthesis: Notl primer-adapter (Life Tech., 2 gl, 0.5 Mg/4l) was added to a sterile 1.5 ml microcentrifuge tube 15 to which was added poly A+ mRNA ( 7 yl, 51.g). The reaction tube was heated to 70oC for 5 minutes or time sufficient to denature the secondary structure of the mRNA. The reaction was then chilled on ice and 5X First strand buffer (Life Tech., 4 gl), 0.1 M DTT (2 41) and 10 mM dNTP Mix (Life Tech., I l) were added and then heated to 37'C for 2 minutes to equilibrate the temperature. SUPERSCRIPT II® reverse transcriptase (Life Tech., 5 4l) was then added, the reaction tube mixed well and incubated at 37oC for 1 hour, and 20 terminated by placement on ice. The final concentration of the reactants was the following: 50 mM Tris-HCI (pH 8.3); 75 mM KCI; 3 mM MgCI 2 ; 10 mM DTT; 500 mM each dATP, dCTP, dGTP and dTTP; 50 mg/ml Notl primer-adapter; 5 mg (250 mg/ml) mRNA; 50,000 U/ml SUPERSCRIPT II® reverse transcriptase. Second strand synthesis: While on ice, the following reagents were added to the reaction tube from the first strand synthesis, 25 the reaction well mixed and allowed to react at 16'C for 2 hours, taking care not to allow the temperature to go above 16oC: distilled water (93 ml); 5X Second strand buffer (30 ml); dNTP mix (3 ml); 10 U/ml E. Coli DNA ligase (1 ml); 10 U/ml E. coli DNA polymerase I (4 ml); 2 U/ml E. coli RNase H (1 ml). 10 U T4 DNA Polymerase (2 ml) was added and the reaction continued to incubate at 16'C for another 5 minutes. The final concentration of the reaction was the following: 25 mM Tris-HCI (pH 7.5); 100 mM KCI; 5 mM MgC1 2 ; 10 30 mM (NH 4

)

2

SO

4 ; 0.15 mM b-NAD+; 250 mM each dATP, dCTP, dGTP, dTTP; 1.2 mM DTT; 65 U/ml DNA ligase; 250 U/ml DNA polymerase I; 13 U/ml RNase H. The reaction was halted by placement on ice and by addition of 0.5 M EDTA (10 ml), then extracted through phenol:chloroform:isoamyl alcohol (25:24:1, 150 ml). The aqueous phase was removed, collected and diluted into SM NaCI (15 ml) and absolute ethanol ( 20'C, 400 ml) and centrifuged for 2 minutes at 14,000 x g. The supernatant was carefully removed from the 35 resulting DNA pellet, the pellet resuspended in 70% ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000 x g. The supernatant was again removed and the pellet dried in a SPEEDVACTM drier. -45 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Adapter addition The following reagents were added to the cDNA pellet from the Second strand synthesis above, and the reaction was gently mixed and incubated at 16oC for 16 hours: distilled water (25 ml); 5X T4 DNA ligase buffer (10 ml); Sall adapters (10 ml); T4 DNA ligase (5 ml). The final composition of the reaction was the 5 following: 50 mM Tris-HCl (pH 7.6); 10 mM MgCI 2 ; 1 mM ATP; 5% (w/v) PEG 8000: 1 mM DTT; 200 mg/ml Sal I adapters; 100 U/ml T4 DNA ligase. The reaction was extracted through phenol:chloroform:isoamyl alcohol (25:24:1, 50 ml), the aqueous phase removed, collected and diluted into 5M NaCI (8 ml) and absolute ethanol (-20oC, 250 ml). This was then centrifuged for 20 minutes at 14,000 x g, the supernatant removed and the pellet was resuspended in 0.5 ml 70% ethanol, and centrifuged again for 10 2 minutes at 14,000 x g. Subsequently, the supernatant was removed and the resulting pellet dried in a SPEEDVACTM drier and carried on into the next procedure. Enzymatic digestion: To the cDNA prepared with the Sal I adapter from the previous paragraph was added the following reagents and the mixture was incubated at 37oC for 2 hours: DEPC-treated water (41 ml); Not I restriction 15 buffer (REACT, Life Tech., 5 ml), Not 1 (4 ml). The final composition of this reaction was the following: 50 mM Tris-HCI (pH 8.0): 10 mM MgCI 2 ; 100 mM NaCI: 1,200 U/ml Not 1. Gel isolation of cDNA: The cDNA was size fractionated by acrylamide gel electrophoresis on a 5% acrylamide gel, and any fragments which were larger than I kb, as determined by comparison with a molecular weight marker, were 20 excised from the gel. The cDNA was then electroeluted from the gel into 0.1 x TBE buffer (200 ml) and extracted with phenol:chloroform:isoamyl alcohol (25:24:1, 200 ml). The aqueous phase was removed, collected and centrifuged for 20 minutes at 14,000 x g. The supernatant was removed from the DNA pellet which was resuspended in 70% ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000 x g. The supernatant was again discarded, the pellet dried in a speedvac and resuspended in distilled water (15 ml). 25 Ligation of cDNA into pRK5B vector: The following reagents were added together and incubated at 16 oC for 16 hours: 5X T4 ligase buffer (3 ml); pRK5B, XhoI, NotI digested vector, 0.5 mg, 1 ml); cDNA prepared from previous paragraph (5 ml) and distilled water (6 ml). Subsequently, additional distilled water (70 ml) and 10 mg/ml tRNA (0.1 ml) were added and the entire reaction was extracted through phenol:chloroform:isoamyl alcohol (25:24:1). The 30 aqueous phase was removed, collected and diluted into 5M NaCI (10 ml) and absolute ethanol (-20'C, 250 ml). This was then centrifuged for 20 minutes at 14,000 x g, decanted, and the pellet resuspended into 70% ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000 x g. The DNA pellet was then dried in a speedvac and eluted into distilled water (3 ml) for use in the subsequent procedure. Transformation of library ligation into bacteria: 35 The ligated cDNA/pRK5B vector DNA prepared previously was chilled on ice to which was added electrocompetent DH10B bacteria (Life Tech., 20 ml). The bacteria vector mixture was then electroporated as per the manufacturers recommendation. Subsequently SOC media (1 ml) was added and the mixture was incubated at 37oC for 30 minutes. The transformants were then plated onto 20 standard 150 mm LB plates -46 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 containing ampicillin and incubated for 16 hours (37°C) to allow the colonies to grow. Positive colonies were then scraped off and the DNA isolated from the bacterial pellet using standard CsCl-gradient protocols (see, for example, Ausubel et al., supra, 2.3.1.). Example 3: Isolation of full-length ilp 5 The full length nucleic acid sequence of ilp was obtained by screening a plasmid cDNA library (prepared from uterine mRNA as described in Example 2) by colony hybridization using oligonucleotides designed based on the EST sequences from Incyte, Inc (Incyte EST INC2328985 (SEQ ID NO: 14) and Incyte EST INC778319 (SEQ ID NO:15)). The primer oligonucleotide sequences are indicated in Fig. I by overlining or underlining of sense and antisense strands, respectively as 5' 10 CACATTCAGTCCTCAGCAAAATGAA-3' (SEQ ID NO:1l); 5' GAGAATAAAAACAGAGTGAAAATGGAGCCCTTCATTTTGC-3' (SEQ ID NO:12); and 5' CTCAGCTTGCTGAGCTTGAGGGA-3' (SEQ ID NO:13). The sequence of the cDNA obtained by this procedure were determined by standard techniques. The nucleic acid and amino acid sequences are shown in Fig.1. 15 Example 4: Homologv Searching of cDNA Clones and Deduced Protein The cloned nucleic acid sequence and deduced amino acid sequence obtained as described above were compared to sequences in the GenBank sequence database using a "BLAST" search algorithm for determining regions of homology. The three parameters that determine how the sequence comparisons were run were window size, window offset, and error tolerance. Using a combination of these three parameters, the 20 DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith-Waterman alignments were used to display the results of the homology search. Peptide and protein sequence homologies were ascertained using the "ALIGN" program in a way 25 similar to that used in DNA sequence homologies. Pattern Specification Language and parameter windows were used to search protein databases for sequences containing regions of homology which were scored with an initial value. Dotmatrix homology plots were examined to distinguish regions of significant homology from chance matches. The ilp nucleic acid sequence and the ILP amino acid sequence were homologous to but clearly 30 different from any known polypeptide molecule, and therefore the ILP constitutes a novel member of the insulin family of proteins. The complete nucleotide sequence for the ILP gene is shown as SEQ ID NO: 1. When all three possible predicted translations of the sequence were searched against protein databases such as SwissProt and PIR, no exact matches were found to the possible translations of ilp. Fig. 2 shows the comparison of ILP with other 35 insulin and insulin-like polypeptides. The substantial regions of homology among these molecules include the definitive conserved cysteine residues. A hydrophobicity analysis of pro-ILP (SEQ ID NO:2) is shown in Fig. 3. The plot indicates that ILP contains a hydrophobic region at the N-terminus characteristic of a signal sequence. The molecule is otherwise -47 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 lacking in significant hydrophobicity suggesting that ILP is likely to be a secreted protein and does not contain a membrane anchoring or transmembrane domain. A phylogenetic analysis (Fig. 4) shows that ILP is closely related to other well characterized human insulin and insulin-like polypeptides. The most related of these molecules cluster together at the right hand 5 side of the figure. The insulin-like molecules share several characteristics. They are each secreted proteins, and each possesses a similar arrangement of six conserved cysteine residues. Numerous additional amino acids are also generally conserved between members of the family indicating an evolutionary relationship. Amino acid changes that affect the predicted processing of ILP to a mature form (particularly amino acids 47, 48, 107, and 10 108 (R, R, K, and K respectively)) are likely to have significant impact on function. Example 5: ILP polvpeptide structure The mature insulin molecule, like other members of the insulin polypeptide family, is made up of two amino acid chains, the A chain and the B chain, encoded within the full length sequence of the gene. Based on homology information between ILP and other members of the insulin family, a determination was made 15 as to the number of polypeptide chains the mature ILP contains and whether those chains are covalently linked. It was determined by sequence comparisons that the mature ILP polypeptide is made up of an A chain and a B chain. Another standard method of determining the number of chains and covalent crosslinking is to deduce it from the number of amino-terminal residues present per molecule of protein such as by reaction of the a 20 amino group of a protein chain with 2,4-dinitro fluorobenzene (DNFB) to form yellow 2,4-dinitrophenyl derivatives, followed by acid hydrolysis and quantitation of the number of terminal amino acid residues (see for example, Lehninger, A.L. ed., Biochemistry, 2nd. ed., Worth Publishers, Inc., NY, (1975) pp 102-105). If there are no covalent cross-linkages between the chains, they may be dissociated by treating the protein with acid or base or with high concentrations of salt or urea. The dissociated chains may then be separated and 25 purified by electrophoresis or chromatography. If the chains are covalently cross-linked by the -S-S- bridge of a cystine molecule or if a single chain has an intrachain -S-S- linkage, these linkages must first be cleaved. For example, in the case of insulin the polypeptides contain 2 peptide chains cross-linked by two -S-S- bridges. In addition, the A chain has an intrachain -S-S- cross-linkage between positions 6 and 11. Such -S-S- cross linkages may be cleaved by oxidation with performic acid, which converts the two cystine half-residues into 30 cysteic acid residues. The chains may then be separated, and each hydrolyzed. The positions of the cysteic acid residues in the chains can ultimately be determined from the positions of the peptide fragments containing the cysteic acid residues. It is predicted that a mature ILP of the invention contains two chains linked by 2 interchain -S-S- cross-linkages between residue I1I of the B chain and residue 14 of the A chain; between residue 23 of the B chain and residue 27 of the A chain; and one intrachain linkage between residues 13 and 35 18 of the A chain. Example 6: Use of the ILP Gene Secquence in Antisense Analysis Knowledge of the correct, complete cDNA sequences of novel expressed genes encoding an insulin like polypeptide will enable their use in antisense technology in the investigation of gene function. Either -48 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 oligonucleotides, genomic or cDNA fragments comprising the antisense strand of ilp can be used either-in vitro or in vivo to inhibit expression of the specific protein. Such technology is now well known in the art, and probes can be designed at various locations along the nucleotide sequence. By treatment of cells or whole test animals with such antisense sequences, the gene of interest can be effectively turned off. Frequently, the 5 function of the gene can be ascertained by observing behavior at the cellular, tissue or organismal level (e.g. lethality, loss of differentiated function, changes in morphology, etc.). In addition to using sequences constructed to interrupt transcription of the open reading frame, modifications of gene expression can be obtained by designing antisense sequences to intron regions, promoter/enhancer elements, or even to trans-acting regulatory genes. Similarly, inhibition can be achieved 10 using Hogeboom base-pairing methodology, also known as "triple helix" base pairing. Example 7: Transgenic and Knockout Animals Nucleic acids which encode novel ILP from human or homologous sequences from non-human species, such as the murine ILP, can be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic 15 animal (e.g., a mouse) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, murine cDNA encoding ILP or an appropriate sequence thereof can be used to clone genomic DNA encoding ILP in accordance with established techniques and the genomic sequences used to generate transgenic animals that 20 contain cells which express DNA encoding ILP. Methods for generating transgenic animals, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, each herein incorporated by reference in its entirety. Typically, particular cells, such as retina, liver, pancreas, colon, uterus cells would be targeted for ILP transgene incorporation with tissue specific enhancers, which could result in altered cellular expression of the ILP. Transgenic animals that 25 include a copy of a transgene encoding ILP introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding ILP. Such animals can be used as tester animals for reagents thought to confer protection from, for example, diseases associated with abnormal metabolic processes, for example, related to increased ILP levels. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the disease, compared to 30 untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the disease. Alternatively, the non-human homologues of ILP can be used to construct an ILP "knock out" animal which has a defective or altered gene encoding ILP as a result of homologous recombination between the endogenous gene encoding ILP and altered genomic DNA encoding ILP introduced into an embryonic cell of the animal. For example, murine cDNA encoding ILP can be used to clone genomic DNA encoding ILP 35 in accordance with established techniques. A portion of the genomic DNA encoding ILP can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see e.g.. Thomas and Capecchi, Cell 51:503 (1987) for a description of homologous -49 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al., Cell 69: 915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem 5 Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be used in the selection of potential therapeutic 10 agents, such as ILP agonists, that restore the cellular processes initiated or maintained by native ILP; or the knockout animals can be used in the study of the effects of ilp mutations. Example 8: Expression of ILP Expression of the ILP gene may be accomplished by subcloning the cDNA into an appropriate expression vector, transfecting this vector into an appropriate expression host cell, and culturing the host cell. 15 The ILP may be intracellularly expressed or secreted into the periplasm or culture medium of the host cell. In this particular case, the cloning vector previously used for the generation of the tissue library also provides for direct expression of the included sequence in E. coli. Upstream of the cloning site, this vector contains a promoter for 3-galactosidase, followed by sequence containing the amino-terminal Met and the subsequent 7 residues of 3-galactosidase. Immediately following these eight residues is an engineered bacteriophage 20 promoter useful for artificial priming and transcription and a number of unique restriction sites, including EcoRI, for cloning. Induction of the isolated bacterial strain with IPTG using standard methods will produce a fusion protein corresponding to the first seven residues of P-galactosidase, about 15 residues of "linker", and the peptide encoded within the cDNA. Since cDNA clone inserts are generated by an essentially random process. 25 there is one chance in three that the included cDNA will lie in the correct frame for proper translation. If the cDNA is not in the proper reading frame, it can be obtained by deletion or insertion of the appropriate number of bases by well known methods including in vitro mutagenesis, digestion with exonuclease III or mung bean nuclease, or oligonucleotide linker inclusion. The ilp cDNA can be shuttled into other vectors known to be useful for expression of protein in specific hosts. Oligonucleotide amplimers containing cloning sites as well 30 as a segment of DNA sufficient to hybridize to stretches at both ends of the target cDNA (25 bases) can be synthesized chemically by standard methods. These primers can then be used to amplify the desired gene segments by PCR. The resulting new gene segments can be digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternatively, similar gene segments can be produced by digestion of the cDNA with appropriate restriction enzymes and filling in the missing gene 35 segments with chemically synthesized oligonucleotides. Segments of the coding sequence from more than one gene can be ligated together and cloned in appropriate vectors to maximize expression of recombinant sequence. -50 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae, and bacteria such as E. coli. For each of these cell systems, a useful expression vector may also include an origin of replication to allow propagation in bacteria and a selectable 5 marker such as the P-lactamase antibiotic resistance gene to allow selection in bacteria. In addition, the vectors may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells. Vectors for use in eukaryotic expression hosts may require RNA processing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest. Suitable vectors may also contain signal sequences upstream and in-frame of the inserted DNA such 10 that a cleavable signal sequence is fused to the desired protein for secretion into the cell culture medium followed by cleavage of the signal sequence and purification of the protein. Additionally, the vector may contain promoters or enhancers which increase gene expression. Such promoters are host specific and include MMTV, SV40, or metallothionine promoters for CHO cells; trp, lac, tac or T7 promoters for bacterial hosts, or alpha factor, alcohol oxidase or PGH promoters for yeast. 15 Transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, may be used in mammalian host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced ILP can be recovered from the conditioned medium and analyzed using chromatographic methods known in the art. In the following exemplifications of ILP expression in various hosts, ILP refers to pro-ILP encoded 20 by nucleic acid sequence SEQ ID NO: I and encoding the amino acid sequence SEQ ID NO:2 (see Fig. 6); mature ILP encoded by nucleic acid sequences SEQ ID NOS: 18 (encoding the A chain) and 19 (encoding the B chain) and encoding amino acid sequences SEQ ID NOS:9 (A chain) and 10 (B chain) (Fig. 1) covalently linked by disulfide bonds; an ILP C-peptide encoded by nucleic acid sequence SEQ ID NO:20 and encoding an amino acid sequence SEQ ID NO:21 (Fig. 1); or fragments for variants thereof. 25 Expression of ILP in E. coli This example illustrates preparation of an unglycosylated form of ILP by recombinant expression in E. coli. The DNA sequence encoding ILP is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected 30 expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including, for example, the first six 35 STII codons, polyhis sequence, and enterokinase cleavage site, or a lamB signal (USPN 5,324,820)), the ILP coding region, lambda transcriptional terminator, and an argU gene. The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic -51 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing. Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are 5 then grown to a desired optical density, during which the expression promoter is turned on. After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized ILP protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein. ILP prepared as a signal sequence fusion protein and secreted into the host cell culture 10 medium is processed by cleaving the signal sequence and isolating the protein as described. A method for isolating a recombinant polypeptide expressed in E. coli that can be applied to the isolation of ILP is disclosed in U.S. Patent 5,288,931. Disclosed therein is a method for refolding insoluble, improperly folded IGF-I, wherein the IGF-I, precipitated from prokaryotic host cells, is concurrently solubilized, unfolded, and refolded into a biologically active conformation in a single buffer. 15 Another method for isolating a recombinant polypeptide form E. coli is found in U.S. Patent 5,407,810. Disclosed therein is a method for isolating an exogenous polypeptide in a non-native conformation from cells in which it is expressed. The method involves contacting the polypeptide with a chaotropic agent and preferably a reducing agent and with phase-forming species to form multiple aqueous phases, with one of the phases being enriched in the polypeptide and depleted in the biomass solids and nucleic acids 20 originating from the cells. Expression of ILP in mammalian cells This example illustrates preparation of ILP by recombinant expression in mammalian cells. The vector, pRK5 (see EP 307,247. published March 15, 1989) or pRK5B (Holmes et al., supra, 1991), is employed as the expression vector. Optionally, the ilp DNA (DNA27865) is ligated into pRK5B 25 with selected restriction enzyme sites such as Xbal to allow insertion of the ilp DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is designated DNA27865-1091 and has ATCC deposit number 209296. Optionally, DNA sequences encoding the mature form of ILP or the ILP C peptide may be inserted into a vector. In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) 30 are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 pg DNA27865-1091 DNA is mixed with about 1 pg DNA encoding the VA RNA gene (Thimmappaya et al., Cell, 31:543 (1982)) and dissolved in 500 pl of I mM Tris-HCI, 0.1 mM EDTA, 0.227 M CaCI 2 . To this mixture is added, dropwise, 500 pl of 50 mM HEPES (pH 7.35), 280 mM NaCI, 1.5 mM NaPO 4 , and a precipitate is allowed to form for 10 minutes at 35 250C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37 0 C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days. -52 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 pCi/ml 3 5 S-cysteine and 200 pCi/m 3 5

S

methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period 5 of time to reveal the presence of ILP polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium or cell lysate is tested in selected bioassays. In an alternative technique, an ILP-encoding vector such as DNA27865-1091 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 pg DNA27865-1091 DNA 10 is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 pg/ml bovine insulin and 0.1 plg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed 15 ILP can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography. Where the vector encoding the desired ILP does not secrete the ILP into the culture medium. the cells are lysed and the lysate is processed to recover the desired ILP. In another embodiment, ILP can be expressed in CHO cells. The DNA27865-1091 can be transfected into CHO cells using known reagents such as CaPO 4 or DEAE-dextran. As described above, the cell cultures 20 can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 3 5 S-methionine. After determining the presence of ILP polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed ILP can then be concentrated and purified by any selected method. Under conditions in which the ILP is not secreted into the medium, the 25 desired ILP is recovered from the cell lyste. Epitope-tagged ILP may also be expressed in host CHO cells. The ILP may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged ILP insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the 30 CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium or cell lysate containing the expressed poly-His tagged ILP can then be concentrated and purified by any selected method, such as by Ni 2 +-chelate affinity chromatography. Expression of ILP in Yeast 35 The following method describes recombinant expression of ILP in yeast. First, yeast expression vectors are constructed for intracellular production or secretion of ILP from the ADH2/GAPDH promoter. DNA encoding ILP, a selected signal peptide and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of ILP. For secretion, -53 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 DNA encoding ILP can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, the yeast alpha-factor secretory signal/leader sequence, and linker sequences (if needed) for expression of ILP. Alternatively, the native signal sequence of ILP is employed for secretion of the ILP. Yeast cells, such as S. cerevisiae strain AB 110, can then be transformed with the expression plasmids 5 described above and cultured in selected fermentation media as set forth, for example, in U.S. Patent No. 5,010,003. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain. Recombinant ILP can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. 10 The concentrate containing ILP may further be purified using selected column chromatography resins. Expression of ILP in Baculovirus The following method describes recombinant expression of ILP in baculovirus. The ILP is fused upstream of an epitope tag contained with a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids 15 may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the ILP or the desired portion of the ILP (such as the sequence encoding the extracellular domain ofa transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector. 20 Recombinant baculovirus is generated by co-transfecting the above plasmid and BACULOGOLDTM virus DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28 0 C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A laboratory Manual, Oxford: Oxford University 25 Press (1994). Expressed poly-his tagged ILP can then be purified, for example, by Ni 2 +-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl 2 ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCI), and sonicated 30 twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCI, 10% Glycerol, pH 7.8) and filtered through a 0.45 pm filter. A Ni 2 +-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline A 2 80 with loading buffer, 35 at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate: 300 mM NaCI, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching

A

2 8 0 baseline again, the column is developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or western blot with -54 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Ni 2 +-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His l 0 -tagged ILP are pooled and dialyzed against loading buffer. Alternatively, purification of the IgG tagged (or Fc tagged) ILP can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography. 5 Example 9: Chimeric ILP molecules ILP may be expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS 10 extension/affinity purification system (Immunex Corp., Seattle Wash.). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego Calif.) between the purification domain and the ilp sequence may be useful to facilitate expression of ILP. Example 10: Production of ILP-Specific Antibodies Two approaches are utilized to raise antibodies to ILP (including pro-ILP, mature ILP, or ILP C 15 peptide, or a fragment thereof). Each approach is useful for generating either polyclonal or monoclonal antibodies. In one approach, denatured ILP from the reverse phase HPLC separation is obtained in quantities of 75 mg or more depending upon the capacity of the chromatographic column available in the art at the time of purification. This denatured protein can be used to immunize mice or rabbits using standard protocols; about 100 micrograms are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a 20 rabbit. For identifying mouse hybridomas, the denatured protein can be radioiodinated and used to screen potential murine B-cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg would be sufficient for labeling and screening of several thousand clones. 25 In the second approach, the amino acid sequence of ILP, as deduced from translation of the cDNA. is analyzed to determine regions of high immunogenicity. Oligopeptides comprising hydrophilic regions are synthesized and used in suitable immunization protocols to raise antibodies. Analysis to select appropriate epitopes is described by Ausubel, F.M. et al., supra (1989). The optimal amino acid sequences for immunization are usually at the C-terminus, the N-terminus and those 30 intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation. Typically. selected peptides, about 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431A using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (Ausubel, F.M. et al., supra). If 35 necessary, a cysteine may be introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% BSA, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG. -55 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Hybridomas may also be prepared and screened using standard techniques. For example, hybridomas of interest are detected by screening with detectably labeled ILP to identify those fusions producing the monoclonal antibody with the desired specificity. In a typical protocol, wells of microtiter plates (FAST: Becton-Dickinson. Palo Alto, Calif.) are coated with affinity purified, specific rabbit-anti-mouse (or suitable 5 anti-species Ig) antibodies at 10 mg/ml. The coated wells are blocked with 1% bovine serum albumin (BSA), washed and exposed to supernatants from hybridomas. After incubation the wells are exposed to labeled ILP, 1 mg/ml. Clones producing antibodies will bind a quantity of labeled ILP which is detectable above background. Such clones are expanded and subjected to 2 cycles of cloning at limiting dilution (1 cell/3 wells). Cloned hybridomas are injected into pristine mice to produce ascites, and monoclonal antibody is purified 10 from mouse ascitic fluid by affinity chromatography on Protein A. Monoclonal antibodies with affinities of at least 108 M

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1, preferably 109 M - 1 to 1010 M-1 or stronger, will typically be made by standard procedures as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory New York; and in Goding (1986) Monoclonal Antibodies: Principles and Practice, Academic Press, NYC, both incorporated 15 herein by reference, each in its entirety. Example 11: Diagnostic Test Using ILP-Specific Antibodies Particular anti-ILP antibodies are useful for the diagnosis of prepathologic conditions, and chronic or acute diseases which are characterized by differences in the amount or distribution of ILP. ILP has been found to be expressed in human colon, uterus, liver, placenta, lung and eye and is thus likely to be associated 20 with abnormalities or pathologies which affect these organs. Diagnostic tests for ILP include methods utilizing the antibody and a label to detect ILP in human body fluids, tissues or extracts of such tissues. The polypeptide and antibodies of the present invention may be used with or without modification. Frequently, the polypeptide and antibodies will be labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal. A 25 wide variety of labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, which patents are herein incorporated by reference in their entirety. Also, recombinant 30 immunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567, incorporated herein by reference in its entirety. A variety of protocols for measuring soluble or membrane-bound ILP. using either polyclonal or monoclonal antibodies specific for that ILP, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioreceptor assay (RRA), and fluorescent 35 activated cell sorting (FACS). A two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on ILP is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, D.E. et al. ((1983) J Exp. Med. 158:1211). -56 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 Example 12: Purification Of ILP Using Specific Antibodies Native or recombinant ILP may be purified by a variety of standard techniques in the art of protein purification. For example, pro-ILP, mature BP, or ILP C-peptide is purified by immunoaffinity chromatography using antibodies specific for the ILP. In general, an immunoaffinity column is constructed 5 by covalently coupling the anti-ILP antibody to an activated chromatographic resin. Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to 10 a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturers instructions. Such an immunoaffinity column is utilized in the purification of ILP by preparing a fraction from cells containing ILP in a soluble form. This preparation is derived by solubilization of the whole cell or of a 15 subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble ILP containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown. A soluble ILP-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of ILP (e.g., high ionic strength buffers in the 20 presence of detergent). Then, the column is eluted under conditions that disrupt antibody/ILP binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and ILP is collected. Example 13: Identification of ILP Receptors Purified ILP is useful for characterization and purification of specific cell surface receptors and other 25 binding molecules. Cells which respond to ILP by metabolic changes or other specific responses are likely to express a receptor for ILP. Such receptors include, but are not limited to, receptors associated with and activated by tyrosine and serine kinases. ILP receptors or other ILP-binding molecules may be identified by interaction with radiolabeled ILP. Radioactive labels may be incorporated into ILP by various methods known in the art. A preferred 30 embodiment is the labeling of primary amino groups in ILP with [125] I Bolton-Hunter reagent (Bolton, A.E. and Hunter, W.M. (1973) Biochem J 133:529), which has been used to label other polypeptides without concomitant loss of biological activity (Hebert, C.A. et al. (1991) J. Biol. Chem.266:18989; McColl, S. et al. (1993) J. Immunol. 150:4550-4555). Receptor-bearing cells are incubated with labeled ILP. The cells are then washed to removed unbound ILP, and receptor-bound ILP is quantified. The data obtained using 35 different concentrations of ILP are used to calculate values for the number and affinity of receptors. Labeled ILP is useful as a reagent for purification of its specific receptor. In one embodiment of affinity purification, ILP is covalently coupled to a chromatography column. Receptor-bearing cells are extracted, and the extract is passed over the column. The receptor binds to the column by virtue of its -57 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 biological affinity for ILP. The receptor is recovered from the column and subjected to N-terminal protein sequencing. This amino acid sequence is then used to design degenerate oligonucleotide probes for cloning the receptor gene. In an alternative method, mRNA is obtained from receptor-bearing cells and made into a cDNA 5 library. The library is transfected into a population of cells, and those cells expressing the receptor are selected using fluorescently labeled ILP. The receptor is identified by recovering and sequencing recombinant DNA from highly labeled cells. In another alternative method, antibodies are raised against the surface of receptor bearing cells, specifically monoclonal antibodies. The monoclonal antibodies are screened to identify those which inhibit 10 the binding of labeled ILP. These monoclonal antibodies are then used in affinity purification or expression cloning of the receptor. Soluble receptors or other soluble binding molecules are identified in a similar manner. Labeled ILP is incubated with extracts or other appropriate materials derived from the uterus. After incubation, ILP complexes larger than the size of purified ILP are identified by a sizing technique such as size exclusion 15 chromatography or density gradient centrifugation and are purified by methods known in the art. The soluble receptors or binding protein(s) are subjected to N-terminal sequencing to obtain information sufficient for database identification, if the soluble protein is known, or for cloning, if the soluble protein is unknown. Example 14: Determination of ILP-lnduced Cellular Response The biological activity of ILP is measured, for example, by binding of an ILP of the invention to an 20 ILP receptor. A test compound is screened as an antagonist for its ability to block binding of ILP to the receptor. A test compound is screened as an agonist of the ILP for its ability to bind an ILP receptor and influence the same physiological events as ILP using, for example, the KIRA-ELISA assay described by Sadick, M.D. et al. (Sadick, M.D. et al., Analytical Biochemistry 235:207-214 (1996)) in which activation of a receptor tyrosine kinase is monitored by immuno-capture of the activated receptor and quantitation of the 25 level of ligand-induced phosphorylation. The assay may be adapted to monitor ILP-induced receptor activation through the use of an ILP receptor-specific antibody to capture the activated receptor. Example 15: Drug Screening This invention is particularly useful for screening compounds by using ILP polypeptide or binding fragment thereof in any of a variety of drug screening techniques. The ILP or fragment employed in such a 30 test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between ILP or a 35 fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between ILP and its target cell or target receptors caused by the agent being tested. Thus, the present invention provides methods of screening for drugs or any other agents which can affect ILP-associated disease. These methods comprise contacting such an agent with an ILP or fragment -58 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 thereof and assaying (1) for the presence of a complex between the agent and the ILP or fragment, or(ii) for the presence of a complex between the ILP or fragment and the cell, by methods well known in the art. In such competitive binding assays, the ILP or fragment is typically labeled. After suitable incubation, free ILP or fragment is separated from that present in bound form, and the amount of free or uncomplexed 5 label is a measure of the ability of the particular agent to bind to ILP or to interfere with the ILP/cell complex. Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984, incorporated herein by reference. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to ILP, 10 the peptide test compounds are reacted with ILP and washed. Bound ILP is detected by methods well known in the art. Purified ILP can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support. This invention also contemplates the use of competitive drug screening assays in which neutralizing 15 antibodies capable of binding ILP specifically compete with a test compound for binding to ILP or fragments thereof In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with ILP. Example 16: Rational Drug Design The goal of rational drug design is to produce structural analogs of biologically active polypeptide 20 of interest (i.e., ILP) or of small molecules with which they interact, e.g., agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the ILP polypeptide or which enhance or interfere with the function of the ILP in vivo (c.f. Hodgson, J. (1991) Bio/Technology 2:19-21, incorporated herein by reference in its entirety). In one approach, the three-dimensional structure of the ILP, or of an ILP-inhibitor complex, is 25 determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the ILP must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the ILP may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous ILP-like molecules or to identify efficient inhibitors. Useful examples 30 of rational drug design may include molecules which have improved activity or stability as shown by Braxton, S. and Wells, J.A. ((1992) Biochemistry 31:7796-7801)or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda, S.B. et al. ((1993) J. Biochem. 113:742-746), which references are incorporated herein by reference in their entirety. It is also possible to isolate a target-specific antibody, selected by functional assay, as described 35 above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The -59 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore. By virtue of the present invention, sufficient amount of the ILP may be made available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the ILP amino acid sequence 5 provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography. All documents cited throughout the specification as well as the references cited therein are hereby expressly incorporated by reference in their entirety. While the present invention is illustrated with reference to specific embodiments, the invention is not so limited. It will be understood that further modifications and 10 variations are possible without diverting from the overall concept of the invention. All such modifications are intended to be within the scope of the present invention. Deposit of Material The following materials have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA (ATCC): 15 Material ATCC Dep. No. Deposit Date DNA27865-1091 209296 September 23, 1997 These deposits are made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of 20 deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto 25 according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638). The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a 30 license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any 35 constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting -60 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888 the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. What is claimed is: -61 SUBSTITUTE SHEET (RULE 26)

Claims (39)

1. An isolated nucleic acid molecule comprising the sequence encoding an insulin-like polypeptide (ILP) A chain (SEQ ID NO:18) shown in Fig. 1, or an isolated complement of the nucleic acid molecule.

2. An isolated nucleic acid molecule comprising the sequence encoding an insulin-like polypeptide 5 (ILP) B chain (SEQ ID NO:19) shown in Fig. 1, or an isolated complement of the nucleic acid molecule.

3. An isolated nucleic acid molecule comprising the sequence encoding an insulin-like polypeptide (ILP) C-peptide (SEQ ID NO:20) shown in Fig. 1, or an isolated complement of the nucleic acid molecule.

4. An isolated nucleic acid molecule comprising the sequence shown in SEQ ID NO: 1 shown in Fig. 6, or its complement, wherein the nucleic acid encodes an insulin-like polypeptide (ILP). 10

5. An isolated nucleic acid comprising DNA encoding an insulin-like polypeptide (ILP) having amino acid residues I to 135 (SEQ ID NO:2) of Fig. 6.

6. An isolated native sequence insulin-like polypeptide (ILP) comprising amino acid residues 1 to 135 of Fig. 6 (SEQ ID NO:2).

7. An isolated insulin-like polypeptide (ILP) A chain polypeptide comprising amino acid residues 15 109 to 135 (SEQ ID NO:9) of Fig. 1, and an isolated ILP B chain polypeptide comprising amino acid residues 19 to 48 (SEQ ID NO:10) of Fig. 1, which chains are covalently linked by disulfide bonds.

8. An isolated insulin-like polypeptide (ILP) C-peptide comprising amino acid residues 49 to 108 (SEQ ID NO:21) of Fig. 1.

9. An expression vector comprising the nucleic acid molecule of claim 1. 20

10. An expression vector comprising the nucleic acid molecule of claim 2.

11. An expression vector comprising the nucleic acid molecule of claim 3.

12. An expression vector comprising the nucleic acid molecule of claim 4.

13. An expression vector comprising isolated nucleic acid molecules comprising the sequence encoding the insulin-like polypeptide (ILP) A chain (SEQ ID NO: 18) and the sequence encoding the insulin 25 like polypeptide (ILP) B chain (SEQ ID NO:19) of Fig. 1.

14. A host cell comprising the expression vector of claim 9.

15. The host cell of claim 14 wherein the host cell is selected from the group consisting of a CHO cell, an E. coli cell, and a yeast cell.

16. A host cell comprising the expression vector of claim 10. 30

17. The host cell of claim 16 wherein the host cell is selected from the group consisting of a CHO cell, an E. coli cell, and a yeast cell.

18. A host cell comprising the expression vector of claim 11.

19. The host cell of claim 18 wherein the host cell is selected from the group consisting of a CHO cell, an E. coli cell, and a yeast cell. 35

20. A host cell comprising the expression vector of claim 12.

21. The host cell of claim 20 wherein the host cell is selected from the group consisting of a CHO cell, an E. coli cell, and a yeast cell.

22. A host cell comprising the expression vector of claim 13. -62 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888

23. The host cell of claim 22 wherein the host cell is selected from the group consisting of a CHO cell, an E. coli cell, and a yeast cell.

24. A method for producing an insulin-like polypeptide (ILP), said method comprising: a) culturing the host cell of claim 14 under conditions suitable for expression of the ILP; and 5 b) recovering the ILP from the culture.

25. A method for producing an insulin-like polypeptide (ILP), said method comprising: a) culturing the host cell of claim 16 under conditions suitable for expression of the ILP; and b) recovering the ILP from the culture.

26. A method for producing an insulin-like polypeptide (ILP), said method comprising: 10 a) culturing the host cell of claim 18 under conditions suitable for expression of the ILP; and b) recovering the ILP from the culture.

27. A method for producing an insulin-like polypeptide (ILP), said method comprising: a) culturing the host cell of claim 20 under conditions suitable for expression of the ILP; and b) recovering the ILP from the culture. 15

28. A method for producing an insulin-like polypeptide (ILP), said method comprising: a) culturing the host cell of claim 22 under conditions suitable for expression of the ILP; and b) recovering the ILP from the culture.

29. A method for determining the presence of insulin-like polypeptide (ILP) mRNA in a sample, the method comprising: 20 a) contacting a sample suspected of containing ILP mRNA with a detectable nucleic acid probe that hybridizes under moderate to stringent conditions to ILP mRNA; and b) detecting hybridization of the probe to the sample.

30. The method of claim 29, wherein the sample is a tissue sample and detecting is by in situ hybridization. 25

31. The method of claim 29, wherein the sample is a cell extract and detecting is by Northern analysis.

32. A method of detecting the presence of insulin-like polypeptide (ILP) in a sample, the method comprising: a) contacting a detectable anti-ILP antibody with a sample suspected of containing ILP; and 30 b) detecting binding of the antibody to the sample; wherein the sample is selected from the group consisting of a body fluid, a tissue sample, a cell extract, and a cell culture medium.

33. A chimeric molecule comprising an insulin-like polypeptide (ILP) fused to a heterologous amino acid sequence. -63 SUBSTITUTE SHEET (RULE 26) WO 99/15664 PCT/US98/17888

34. The chimeric molecule of claim 33 wherein the ILP is selected from the group consisting of an isolated polypeptide comprising amino acid residues 1 to 135 (SEQ ID NO:2) of Fig. 6 or a fragment thereof, an isolated polypeptide comprising an A chain comprising amino acid residues 109 to 135 (SEQ ID 5 NO:9) of Fig.1 and a B chain comprising amino acid residues 19 to 48 (SEQ ID NO:10) of Fig. 1, wherein the A and B chain are covalently linked by disulfide bonds or a fragment thereof, and an isolated polypeptide comprising a C chain comprising amino acid residues 49 to 108 (SEQ ID NO:21) of Fig. 1 or a fragment thereof.

35. The chimeric molecule of claim 33 wherein the heterologous amino acid sequence is an epitope 10 tag sequence.

36. The chimeric molecule of claim 33 wherein the heterologous amino acid sequence is a Fc region of an immunoglobulin.

37. An antibody which specifically binds to insulin-like polypeptide (ILP).

38. The antibody of claim 37 wherein the ILP is selected from the group consisting of 15 an isolated polypeptide comprising amino acid residues I to 135 (SEQ ID NO:2) of Fig. 6 or a fragment thereof, an isolated polypeptide comprising an A chain comprising amino acid residues 109 to 135 (SEQ ID NO:9) of Fig. 1 and a B chain comprising amino acid residues 19 to 48 (SEQ ID NO:10) of Fig. 1, wherein the A and B chain are covalently linked by disulfide bonds or a fragment thereof, and 20 an isolated polypeptide comprising a C chain comprising amino acid residues 49 to 108 (SEQ ID NO:21) of Fig. 1 or a fragment thereof.

39. The antibody of claim 37 wherein the antibody is a monoclonal antibody. -64-