EP1017816A1 - Insulin-like polypeptide and uses therefor - Google Patents

Insulin-like polypeptide and uses therefor

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Publication number
EP1017816A1
EP1017816A1 EP98942285A EP98942285A EP1017816A1 EP 1017816 A1 EP1017816 A1 EP 1017816A1 EP 98942285 A EP98942285 A EP 98942285A EP 98942285 A EP98942285 A EP 98942285A EP 1017816 A1 EP1017816 A1 EP 1017816A1
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EP
European Patent Office
Prior art keywords
ilp
polypeptide
seq
cell
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98942285A
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German (de)
French (fr)
Inventor
Austin Gurney
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Genentech Inc
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Genentech Inc
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Publication of EP1017816A1 publication Critical patent/EP1017816A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/64—Relaxins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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 BACKGROUND OF THE INVENTION
  • ILP insulin-like polypeptide
  • 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 proinsu n to produce mature insulin
  • a third amino acid chain (C-peptide) is cleaved from proinsu n to produce mature insulin
  • An important effect of mature insulin is its ability to increase the rate of glucose trans ⁇ ort 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
  • the rate of glucose transport into cells may be as great as five times normal Thus, the rate of glucose transport for many tissues can be altered as much as 20-fold
  • Insulin promotes glucose transport into cells by stimulating a process of facilitated diffusion m 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
  • insulin enhances glucose transport into the heart and certain smooth muscle organs, such as the uterus
  • glycogen stores in skeletal muscle increase markedly, and there is a moderate enhancement of glycogen in the skin, glands, and other tissues
  • 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
  • a rapid and potent 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
  • IGF-I A related molecule, 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 range of cell types and can mediate the effects of growth hormone on skeletal growth Most tissues and especially the liver produce IGF-I 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.
  • 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-I and IGF-II, are chemically related to human promsulin in that they contain A and B domains connected by a C domain region, and have high homology to promsulin The IGFs further contain a D domain at the C-terminus that is not found in promsulin 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
  • IGFBP- 1 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 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
  • PSF prostacyclin-stimulatmg factor
  • ESM-1 endothelial cell-specific molecule
  • 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 other surface of plasma membranes in manv different cell types Binding to IGF type 2- and insulin receptors also seems to be of importance
  • rhIGF-I recombinant human IGF-I
  • body fuel metabolism see, for example Boulware, S D et al (1992) Am J Physiol 262 E130-E133 and references cited therein
  • rhIGF-I has been shown to produce hypoglycemia, even when infused simultaneously with anti-insulin serum While both insulin and rhIGF-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 C n 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
  • IGF-I insulin growth factor-I
  • hypophysectomized rats Guler et al , Endocrinology, JJ8 Supp 129 abstract, Skottner et al , (1987) J Endoc ⁇ nol JJ2 123-132, Guler et al , (1988) PNAS USA 85 4889-4893, Froesch et al , in Endocrinology Intl Congress Series 655, Lab ⁇ e and Proulx, eds Amsterdam Excerpta Medica, 1984) diabetic rats (Scheiwiller et al (1986) Nature 323 169- 171 ( 1986)).
  • Reiaxin 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 reiaxin were considered to be a lengthening of the pubic ligaments, widening of the pelvis, dampening of uterine contractions, and softening and dilating of the cervix (Wade, J D and Trege
  • Reiaxin receptors have been observed in three tissues by autoradiography using J ⁇ P-Iabeled synthetic reiaxin, which tissues include uterus, brain, and heart Osheroff, P and Ho, W -H (1993) J Biol Chem 268 15193-15199) Reiaxin binding regions were observed in brain m regions associated control of cardiovascular functions such as blood pressure and fluid and electrolyte homeostasis Reiaxin has been shown to be more potent than angiotensin II or endothe n, suggesting that it may play a role in cardiovascular disorders (Summers, R J et al in "Recent Progress in Reiaxin 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 prevented or attenuated vascular and neural (electrophys ⁇ olog ⁇ cal)dysfunct ⁇ on and impaired Na+ - and K+ - dependent adenosme t ⁇ phosphate activity in tissues of diabetic rats (Ido.
  • An aspect of the subject invention provides a nucleic acid sequence (SEQ ID NO 1 ) of a gene, pro-dp, 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), which is a member of the msulin/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 ammo 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)
  • Another aspect of the invention includes a method for determining expression of p in a cell
  • the diagnostic test comprises providing a cell extract or a tissue sample containing cells suspected of expressing tip 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 1 (Fig 6) or a fragment thereof
  • Another aspect of the invention includes a method of diagnosing a physiologic or pathologic 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
  • 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 lip or nucleic acid encoding mature A.
  • the invention encompasses a transgenic animal comprising an altered tip 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)
  • a transgenic animal of the invention 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
  • compositions comprising an ILP covalently linked (A and B chains) and/or an ILP C-peptide as defined herein in admixture with a pharmaceutically 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 m toxicokinetics" in Toxicokmetics and New Drug Development. Yacobi et al (eds), Pergamon Press, NY, pp 42-96, each of which references are herein incorporated by reference in its entirety)
  • 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
  • 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
  • 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
  • the ILP-encoding or ILP C-peptide-encodmg 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 expression cassette, vectors encoding the expression cassette and host cells comprising the expression cassette Preferred host cells include, but
  • a host cell expressing an ILP or ILP agonist or an ILP C-peptide 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 m U S Patents 4,892,538, and 5,01 1,472, WO 92/19195, WO 95/05452, or Aeischer et al (1996) Nature 2 696-699, for example, which references are herein incorporated by reference in their entirety
  • 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 lonotropic activity, cardiac chronotropic activity, and collagen deposition
  • Fig 1 depicts the nucleotide sequence for the dp (SEQ ID NO 1 ) and the predicted amino acid 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 overhning
  • a and B chains are indicated by the encircled numbers 1 through 6 below the cysteine residues within the sequence, where the linkages are between 1 and 4, 2 and 6, and 3 and 5
  • Primer oligonucleotides IN2328985 f, IN2328985 p, and IN2328985 r, designed based on homology to relaxm and used to isolate the full length dp, are indicated by overhning and underlining
  • Fig 2 shows the ammo acid sequence alignment of ILP (SEQ ID NO 2) with other polypeptide of the insulin/IGF family h-Insuhn (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 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
  • Fig 4 shows a relatedness tree of some human insulin/IGF polypeptide family members (including
  • GenBank accession numbers h-prelGFl (P70277), h-preIGF2 (P01344), h-prepro insulin (PI 0042), 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 INC2328985), and Incyte EST INC7783 19 (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 1
  • the deduced ammo acid sequence of pro-ILP is indicated as SEQ ID NO 2
  • uterine-expressed insulin-like polypeptide or “insulin-like polypeptide” or “ILP” refers to a naturally occurring ILP or active fragments thereof, having the ammo acid sequence shown in SEQ
  • the ILP polypeptide may be encoded by dp having the nucleic acid sequence shown in SEQ ID NO 1 (Fig 6) encoded within SEQ ID NO 22 shown in Fig 1 (SEQ ID NO 1 within Genentech DNA 27865 (Fig 6).
  • ILP may also be defined as the polypeptide encoded by an mRNA transcribed from the nucleic acid sequence of SEQ ID NO 1
  • 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 ammo acid sequence of the ILP (SEQ ID NO 2)
  • 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 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
  • novel native human ILPs of the present invention are about 1 14 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 ammo chains, an A chain (SEQ ID NO 9) and a B chain (SEQ ID NO 10) linked by disulfide bonds
  • ILP is associated with native glycosylation, or other post-translational de ⁇ vatization
  • 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, 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
  • 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
  • “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 occurring ILP chemical modifications such as ubiquitmation, labeling (e g .
  • ILP fragments regions within the sequence of a mature native polypeptide
  • ILP fragments will have a consecutive sequence of at least 10 and more preferably at least 20, ammo 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
  • the ILP fragment of the invention has sufficient length to displav biologic and/or lmmunologic activity Fragment can also include a portion of each of the A and B chains (SEQ ID Nos 9 and 10) linked by one or more disulfide bonds Similarly, with regard to nu
  • Ohgonucleotides 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.
  • 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, 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
  • the non-peptide analogs of the native novel ILPs of the present 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
  • amino acid sequence variants of the present invention have at least about 60% amino acid sequence identity, more preferably at least about 75 % amino acid sequence identity and most preferably at least about 90% amino acid sequence identity with a native ILP of the present invention
  • 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)
  • 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
  • chimeric ILP 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
  • chimeric ILPs include immunoadhesins and epitope tagged ILP
  • 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)
  • 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 protemaceous or non-protemaceous de ⁇ vatizing agent, fusions to heterologous polypeptide sequences, and post-translational modifications
  • Covalent modifications are traditionally introduced by reacting targeted amino acid residues with an organic de ⁇ vatizing 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 cells on the expressed polypeptide Glutaminyl and asparaginyl residues are frequently post-translational ly 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
  • 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
  • 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
  • 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 ammo acid residues in the candidate sequence that are identical with the residues 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 m the art For example, the sequences disclosed herein were analyzed using "ALIGN”, Genentech, Inc 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
  • amino acid and “amino acids” refer to all naturally occurring L- ⁇ -amino acids
  • D-ammo acids may be present in the polypeptide or peptides of the present invention in order to facilitate conformational restriction
  • a D amino acid cysteine may be provided at one or both termini of a peptide functional 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
  • amino acid sequence variant refers to molecules with some differences in their amino acid sequences as compared to a native amino acid sequence
  • Substitutional variants are those that have at least one am o 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 ammo 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 either the ⁇ -carboxv or ⁇ -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 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 heterotetrame ⁇ c 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 the heavy chains of different immunoglobuhn isotypes Each heavy and light chain also has regularly spaced mtrachain disulfide bridges Each heavy chain has at one end a variable domain (V j ) followed by a number of constant domains Each light chain has a variable domain at one and (V ⁇ ) 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 acid residues are believed to form an interface between the light and heavy chain variable domains (Clothia et l , ] Mol Biol 186, 651 -663 (1985). Novotn
  • the light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda ( ⁇ ) based on the ammo acid sequences of their constant domains
  • immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e g IgG-1 , IgG- 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 ⁇ , delta, epsilon, ⁇ , and ⁇ , respectively
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known
  • antibody is used in the broadest sense and specifically covers single monoclonal 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, l e , the individual antibodies comprising the population are identical 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
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hyb ⁇ doma method first described by Kohler & Milstein, Nature 256 495 (1975), or may be made 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
  • humanized forms of non-human (e g murine) antibodies are specific chimeric immunoglobulins, immunoglobuhn 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 immunoglobuhn
  • 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, affinity and capacity
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity
  • Fv framework region (FR) residues of the human immunoglobuhn are replaced by corresponding non-human FR residues
  • the humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or FR sequences
  • neutralizing antibody an antibody molecule as herein defined which is able- to block: or significantly reduce an effector function of native sequence ILP
  • 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 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 cha ⁇ n(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 , Morrison et al , Proc Natl Acad Sci USA 8 L 6851 -6855 (1984))
  • 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 include a promoter, optionally an operator sequence, a ⁇ bosome binding site, and possibly
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence
  • DNA for a presequence or a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotem 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
  • a ⁇ bosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation
  • "operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase
  • enhancers do not have to be contiguous Linking is accomplished by gation at convenient restriction sites If such sites do not exist, then synthetic ohgonucleotide adaptors or linkers are used in accord with conventional practice "Ohgonucleotides” are short-length, single- or double
  • solid phase is meant a non-aqueous matrix to which a reagent of interest (e g , ILP or an antibody thereto) can adhere
  • a reagent of interest e g , ILP or an antibody thereto
  • solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysaccha ⁇ des (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones
  • the solid phase can 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 U S Patent No 4,275.149, herein incorporated by reference in its entirety
  • transfection 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 into the host cell genome, or may exist as an extrachromosomal element
  • the preferred methods of transfection of the cells with D A is the calcium treatment method described by Cohen et al , Proc Nad Acad Sci US A 69 21 10- 21 14 (1972) or the polyethylene glycol method of Chung et al , Nuc Acids Res 16 3580 (1988)
  • yeast are used as the host, transfection is generally accomplished using polyethylene glycol, as taught by Hinnen, Proc Natl Acad Sci USA , 75 1929-1933 (1978)
  • transfection generally is carried out by the calcium phosphate precipitation method, Graham et al , Virology 52 546 (1978).
  • 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 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
  • 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 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
  • DHFR dihydrofolate reductase
  • 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 1 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 co
  • Hybridization is preferably performed under "stringent conditions" which means ( 1 ) employing low ionic strength and high temperature for washing, for example, 0 015 sodium chlo ⁇ de/0.0015 M sodium 0 c ⁇ trate/0 1% sodium dodecyl sulfate at 50 C C, or (2) employing during hybridization a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide with 0 1% bovine serum album ⁇ n/0 1% F ⁇ coll/0 1% polyv ⁇ nylpyrrol ⁇ done/50 nM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C
  • formamide for example, 50% (vol/vol) formamide with 0 1% bovine serum album ⁇ n/0 1% F ⁇ coll/0 1% polyv ⁇ nylpyrrol ⁇ done/50 nM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 m
  • a "signal or leader sequence” can direct the polypeptide through the membrane of a 0 cell
  • 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 ammo 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 5 to display biologic and/or lmmunologic activity
  • Immunoadhesins or "ILP - immunoglobuhn chimeras” are chimeric antibody-like molecules that combine the functional doma ⁇ n(s) of a binding protein (usually a receptor, a cell-adhesion molecule or a ligand) with the an immunoglobuhn sequence
  • a binding protein usually a receptor, a cell-adhesion molecule or a ligand
  • the most common example of this type of fusion protein combines the hinge and Fc regions of an immunoglobuhn (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 " "Treatment” refers to both therapeutic treatment and prophylactic or preventative measures
  • “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 like Preferabh .
  • 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
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide, proteins, such as serum albumin, gelatin or immunoglobulins. hydrophilic polymers such as polyvinylpyrrohdone. amino acids such as glycine, glutamine.
  • arginine or lysine monosaccha ⁇ des, disaccha ⁇ des, and other carbohydrates including glucose, mannose, or dext ⁇ ns, chelating agents such as EDTA, sugar alcohols such as manmtol or sorbitol. salt-forming counte ⁇ ons such as sodium, and/or nonionic surfactants such as TweenTM, polyethylene glycol (PEG), and PluronicsTM
  • the native ILPs of the present invention may be isolated from cDNA or genomic libraries
  • a suitable cDNA library can be constructed by obtaining polyadenylated mRNA from cells known 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) genomic library
  • probes either cDNA or genomic. are screened with probes designed to identify the gene of interest or the protein encoded by it
  • suitable probes include monoclonal and polyclonal antibodies that recognize and specifically bind to an ILP of the invention
  • suitable probes include carefully selected ohgonucleotide probes (usually of about 20-80 bases in length) that 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, ohgonucleotides, cDNAs.
  • 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 Laborator y Manual.
  • DNA encoding an ILP of the present invention is isolated b ⁇ using carefully selected o gonucleotide sequences to screen cDNA libraries from various tissues, the ohgonucleotide 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 ohgonucleotides may be degenerate at one or more positions The use of degenerate ohgonucleotides is of particular importance where a library is screened from a species in which preferential codon usage is not known
  • the ohgonucleotide must be labeled such that it can be detected upon hybridization to DNA in the library being screened
  • T e preferred method of labeling is to use ATP (e g , ⁇ 32 P) and polynucleotide kinase to radiolabel the 5' end of the ohgonucleotide
  • ATP e g , ⁇ 32 P
  • polynucleotide kinase to radiolabel the 5' end of the ohgonucleotide
  • other methods may be used to label the ohgonucleotide 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
  • cDNAs from other species can also be obtained by cross-species hybridization
  • human or other mammalian cDNA or genomic libraries are probed by labeled ohgonucleotide sequences selected from known ILP sequences (such as murine or human sequences) in accord with known criteria
  • the probe sequence should be sufficient in length and sufficiently unambiguous that false positives are minimized
  • TypicalK a J P-labeled ohgonucleotide having about 30 to 50 bases is sufficient, particularly if the ohgonucleotide 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 Once the sequence is known, the gene encoding a particular ILP can also be obtained
  • nucleic acid encoding a novel ILP is available, it is generally gated into a rep cable expression vector for further cloning (amplification of the DNA), or for expression
  • 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.
  • each vector contains 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 hgation techniques Isolated plasmids or DNA fragments are cleaved, tailored, and rehgated in the form desired to generate the plasmids required
  • the hgation mixtures are commonly used to transform E coli cells, e g E coli K12 strain 294 (ATCC 31 ,446) and successful transformants selected by ampicil n or tetracyc ne resistance where appropriate Plasmid
  • 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 prokaryotes such as E coli B, E coli XI 776 (ATCC 31 ,537), E coli W31 10 (ATCC 27,325), Pseudomonas species, or Serratia Marcesans are suitable
  • 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
  • S pombe Beach and Nurse, Nature 290 140 ( 1981 )
  • Kluvveromvces lactis de Louvencourt L et al , ] Bactenol 154 737-742 (1983)
  • yarrowia EP 402,226)
  • Pichia pastons EP 183,070
  • Tnchoderma reesia EP 244,234)
  • Neurospora crassa Case et al , Proc Natl Acad Sci USA 76 5259-5263 (1979)
  • Aspergillus hosts such as A nidulans (Ballance et al
  • Suitable host cells may also derive from multicellular organisms Such host cells are capable of complex processing and glycosylation activities
  • any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture, although cells from mammals such as humans are preferred
  • invertebrate cells include plants and insect cells Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (cate ⁇ illar), 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 315 592-594 (1985)
  • a variety of such viral strains are publicly available, e g the L- l variant of Autographa califor ica NPV and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as 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
  • regulatory and signal sequences compatible with plant cells are available, such as the nopahne synthase promoter and polyadenylation signal sequences Depicker et al , J Mol Appl Gen 1 561 (1982)
  • 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
  • HB 8065 mouse mammary tumor (MMT 060562 ATCC CCL51), TRI cells (Mather et al , Annals N Y Acad 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
  • transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, m 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
  • transient expression systems are particularly useful in the invention for pu ⁇ oses of identifying analogs and variants of a native ILP of the invention
  • Prokaryote cells used to produced the ILPs of this invention are cultured in suitable media as describe generally in Sambrook et al , supra
  • Mammalian cells can be cultured in a variety of media
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for cultu ⁇ ng the host cells
  • any of the media described in Ham and Wallace, Meth Enzymol 58, 44 (1979), Barnes and Sato, Anal Biochem 102 are suitable for cultu ⁇ ng the host cells.
  • 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 ' M drug) trace elements (defined as inorganic 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
  • 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 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 m situ hybridization, using an appropriately labeled probe, based on the sequences provided herein
  • Various labels may be employed, most commonly radioisotopes, particularly J 32 P
  • other techniques may also be employed, such as using biotm-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 radionuchdes, fluorescers, enzymes, or the like
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes
  • Antibodies useful for lmmunohistochemical staining and/or assay of sample fluids may be either 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
  • Ammo 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
  • the ammo 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
  • 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
  • Naturally-occurring amino acids are divided into groups based on common side chain properties
  • 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 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 chain
  • substitutions which in general are expected to produce the greatest changes m 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.
  • a hydrophobic residue e g ieucyl, isoleucyl, phenylalanyl. valyl or alanyl
  • a cysteine or proline is substituted for (or by) any other residue
  • a residue having an electropositive side chain e g lysyl, arginyl, or histidyl
  • an electronegative residue e g , glutamyl or aspartyl
  • a residue having a bulky side chain e g , phenylalanme.
  • 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)
  • substitutions that affect the processing of the ILP of the ILP are expected to have significant effects
  • 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
  • Ammo 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 mtrasequence 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 1 to 3 residues
  • An example of a terminal insertion includes fusion of a heterologous N-terminal signal sequence to the N-termmus 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
  • ILP molecules include the fusion of the N- or C-lerminus of the ILP molecule to immunogenic polypeptide, e g bacterial polypeptide such as beta-lactamase or an enzyme encoded by the E coli t ⁇ locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as immunoglobuhn regions (preferably immunoglobuhn constant regions), albumin, or ferritin, as described in WO 89/02922 published on 6 April 1989
  • immunogenic polypeptide e g bacterial polypeptide such as beta-lactamase or an enzyme encoded by the E coli t ⁇ locus, or yeast protein
  • C-terminal fusions with proteins having a long half-life such as immunoglobuhn regions (preferably immunoglobuhn constant regions), albumin, or ferritin, as described in WO 89/02922 published on 6 April 1989
  • immunologically active derivatives of the novel ILPs containing an 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
  • 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 as t ⁇ LE, ⁇ -glactosidase, viral polypeptide such as he ⁇ es 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 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 acceptable carrier and administered to a subject in order to raise antibodies against the ILP molecule, which antibodies in turn are useful as diagnostics, m 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
  • Such screening includes, but is not limited to, arrays of receptor binding
  • the gene encoding an ILP variant can, for example, be 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 nonva ⁇ ant version of the ILP Site-directed (site-specific) mutagenesis allows the production of ILP variants through the use of specific ohgonucleotide 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 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 .
  • the site-specific mutagenesis technique typically employs a phage vector that exists in both a s gle-stranded and double-stranded form
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M 13 phage.
  • template plasmid DNA (1 ⁇ g) 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 deoxynucleotide t ⁇ phosphates and is included m the GeneAmp kits (obtained from Perkin-Elmer Cetus. Norwalk.
  • 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 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))
  • phagemid display method may be useful in making amino acid sequence 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 forming a family of related plasmids.
  • transforming suitable host cells with the plasmids infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein 5) cultu ⁇ ng 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 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
  • the plasmid is under tight control of the transcription regulatory element, and the cultu ⁇ ng conditions are adjusted so that the amount or number of phagemid particles displaying more than one copy of the fusion protein on
  • 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 de ⁇ vatizing 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 residues important for biological activity for immunoassays of the ILP, or for the preparation of anti-ILP antibodies for immunoaffinity purification of the recombinant
  • complete inactivation of the biological activity of the protein after reaction with nmhyd ⁇ n 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 of a peptide fragment containing the modified amino acid residue
  • modifications are within the ordinary skill in the art and are performed without undue experimentation
  • De ⁇ vatization 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
  • cross-linking agents include 1 , 1 -b ⁇ s(d ⁇ azoacetyl)-2- phenylethane, glutaraldehvde, N-hydroxysuccinimide esters, homobifunctional imidoesters, and bifunctional maleimides
  • De ⁇ vatizing agents such as methyl-3-[(p-az ⁇ dophenyl)d ⁇ th ⁇ o]prop ⁇ o ⁇ m ⁇ date yield photoactivatable intermediates which are capable of forming cross-links in the presence of light
  • 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,
  • 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
  • these residues are deamidated under mildly acidic conditions
  • Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, threonyl or tyrosyl residues, methylation of the ⁇ -amino groups of lysine, arginine, and histidme side chains (T E Creighton, Proteins Structure and Molecular Properties.
  • ILPs interleukin-like molecules
  • binding protein usually a receptor, a cell- adhesion molecule or a ligand
  • Ig immunoglobuhn
  • fusion protein combines the hinge and Fc regions of an immunoglobuhn (Ig) with domains of a cell-surface receptor that recognizes a specific ligand
  • This type of molecule is called an "lmmunoadhesin", 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 (1989) Traunecker et al .
  • CD44 (Aruffo et al , Cell 61 1303-1313 (1990)), CD28 and B7 (Linsley ⁇ ?/ ⁇ / , J Exp Med 173 721-730 (1991 )), CTLA-4 (Lisley et al , ] Exp Med 174 561-569 (1991 )).
  • CD22 (Stamenkovic e/ ⁇ / , Cell 66 1 133- 1 144 (1991)), NP receptors (Bennett et al , J Biol Chem 266 23060-23067 (1991 )), IgE receptor ⁇ (Ridgway and Gorman, J Cell Biol 1 15 abstr 1448 (1991 )), IFN- ⁇ R a- and ⁇ -chain (Marsters et al , Proc Natl Acad Sci USA 92 5401 -05 (1995)) trk-A -B, and -C (Shelton et al .
  • nucleic acid encoding the desired ILP 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 immunoglobuhn constant domain sequence, however fusion to the N-terminus of the immunoglobuhn is also possible
  • the encoded chimeric polypeptide will retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobuhn heavy chain Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CHI of the heavy chain or the corresponding region of
  • an ILP polypeptide chain is fused to the hinge region and CH2 and CH3 or CH 1 , hinge.
  • the ILP-immunoglobuhn 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, IgD, 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 may also exist in multimeric form in serum
  • each four unit may be the same or different
  • ILP-immunoglobuhn chimeras within the scope of the invention are schematically diagrammed below (a) AC L -AC L , (b) AC H -[AC H , AC L -AC H , AC L -V H C H , or V L C L -AC H ],
  • Y designates the residue of a covalent cross-linking agent
  • an immunoglobuhn light chain might be present either covalently associated to an ILP- immunoglobuhn heavy chain fusion polypeptide. or directly fused to the ILP polypeptide In the former case.
  • DNA encoding an immunoglobuhn light chain is typically coexpressed with the DNA encoding the ILP- immunoglobuhn heavy chain fusion protein Upon secretion, the hybrid heavy chain and the light chain will be covalently associated to provide an lmmunoglobu n-hke structure comprising two disulf ⁇ de-Iinked immunoglobuhn heavy chain-light chain pairs
  • the immunoglobuhn sequences used in the construction of the immunoadhesins of the present invention are from an IgG immunoglobuhn heavy chain constant domain
  • IgG-1 and IgG-3 immunoglobuhn sequences are preferred
  • IgG-1 and IgG-3 immunoglobuhn sequences are preferred
  • IgG-1 and IgG-3 immunoglobuhn sequences are preferred
  • IgG-1 and IgG-3 immunoglobuhn sequences is preferred.
  • IgG-1 and IgG-3 immunoglobuhn sequences is preferred.
  • IgG-1 immunoglobuhn heavy chain constant domain
  • purification of IgG-3 requires protein G, a significantly less versatile medium
  • other structural and functional properties of immunoglobulins should be considered when choosing the Ig fusion partner for a particular lmmunoadhesin construction
  • 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
  • ILP-Ig 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 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
  • IgG isotypes with fewer serologically-defined allotypes are preferred
  • IgG-1 has only four serologically-defined allotypic sites, two of which (Gl m and 2) are located in the Fc region, and one of these sites G lm l , is non- lmmunogenic
  • there are 12 serologically-defined allotypes in IgG-3 all of which are in the Fc region, only three of these sites (G3m5, 1 1 and 21 ) have one allotype which is nonimmunogenic
  • ILP-Ig immunoadhesins are most conveniently constructed by fusing the cDNA sequence encoding the ILP portion in-frame to an Ig cDNA sequence
  • 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 e. ⁇ t .
  • Ig regulatory sequences for expression cDNAs encoding IgG heavy-cha constant regions can be isolated based on published sequence from cDNA libraries derived from spleen or peripheral blood lymphocytes, by - hybridization or by polymerase chain reaction (PCR) techniques.
  • Nonproteinaceous polymer ordinarily is a hydrophilic synthetic polymer, i.e., a polymer not otherwise found in nature.
  • hydrophilic polyvinyl polymers fall within the scope of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone.
  • polyalkylene ethers such as polyethylene glycol (PEG); 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 heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose.
  • the polymer prior to cross-linking need not be, but preferably is, water soluble, but the final conjugate must be water soluble.
  • the polymer should not be highly immunogenic in the conjugate form, nor should it possess viscosity that is incompatible with intravenous infusion or injection if it is intended to be administered by such routes.
  • the polymer contains only a single group which is reactive. This helps to avoid cross- linking of protein molecules.
  • 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.
  • 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 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 groups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde.
  • Carboxyl groups are de ⁇ vatized by coupling PEG-amine using carbodnmide Polymers are conjugated to ohgosaccha ⁇ de groups by oxidation using chemicals, e g metape ⁇ odate, or enzymes, e g glucose or galactose oxidase.
  • oligosaccharides are particularly advantageous because, in general, there are fewer substitutions than ammo acid sites for de ⁇ vatization, and the ohgosaccha ⁇ de products thus will be more homogeneous
  • the oligosaccha ⁇ de substituents also are optionally modified by enzyme digestion to remove sugars, e g by neuraminidase digestion, prior to polymer de ⁇ vatization
  • the polymer will bear a group which is directly reactive with an amino acid side chain, or the N- or C-terminus of the polypeptide linked, or which is reactive with the multifunctional cross-linking agent
  • polymers bearing such reactive groups are known for the preparation of immobilized proteins
  • Cyanogen bromide activation is a particularly useful procedure to employ in crosslinking polysaccha ⁇ des
  • 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 de ⁇ vatization reaction
  • Water soluble in reference to the polymer conjugate means that the conjugate is soluble m physiological fluids such as blood
  • the degree of substitution with such a polymer will vary depending upon the number of reactive sites 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, hydrophihcity and other characteristics of the polymer, and the particular protein de ⁇ vatization sites chosen
  • the conjugate contains about from 1 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 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
  • PEG polymer
  • the polymer is cross-linked by a wide variety of methods known m the art for the covalent modification of proteins with nonproteinaceous polymers such as PEG Certain of these methods, however, are not preferred for the pu ⁇ oses 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 dnmidazole chemistry (Beauchamp et al .
  • the long half-life conjugates of this invention are separated from the unreacted starting materials by 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
  • novel ILPs may be entrappe ⁇ 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 disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A , Ed (1980)
  • Polyclonal antibodies to an ILP, or fragment of the ILP, of the present invention generally are raised in animals by multiple subcutaneous (sc) or mtraperitoneal (lp) injections of the ILP and an adjuvant
  • sc subcutaneous
  • lp mtraperitoneal
  • Animals are immunized against the immunogenic conjugates or derivatives by combining approximately 1 mg or 1 ⁇ g of conjugate (for rabbits or mice, respectively) with three volumes of Freud's complete adjuvant and injecting the solution mtradermally 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 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 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
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, I e .
  • the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts
  • the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies
  • the anti-ILP monoclonal antibodies of the invention may be made using the hyb ⁇ doma method first described by Kohler & Milstem, Nature 256 495 (1975), or may be made by recombinant DNA methods (Cabilly. et al , 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 ohgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies)
  • the hyb ⁇ doma cells of the invention serve as a preferred source of such DNA
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobuhn 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 8 .
  • chimeric or “hybrid” antibodies are prepared that have the binding specificity of an ILP monoclonal antibody of the invention
  • 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 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
  • lmmunotox s may be constructed using a disulfide exchange reaction or by forming a thioether bond
  • suitable reagents for this pu ⁇ ose include lminothiolate and methyI-4-mercaptobuty ⁇ m ⁇ date
  • 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
  • the detectable moiety may be a radioisotope, such as - H, C, j2 P, S, or
  • a fluorescent or chemiluminescent compound such as fluorescem isothiocyanate. rhodamine, or lucife ⁇ n. biotin, radioactive isotopic labels, such as, e g , ' 5 I, J ⁇ P, 4 C, or 1 H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase
  • the antibodies of the present invention may be employed m any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (see, for example, Zola, Monoclonal Antibodies A Manual of Techniques, pp 147-158 (CRC Press, Inc , 1987)
  • 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 "import” variable domain
  • Humamzation 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 .
  • humanized antibodies are chimeric antibodies (Cabilly, supra), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species
  • 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
  • humanized antibodies 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 immunoglobuhn models are commonly available and are familiar to those skilled m the art
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobuhn sequences Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobuhn sequence l e the analysis of residues that influence the ability of the candidate immunoglobuhn to bind its antigen
  • 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 ant ⁇ gen(s), is achieved
  • the CDR residues are directly and most substantially involved m influencing antigen binding (see, for example, WO 92/22653)
  • Jakobovits et al Proc Natl Acad Sci USA 90, 2551-255 (1993). Jakobovits et al . Nature 362, 255-258
  • Bispecific antibodies are monoclonal, preferably human or humanized antibodies that have binding specificities for at least two different antigens
  • 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
  • the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobuhn 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 immunoglobuhn heavy and light chains these hyb ⁇ domas (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 which is usually done by affinity chromatography steps, is rather cumbersome and the product yields are low Similai procedures are disclosed in PCT application publication No WO
  • 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 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- linking techniques
  • kits 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
  • the reagents for these assays can be provided in a kit, i e , a packaged combination of reagents, for combination with the sample to be tested
  • the components of the kit will normally be provided in predetermined ratios
  • a kit may comprise the antibody or ILP (DNA or polypeptide or fragment thereof) labeled directly or indirectly with a suitable label
  • 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)
  • substrates and cofactors required by the enzyme e g a substrate precursor which provides the detectable chromophore or fluorophore
  • other additives may be included such as stabilizers, buffers and the like
  • the relative amounts of the various reagents may be varied widely to provide for concentrations
  • Amino acid sequence variants of the native ILPs of the present invention may be employed 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 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
  • the ILPs of the present invention provides valuable sequence motifs which can be 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
  • 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
  • sequences from an ILP disclosed herein may be substituted or inserted into the ammo acid sequences of other insulin family members
  • 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
  • a sample such as a body fluid or tissue sample
  • the present invention provides a nucleotide sequence uniquely identifying a novel insuhn-like polypeptide which is expressed, for example, in colon and uterus As a result of expression in these organs, the nucleic acid, dp, 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, adenocarcinoma, colitis inflammatory bowel disease, pelvic inflammatory disease, gastrointestinal bleeding, Crohn's disease, abnormal uterine contraction, constipation, irritable bowel syndrome, diabetes, and obesity
  • nucleotide sequences encoding ILP have numerous applications in techniques known to those skilled in the art of molecular biology These techniques include use as 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
  • 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
  • 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 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 of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence
  • Nucleotide sequences encoding ILP may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques (cf Sambrook J et al (1989) Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory, New York)
  • Useful nucleotide sequences for joining to p 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, probe generation vectors, sequencing vectors, and the like
  • 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 i/ -specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding ILP Such probes may also be used for the detection of similar insu n-like peptide encoding sequences
  • the hybridization probes of the subject invention may be derived from the nucleotide sequences of SEQ ID NO 1 or its complement or from genomic sequences including promoters, enhancer elements and introns of naturally occurring dp Hybridization probes may be labeled by a variety of reporter groups including radionuchdes such as J ⁇ P or J J 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 ohgonucleotides based upon the nucleotide sequences which encode ILP
  • probes used in PCR may 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
  • RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides
  • the nucleotide sequence can be used to construct an assay to detect disease associated with abnormal levels of expression of dp
  • 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 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 dp expression and the presence of disease
  • the nucleotide sequence for tip can be used to construct hybridization probes for mapping that gene
  • 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
  • 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 be found in genome issue of Science ( 1994) 265 1981 Correlation between the location of tip 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
  • Nucleotide sequences encoding ILP may be used to produce purified ILP using well known methods of recombinant DNA technology (see, for example.
  • ILP may be expressed in a variety of host cells, either prokaryotic or eukaryotic Host cells may be from the same species in which p 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 cell may be secreted or may be contained intracellularly, depending on the particular genetic construction used
  • 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 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 431 A 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 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 ammo acids, preferably at least 10 ammo acids They should mimic a portion of the am o acid sequence of the protein and may contain the entire amino acid sequence of ILP Short stretches of ILP amino acid
  • 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 production of synthetic antibodies or other specific-binding molecules such as the screening of recombinant immunoglobuhn 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
  • 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 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 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 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 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" m Toxicokmetics and New Drug Development.
  • ILP or ILP agonist or antagonist to be employed therapeutical ly 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 ⁇ g kg/day to 10 mg/kg/day
  • the 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 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/i munogenicity, these and other characteristics may aid in defining an effective carrier 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 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 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 formulations (such as hposomally encapsulated ILP or PEGylated ILP or ILP polymeric microspheres
  • 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 ⁇ g/kg/day to 10 mg/kg/day, depending upon the route of administration
  • 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.
  • the sustained release formulations of these proteins were developed using poly-lactic-coglycohc acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties
  • PLGA poly-lactic-coglycohc acid
  • the degradation products of PLGA, lactic and glyco c acids, can be cleared quickly within the human body
  • the degradabihty 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/glyco de polymer," in M Chasm and R Langer (Eds ).
  • Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker New York. 1990), pp 1-41
  • 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
  • ILP ILP receptors
  • ILP antagonists where the expression of ILP is increased in the diseased state
  • the nucleic acid sequence of the relaxm molecule was 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 reiaxin nucleic acid sequence, and represent sequences within a gene of an insu n-like polypeptide (ILP)
  • the EST corresponding to SEQ ID NO 15 was used to clone the full length ILP gene
  • the full length ILP gene sequence was cloned using ohgonucleotide 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 ohgonucleot
  • 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 is then hgated 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 bacte ⁇ ophage particles and subsequent infection of £ 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 size of the genome and the size of the cloned fragments (see, Clark and Carbon.
  • 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 reverse transc ⁇ ptase to synthesize complementary cDNA strands from the mRNA template
  • dscDNA double stranded cDNA
  • the dscDNA copy of the mRNA is methylated and equipped with suitable (usually £coRI) linkers
  • reverse transc ⁇ ptase-producing cells are transformed with vectors in which the 5' end of a mRNA molecule having a 5' ohgonucleotide cap is hgated to a s gle-stranded 5' overhang complementary to the ohgonucleotide cap, and the 3' end of the mRNA molecule is hgated to a single-stranded 3' overhang complementary to the 3' end of the mRNA molecule, so that the reverse transc ⁇ ptase produced by the cell converts the mRNAs into dscDNAs to form a cDNA library
  • a cDNA library may be prepared such that the library is enriched in signal sequences This library is enriched in am o terminal signal sequences which are within a clo
  • the 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
  • a starch degrading enzyme e g amylase
  • 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
  • the method involves transforming non-amylolytic yeast cells with exogenous DNA containing the coding sequence of a randomly selected, unidentified mammalian signal peptide hgated to DNA encoding an amylase, which amylase lacks a functional native signal peptide
  • 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 the ATG start codon is eliminated or mut
  • 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
  • random DNA ohgonucleotide primers are used 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 hgated to an adapter ohgonucleotide 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 hgated into the amylase expression vector described above which has been digested with enzvmes compatible with the first and second restriction sites of the cDNA Selected clones are used to
  • Not ⁇ primer-adapter (Life Tech , 2 ⁇ l, 0 5 ⁇ g/ ⁇ l) was added to a sterile I 5 ml microcent ⁇ fuge tube to which was added poly A+ mRNA (7 ⁇ l, 5 ⁇ g) The reaction tube was heated to 70°C 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 ⁇ l), 0 1 M DTT (2 ⁇ l) and 10 mM dNTP Mix (Life Tech , 1 ⁇ l) were added and then heated to 37°C for 2 minutes to equilibrate the temperature SUPERSCRIPT II® reverse transc ⁇ ptase (Life Tech , 5 ⁇ l) was then added, the reaction tube mixed well and incubated at 37°C for 1 hour, and terminated by placement on ice The final concentration of the reactants was the following 50 mM T ⁇ s-HCl (pH 8 3), 75 mM KC1, 3
  • the following reagents were added to the cDNA pellet from the Second strand synthesis above, and the reaction was gently mixed and incubated at 16°C for 16 hours distilled water (25 ml), 5X T4 DNA hgase buffer (10 ml), Sail adapters (10 ml), T4 DNA hgase (5 ml)
  • the final composition of the reaction was the following 50 mM T ⁇ s-HCl (pH 7 6), 10 mM MgCl 2 , 1 mM ATP, 5% (w/v) PEG 8000, 1 mM DTT, 200 mg/ml Sal I adapters.
  • the cDNA was size fractionated by acrylamide gel electrophoresis on a 5% acrylamide gel, and any fragments which were larger than 1 kb, as determined by comparison with a molecular weight marker, were 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 m 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) Ligation of cDNA into pRK5B vector
  • 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 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
  • the dp nucleic acid sequence and the ILP amino acid sequence were homologous to but clearly 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
  • 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 dp
  • Fig 2 shows the comparison of ILP with other insulin and insuhn-like polypeptides The substantial regions of homology among these molecules include the definitive conserved cysteine residues
  • Fig 3 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 lacking in significant hydrophobicity suggesting that ILP is likely to be a secreted protein and does not contain a membrane anchoring or transmembrane domain
  • FIG. 4 A phylogenetic analysis (Fig 4) shows that ILP is closely related to other well characterized human insulin and sulin-hke polypeptides The most related of these molecules cluster together at the right hand side of the figure
  • the msuhn-hke molecules share several characteristics They are each secreted proteins, and each possesses a similar arrangement of six conserved cysteine residues Numerous additional ammo 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 108 (R, R, K, and K respectively)) are likely to have significant impact on function Example 5 ILP polypeptide structure
  • the mature insulin molecule like other members of the insulin polypeptide family, is made up of two am o 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 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 ⁇ - ammo group of a protein chain with 2,4-dm ⁇ tro fluorobenzene (DNFB) to form yellow 2,4-d ⁇ n ⁇ trophenyl derivatives, followed by acid hydrolysis and quantitation of the number of terminal ammo 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 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 l
  • Nucleic acids which encode novel ILP from human or homologous sequences from non-human species 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 animal e g , a mouse
  • a transgenic animal 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
  • 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 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
  • 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
  • murine cDNA encoding ILP can be used to clone genomic DNA encoding ILP 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
  • several kilobases of unaltered flanking DNA are included in the vector (see e g Thomas and Capecchi.
  • 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 Cells A Practical Approach, E J Robertson, ed (IRL, Oxford, 1987), pp 1 13-152)
  • 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 m their germ cells can be identified by standard techniques and used to breed animals in which
  • Expression of the ILP gene may be accomplished by subcloning the cDNA into an appropriate expression vector, transfectmg this vector into an appropriate expression host cell, and cultu ⁇ ng the host cell
  • the ILP may be intracellularly expressed or secreted into the pe ⁇ plasm or culture medium of the host cell
  • the cloning vector previously used for the generation of the tissue library also pro ⁇ ides for direct expression of the included sequence in £ coli Upstream of the cloning site, this vector contains a promoter for ⁇ -galactosidase, followed by sequence containing the ammo-terminal Met and the subsequent 7 residues of ⁇ -galactosidase Immediately following these eight residues is an engineered bacte ⁇ ophage promoter useful for artificial priming and transcription and a number of unique restriction sites, including £c ⁇ RI. for cloning
  • cDNA clone inserts are generated by an essentially random process 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 ohgonucleotide linker inclusion
  • the dp cDNA can be shuttled into other vectors known to be useful for expression of protein in specific hosts Ohgonucleotide amp mers containing cloning sites as w ell as a segment of DNA sufficient to hybridize to stretches at both ends of the target cDNA (25 bases) can be synthesized
  • Suitable vectors may also contain signal sequences upstream and in-frame of the inserted DNA such 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
  • the vector may contain promoters or enhancers which increase gene expression
  • 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 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
  • ILP refers to pro-ILP encoded by nucleic acid sequence SEQ ID NO 1 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 Expression of ILP in £ coli
  • This example illustrates preparation of an unglycosylated form of ILP by recombinant expression in
  • 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 expression vector
  • a variety of expression vectors may be employed
  • An example of a suitable vector is pBR322 (derived from £ coli, see Bolivar et al , Gene, 2 95 (1977)) which contains genes for ampicillin and tetracyclme resistance
  • the vector is digested with restriction enzyme and dephosphorylated
  • the PCR amplified sequences are then hgated into the vector
  • the vector will preferably include sequences which encode for an antibiotic resistance gene, a t ⁇ promoter, a polyhis leader (including, for example, the first six STII codons, polyhis sequence, and enterokmase cleavage site, or a lamB signal (USPN 5,324,820)), the ILP coding region, lambda transc ⁇ ptional terminator, and an argU gene
  • the hgation mixture is then used to transform a selected £ coli strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic 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 then grown to a desired optical density, during which the expression promoter is turned on
  • the cells can be harvested by cent ⁇ fugation
  • the cell pellet obtained bv the cent ⁇ fugation 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 medium is processed by cleaving the signal sequence and isolating the protein as described
  • a method for isolating a recombinant polypeptide expressed in £ 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
  • 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 originating from the 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
  • the dp DNA (DNA27865) is hgated into pRK5B with selected restriction enzyme sites such as Xbal to allow insertion of the dp DNA using hgation methods such as described in Sambrook et al , supra
  • the resulting vector is designated DNA27865- 1091 and has ATCC deposit number 209296
  • DNA sequences encoding the mature form of ILP or the ILP C- peptide may be inserted into a vector
  • the selected host cells may be 293 cells
  • Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates m medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics
  • About 10 ⁇ g DNA27865-1091 DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene (Thimmappaya et al , Cell, 31 543 (1982)) and dissolved in 500 ⁇ l of 1 mM T ⁇ s-HCl, 0 1 mM EDTA, 0 227 M CaCl 2
  • 500 ⁇ l of 50 mM HEPES pH 7 35
  • 280 mM NaCl 1 5 mM NaP0 4
  • a precipitate is allowed to form for 10 minutes at 25°C
  • the precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C
  • the culture medium is aspirated off and 2 m
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml 35 S-cysteme and 200 ⁇ O/mr- > S- methiomne
  • culture medium alone
  • 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 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
  • an ILP-encod g 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 ⁇ g DN A27865- 1091 DNA is added The cells are first concentrated from the spinner flask by cent ⁇ fugation 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-mtroduced into the spinner flask containing tissue culture medium, 5 ⁇ g/ml bovine insulin and 0 1 ⁇ g/ml bovine transfer ⁇ n After about four days, the conditioned media is centrifuged and filtered to remove cells and debris The sample containing expressed ILP can then be concentrated and pur
  • ILP can be expressed in CHO cells
  • the DN A27865- 1091 can be transfected into CHO cells using known reagents such as CaPO ⁇ or DEAE-dextran
  • the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 3 S-methionme
  • the culture medium may be replaced with serum free medium
  • 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 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
  • the 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 - c helate affinity chromatography
  • 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
  • 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
  • the native signal sequence of ILP is employed for secretion of the ILP
  • Yeast cells such as S cerevisiae strain AB 1 10, can then be transformed with the expression plasmids 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% t ⁇ chloroacetic 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 cent ⁇ fugation and then concentrating the medium using selected cartridge filters
  • 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
  • epitope tags include poly-his tags and immunoglobuhn tags (like Fc regions of IgG)
  • plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL 1393 (Novagen)
  • the ILP or the desired portion of the ILP (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may inco ⁇ orate flanking (selected) restriction enzyme sites
  • the product is then digested with those selected restriction enzymes and subcloned into the expression vector
  • Recombinant baculovirus is generated by co-transfectmg the above plasmid and BACULOGOLD M virus DNA (Pharmingen) into
  • Expressed poly-his tagged ILP can then be purified, for example, by Nr 9+ -chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 ceils 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.
  • IgG tagged (or Fc tagged) ILP can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography 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
  • 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 immunoglobuhn, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Co ⁇ , Seattle Wash )
  • the inclusion of a cleavable linker sequence such as Factor XA or enterokmase (Invitrogen, San Diego Calif ) between the purification domain and the dp sequence may be useful to facilitate expression of ILP Example 10 Production of ILP-Specific Antibodies
  • ILP immunoglobulin-like protein
  • pro-ILP pro-ILP
  • mature ILP mature ILP
  • ILP C- peptide or a fragment thereof
  • 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 rabbit
  • the denatured protein can be radioiodmated and used to screen potential murine B-cell hyb ⁇ domas 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
  • the amino acid sequence of ILP as deduced from translation of the cDNA. is analyzed to determine regions of high immunogenicity
  • Ohgopeptides 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-termmus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation
  • peptides typically, about 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A 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)
  • KLH keyhole limpet hemocyanin
  • M-maleimidobenzoyl-N-hydroxysuccinimide ester Ausubel, F M et al , supra
  • 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 w ith antisera, washing and reacting with labele
  • 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 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
  • Suitable labels include radionuchdes 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 inco ⁇ orated by reference in their entirety
  • recombinant immunoglobulins may be produced as shown in U S Pat No 4,816,567, inco ⁇ orated here
  • Native or recombinant ILP may be purified by a variety of standard techniques m the art of protein purification
  • pro-ILP, mature BP, or ILP C-peptide is purified by immunoaffinity chromatography using antibodies specific for the ILP
  • an immunoaffinity column is constructed 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 )
  • monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A
  • Partially purified immunoglobuhn is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology) The antibody is coupled to the resin, the resm is blocked, and the derivative resin is washed according to the manufacturer s 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 solubihzation of the whole cell or of a subcellular fraction obtained via differential cent ⁇ fugation by the addition of detergent or bv 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 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.
  • a chaotrope such as urea or thiocyanate ion
  • Purified ILP is useful for characterization and purification of specific cell surface receptors and other 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-b ding molecules may be identified by interaction with radiolabeled ILP Radioactive labels may be inco ⁇ orated into ILP by various methods known in the art
  • a preferred embodiment is the labeling of primary ammo groups in ILP with L I 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 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
  • 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 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 ohgonucleotide probes for cloning the receptor gene
  • mRNA is obtained from receptor-bearing cells and made into a cDNA 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
  • antibodies are raised against the surface of receptor bearing cells, specifically monoclonal antibodies
  • the monoclonal antibodies are screened to identify those which inhibit 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 chromatography or density gradient cent ⁇ fugation and are purified by methods known in the art The soluble receptors or binding prote ⁇ n(s) are subjected to N-termmal 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-Induced Cellular Response
  • the biological activity of ILP is measured, for example, by binding of an ILP of the invention to an 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.
  • the assay may be adapted to monitor ILP-mduced receptor activation through the use of an ILP receptor-specific antibody to capture the activated receptor
  • 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 test may either be free m solution, affixed to a solid support, borne on a cell surface or located mtracellularly
  • 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 m 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 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
  • the present invention provides methods of screening for drugs or any other agents which can affect ILP-associated disease
  • methods comprise contacting such an agent with an ILP or fragment thereof and assaying (I) 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.
  • 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 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. inco ⁇ orated 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, 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 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 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 9: 19-21 , inco ⁇ orated herein by reference in its entirety).
  • the three-dimensional structure of the ILP, or of an ILP-inhibitor complex is 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 of rational drug design may include molecules which have improved activity or stability as shown by Braxton, S. and Wells, J.A.
  • a target-specific antibody selected by functional assay, as described 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 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

Abstract

The present invention provides nucleotide and amino acid sequences that identify and encode a novel expressed insulin-like polypeptide (ILP) from, for example, colon and uterine cells. The present invention also provides for antisense molecules to the nucleotide sequences which encode ILP, expression vectors for the production of purified ILP, antibodies capable of binding specifically to ILP, hybridization probes or oligonucleotides for the detection of ILP-encoding nucleic acid sequences, genetically engineered host cells for the expression of ILP, diagnostic tests for insulin-like activation based on ILP-encoding nucleic acid molecules and antibodies capable of binding specifically to the protein.

Description

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 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 proinsu n to produce mature insulin An important effect of mature insulin is its ability to increase the rate of glucose transϋort 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 can be altered as much as 20-fold
Insulin promotes glucose transport into cells by stimulating a process of facilitated diffusion m 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 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 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 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 melhtus, 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 range of cell types and can mediate the effects of growth hormone on skeletal growth Most tissues and especially the liver produce IGF-I 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 Endocπnol 84 681 -696, and Uthne et al (1974) J Chn Endocπnol Metab 39 548-554) 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-I and IGF-II, are chemically related to human promsulin in that they contain A and B domains connected by a C domain region, and have high homology to promsulin The IGFs further contain a D domain at the C-terminus that is not found in promsulin 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 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-stimulatmg 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-1 is described in Lassalle et al , J Biol Chem , 271 20458-20464 ( 1996) For other identified IGFBPs, see, e g , 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 1 176-1 185 (1988), Bπnkman et al . The EMBO J , 7 2417- 2423 (1988), Lee et al , Mol Endocπnol , 2 404-41 1 (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 , £I£ 229-235 (1988), WO 89/08667 published 21 September 1989, WO 89/09792 published 19 October 1989, and Bmkert et al 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 other surface of plasma membranes in manv 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-E133 and references cited therein) In normal fasted rats, rhIGF-I has been shown to produce hypoglycemia, even when infused simultaneously with anti-insulin serum While both insulin and rhIGF-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 C n 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 humans (Guler et al (1987) N Engl J Med 3J7 137-140 ) Additionally, IGF-I promotes growth m several metabolic conditions characterized by low IGF-I levels, such as hypophysectomized rats (Guler et al , Endocrinology, JJ8 Supp 129 abstract, Skottner et al , (1987) J Endocπnol JJ2 123-132, Guler et al , (1988) PNAS USA 85 4889-4893, Froesch et al , in Endocrinology Intl Congress Series 655, Labπe and Proulx, eds 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 1st European Congress of Endocrinology 103 abstract 12-390 (Copenhagen, 1987)) An additional use 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 ammo 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 response to rhIGF-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 (Tπpathi 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) Reiaxin 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 reiaxin were considered to be a lengthening of the pubic ligaments, widening of the pelvis, dampening of uterine contractions, and softening and dilating of the cervix (Wade, J D and Tregear, G W ( 1997) supra) However, later studies indicated that reiaxin had a wider physiological role as capable of causing changes in fluid balance via the relationship between plasma osmolahty and arginine vasopressm (Weisenger, R S et al (1995) J Endocrinology 137 505-510), changes in heart rate (chronotropic activity) and heart muscle contractility (lonotropic activity) (Kakouπs. H et al (1995) Lancet 339 1076-1978), and is present in male seminal plasma (Winslow, J W et al (1992) Endocrinology ( 1992) 130 2660-2668)
Reiaxin receptors have been observed in three tissues by autoradiography using J~P-Iabeled synthetic reiaxin, which tissues include uterus, brain, and heart Osheroff, P and Ho, W -H (1993) J Biol Chem 268 15193-15199) Reiaxin binding regions were observed in brain m regions associated control of cardiovascular functions such as blood pressure and fluid and electrolyte homeostasis Reiaxin has been shown to be more potent than angiotensin II or endothe n, suggesting that it may play a role in cardiovascular disorders (Summers, R J et al in "Recent Progress in Reiaxin 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 prevented or attenuated vascular and neural (electrophysιologιcal)dysfunctιon and impaired Na+ - and K+ - dependent adenosme tπphosphate activity in tissues of diabetic rats (Ido. Y et al (1997) Science 277 563- SUMMARY OF THE INVENTION An aspect of the subject invention provides a nucleic acid sequence (SEQ ID NO 1 ) of a gene, pro-dp, 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), which is a member of the msulin/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 ammo 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 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 "Up gene" referring to SEQ ID NO 1 (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 as antibodies (including monoclonal antibodies) to the polypeptides Further embodiments include a chimeric molecule comprising an ILP polypeptide fused to a heterologous ammo 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 immunoglobu n
Another aspect of the invention includes a method for determining expression of p in a cell Preferably the diagnostic test comprises providing a cell extract or a tissue sample containing cells suspected of expressing tip 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 1 (Fig 6) or a fragment thereof
Another aspect of the invention includes a method of diagnosing a physiologic or pathologic 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 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 lip 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 In a further aspect the invention encompasses a transgenic animal comprising an altered tip 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 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 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 m toxicokinetics" in Toxicokmetics and New Drug Development. Yacobi et al (eds), Pergamon Press, 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 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-encodmg 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 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 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 m U S Patents 4,892,538, and 5,01 1,472, WO 92/19195, 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 lonotropic activity, cardiac 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
- 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 dp (SEQ ID NO 1 ) and the predicted amino acid 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 overhning The cysteine residues that are predicted to participate in disulfide bond linkages between the
A and B chains are indicated by the encircled numbers 1 through 6 below the cysteine residues within the sequence, where the linkages are between 1 and 4, 2 and 6, and 3 and 5 Primer oligonucleotides IN2328985 f, IN2328985 p, and IN2328985 r, designed based on homology to relaxm and used to isolate the full length dp, are indicated by overhning and underlining
Fig 2 shows the ammo acid sequence alignment of ILP (SEQ ID NO 2) with other polypeptide of the insulin/IGF family h-Insuhn (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 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 Fig 4 shows a relatedness tree of some human insulin/IGF polypeptide family members (including
GenBank accession numbers) h-prelGFl (P70277), h-preIGF2 (P01344), h-prepro insulin (PI 0042), 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 INC2328985), and Incyte EST INC7783 19 (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 1 The deduced ammo 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 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 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 ammo acid sequence shown in SEQ
-6- SUBST1TUTE SHEET (RULE 26) ID NO 2 The ILP polypeptide may be encoded by dp having the nucleic acid sequence shown in SEQ ID NO 1 (Fig 6) encoded within SEQ ID NO 22 shown in Fig 1 (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 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 ammo 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 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 polypeptide which has been isolated from a mammal The ILP can also be "native sequence ILP" insofar as it has the same ammo 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 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 1 (SEQ ID NO 2). mature human ILP (SEQ ID NOS 9 and 10) and ILP C-peptide (SEQ ID NO 21 ) The novel native human ILPs of the present invention are about 1 14 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 ammo 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 deπvatization 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, 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 "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 occurring ILP chemical modifications such as ubiquitmation, labeling (e g . with radionuclides, various enzymes, etc ), pegylation (deπvatization 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 am o acid sequence and glycosylation variants, and covalent modifications of a native polypeptide 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, ammo 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 displav biologic and/or lmmunologic activity Fragment can also include a portion of each of the 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 hybridization procedures, such as amplification or identification of portions of mRNA or DNA molecules
"O gonucleotides" or "nucleic acid probes" are prepared based on the cDNA sequence which encodes
ILP provided by the present invention Ohgonucleotides 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 1 kb After appropriate 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, 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 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 % 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) 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 The term "percent ammo 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 am o 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 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 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 protemaceous or non-protemaceous deπvatizing agent, fusions to heterologous polypeptide sequences, and post-translational modifications Covalent modifications are traditionally introduced by reacting targeted amino acid residues with an organic deπvatizing 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 cells on the expressed polypeptide Glutaminyl and asparaginyl residues are frequently post-translational ly 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 α-amino groups of lysine, arginine, and histidme side chains (T E Creighton, Proteins Structure and Molecular 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- SUBST1TUTE SHEET (RULE 26) 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 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 ammo acid residues in the candidate sequence that are identical with the residues 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 m the art For example, the sequences disclosed herein were analyzed using "ALIGN", Genentech, Inc 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 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 prevents or interferes with binding and/or activation of an ILP receptor by ILP) Such molecuies 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 ILP gene, thereby inhibiting its expression and biological activity
Ordinarily the terms "amino acid" and "amino acids" refer to all naturally occurring L-α-amino acids In some embodiments, however, D-ammo 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 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 He I isoleucine
Thr T threonine Leu L leucine Ser S serine Tyr Y tyrosine
Glu E glutamic acid Phe F phenylalanine
Pro P proline His H histidme
Gl> G glycine Lys K lysine
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 "ammo acid sequence variant" refers to molecules with some differences in their amino acid sequences as compared to a native amino acid sequence
Substitutional variants are those that have at least one am o 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 ammo 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 either the α-carboxv or α-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 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 heterotetrameπc 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 the heavy chains of different immunoglobuhn isotypes Each heavy and light chain also has regularly spaced mtrachain disulfide bridges Each heavy chain has at one end a variable domain (V j) followed by a number of constant domains Each light chain has a variable domain at one and (V^) 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 acid residues are believed to form an interface between the light and heavy chain variable domains (Clothia et l , ] 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 (λ) based on the ammo acid sequences of their constant 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, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e g IgG-1 , IgG- 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 α, delta, epsilon, γ, and μ, 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 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, l e , the individual antibodies comprising the population are identical 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 hybπdoma method first described by Kohler & Milstein, Nature 256 495 (1975), or may be made 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, immunoglobuhn 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 immunoglobuhn 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, affinity and capacity In some instances, Fv framework region (FR) residues of the human immunoglobuhn 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 substantially all of the CDR regions correspond to those of a non-human immunoglobuhn and all or substantially all of the FR residues are those of a human immunoglobuhn consensus sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobuhn constant region (Fc), typically that of a human immunoglobuhn 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 Biol 2 593-596 (1992), and EP-B-451 216 published 24 January 1996), which references are herein incorporated by reference m their entirety By "neutralizing antibody" is meant an antibody molecule as herein defined which is able- to 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 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 chaιn(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 , Morrison et al , Proc Natl Acad Sci USA 8 L 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, 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 "rep cable 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 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 m 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 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 πbosome 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 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 preprotem 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 πbosome 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 a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by gation at convenient restriction sites If such sites do not exist, then synthetic ohgonucleotide adaptors or linkers are used in accord with conventional practice "Ohgonucleotides" 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 ( 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), polysacchaπdes (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones In certain embodiments, depending on the context, the solid phase can 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 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 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 D A is the calcium treatment method described by Cohen et al , Proc Nad Acad Sci US A 69 21 10- 21 14 (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 Natl Acad Sci USA , 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 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 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 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- SUBST1TUTE SHEET (RULE 26) 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 1 , within Generrech 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 1 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 chloπde/0.0015 M sodium 0 cιtrate/0 1% sodium dodecyl sulfate at 50CC, or (2) employing during hybridization a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide with 0 1% bovine serum albumιn/0 1% Fιcoll/0 1% polyvιnylpyrrolιdone/50 nM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C 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 5 salmon sperm DNA (50 μg/ml), 0 1% SDS, and 10% dextran sulfate at 42°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 0 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 ammo 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 5 to display biologic and/or lmmunologic activity
"Immunoadhesins" or "ILP - immunoglobuhn chimeras" are chimeric antibody-like molecules that combine the functional domaιn(s) of a binding protein (usually a receptor, a cell-adhesion molecule or a ligand) with the an immunoglobuhn sequence The most common example of this type of fusion protein combines the hinge and Fc regions of an immunoglobuhn (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 " "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 like Preferabh . 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 ascorbic acid, low molecular weight (less than about 10 residues) polypeptide, proteins, such as serum albumin, gelatin or immunoglobulins. hydrophilic polymers such as polyvinylpyrrohdone. amino acids such as glycine, glutamine. asparagine, arginine or lysine, monosacchaπdes, disacchaπdes, and other carbohydrates including glucose, mannose, or dextπns, chelating agents such as EDTA, sugar alcohols such as manmtol or sorbitol. salt-forming counteπons such as sodium, and/or nonionic surfactants such as Tween™, polyethylene glycol (PEG), and Pluronics™
General Procedures for the Production of an ILP by Recombinant DNA Technology
A Identification and isolation of nucleic acid encoding a novel insuhn-hke polypeptide. 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 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) 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 ohgonucleotide probes (usually of about 20-80 bases in length) that 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, ohgonucleotides, cDNAs. or fragments thereof that encode the same or a similar gene, and/or homologous genomic DNAs oi 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 If DNA encoding an ILP of the present invention is isolated b\ using carefully selected o gonucleotide sequences to screen cDNA libraries from various tissues, the ohgonucleotide 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 ohgonucleotides may be degenerate at one or more positions The use of degenerate ohgonucleotides is of particular importance where a library is screened from a species in which preferential codon usage is not known
The ohgonucleotide must be labeled such that it can be detected upon hybridization to DNA in the library being screened T e preferred method of labeling is to use ATP (e g , γ32P) and polynucleotide kinase to radiolabel the 5' end of the ohgonucleotide However, other methods may be used to label the ohgonucleotide 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 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 ohgonucleotide sequences selected from known ILP sequences (such as murine or human sequences) in accord with known criteria Preferably, the probe sequence should be sufficient in length and sufficiently unambiguous that false positives are minimized TypicalK a J P-labeled ohgonucleotide having about 30 to 50 bases is sufficient, particularly if the ohgonucleotide 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 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 tπester, phosphite phosphoramidite and H-phosphonate methods, PCR and other autopπmer methods, and ohgonucleotide syntheses on solid supports B Cloning and expression of nucleic acid encoding a novel ILP
Once the nucleic acid encoding a novel ILP is available, it is generally gated into a rep cable expression vector for further cloning (amplification of the DNA), or for expression
-17- SUBST1TUTE SHEET (RULE 26) 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 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 hgation techniques Isolated plasmids or DNA fragments are cleaved, tailored, and rehgated in the form desired to generate the plasmids required For analysis to confirm correct sequences in constructed plasmids, the hgation mixtures are commonly used to transform E coli cells, e g E coli K12 strain 294 (ATCC 31 ,446) and successful transformants selected by ampicil n or tetracyc ne resistance where appropriate Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced by the 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 prokaryotes such as E coli B, E coli XI 776 (ATCC 31 ,537), E coli W31 10 (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 available and useful herein, such as S pombe (Beach and Nurse, Nature 290 140 ( 1981 )), Kluvveromvces lactis (de Louvencourt L et al , ] Bactenol 154 737-742 (1983)), yarrowia (EP 402,226), Pichia pastons (EP 183,070), Tnchoderma 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 H2 284-289 (1983), Tilburn et al . Gene 26 205-221 (1983), Yelton et al , Proc Natl Acad Sci USA 81 1470-1474 (1984)) and A mger (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 corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (cateφillar), 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 315 592-594 (1985) A variety of such viral strains are publicly available, e g the L- l variant of Autographa califor ica NPV and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells
Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as 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 nopahne synthase promoter and 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 (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 CVl 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 cellsΛDHFR (CHO, Urlaub and Chasm, Proc Natl Acad Sci USA 77 4216 (1980)), mouse sertolli cells (TM4 Mather, Biol Reprod 23 243-251 (1980)), monkey kidney cells (CVl 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 CCL51), TRI cells (Mather et al , Annals N Y Acad 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 the host cell accumulates many copies of the expression vector and, m 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 puφoses of identifying analogs and 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 1 17,060 and EP 1 17,058 Particularly useful 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 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 of £ coli cells is PET15b (Novagen)
C Cultuπng the Host Cells
Prokaryote cells used to produced the ILPs of this invention are cultured in suitable media as describe generally in Sambrook et al , supra
Mammalian cells can be cultured in a variety of media Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for cultuπng 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 ( 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 ' M drug) trace elements (defined as inorganic 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 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 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 m situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Various labels may be employed, most commonly radioisotopes, particularly J 32 P However, other techniques may also be employed, such as using biotm-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 radionuchdes, fluorescers, enzymes, or the like Alternatively, antibodies may be employed that can recognize specific duplexes, 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 Gene expression, alternatively, may be measured by immunological methods, such as lmmunohistochemical 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 C n Pharm 75, 734-738 (1980)
Antibodies useful for lmmunohistochemical staining and/or assay of sample fluids may be either 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 Ammo Acid Sequence Variants of a Native ILP
Ammo 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 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 ammo 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 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 such modifications is called "alanme 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, lie
(2) neutral hydrophobic cys, ser, thr,
(3) acidic asp, glu, (4) basic asn, gin, his, lys, arg,
(5) residues that influence chain orientation gly, pro, and
(6) aromatic tφ, 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 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 chain The substitutions which in general are expected to produce the greatest changes m 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 ieucyl, 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 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 , phenylalanme. 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 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 Ammo 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 mtrasequence 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 1 to 3 residues An example of a terminal insertion includes fusion of a heterologous N-terminal signal sequence to the N-termmus 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 heφes gD for mammalian cells
Other msertional variants of the native ILP molecules include the fusion of the N- or C-lerminus of the ILP molecule to immunogenic polypeptide, e g bacterial polypeptide such as beta-lactamase or an enzyme encoded by the E coli tφ locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as immunoglobuhn regions (preferably immunoglobuhn constant regions), albumin, or ferritin, as described in WO 89/02922 published on 6 April 1989
Further msertional variants are immunologically active derivatives of the novel ILPs, containing an 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 as tφLE, β-glactosidase, viral polypeptide such as heφes 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 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 acceptable carrier and administered to a subject in order to raise antibodies against the ILP molecule, which antibodies in turn are useful as diagnostics, m 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 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 mutatιon(s), the gene encoding an ILP variant can, for example, be 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 nonvaπant version of the ILP Site-directed (site-specific) mutagenesis allows the production of ILP variants through the use of specific ohgonucleotide 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 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 a s gle-stranded and double-stranded form Typical vectors useful in site-directed mutagenesis include vectors such as the M 13 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 oligodeoxyπbonucleotide directed site-specific mutations in DNA fragments using M13-deπved 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 The PCR amplification technique may also be used to create ammo acid sequence variants of a novel
ILP In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) 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 deoxynucleotide tπphosphates and is included m the GeneAmp kits (obtained from Perkin-Elmer Cetus. Norwalk. CT and Emeryville, CA), and 25 pmole of each ohgonucleotide primer, to a final volume of 50 μl The reaction mixture is overlayered with 35 μl mineral oil The reaction is denatured for 5 minutes at 100°C, placed briefly on ice, and then 1 μl Thermus aquaticus (Taq) DNA polymerase (5 units/μl), purchased from 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 mm 55°C,
30 sec 72°C, then 19 cycles of the following 30 sec 94oC,
30 sec 55°C, 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 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 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 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) cultuπng 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 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 cultuπng conditions are adjusted so that the amount or number of phagemid particles displaying more than one copy of the fusion protein on the 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 of the polypeptide and the transcription regulatory element will be a promoter system Preferred promoter systems are selected from lac Z, λp^, tac T7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof Also, normally the method will employ a helper phage selected from M 13K07, M13R408, M 13-VCS, and Phi X 174 The preferred helper phage is M 13K07 and the preferred coat protein is the M13 Phage gene III coat protein The preferred host is E coli, and protease-deficient strains of £ 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 m Molecular Biology, Ausubel et al eds , supra
F Covalent Modifications 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 deπvatizing 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 residues important for biological activity for immunoassays of the ILP, or for the preparation of anti-ILP antibodies for immunoaffinity purification of the recombinant For example complete inactivation of the biological activity of the protein after reaction with nmhydπn 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 of a peptide fragment containing the modified amino acid residue Such modifications are within the ordinary skill in the art and are performed without undue experimentation
Deπvatization 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 -bιs(dιazoacetyl)-2- phenylethane, glutaraldehvde, N-hydroxysuccinimide esters, homobifunctional imidoesters, and bifunctional maleimides Deπvatizing agents such as methyl-3-[(p-azιdophenyl)dιthιo]propιoιmιdate 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, 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 Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, threonyl or tyrosyl residues, methylation of the α-amino groups of lysine, arginine, and histidme side chains (T E Creighton, Proteins Structure and Molecular Properties. W H Freeman & Co , San Francisco, pp 79-86 (1983)) Further derivatives of the ILPs herein are the so called "immunoadhesins", which are chimeric antibody-like molecules combining the functional domaιn(s) of a binding protein (usually a receptor, a cell- adhesion molecule or a ligand) with the an immunoglobuhn sequence The most common example of this type of fusion protein combines the hinge and Fc regions of an immunoglobuhn (Ig) with domains of a cell-surface receptor that recognizes a specific ligand This type of molecule is called an "lmmunoadhesin", 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 (1989) Traunecker et al . Nature 339 68-70 ( 1989), Zettmeissl e/ α/ , DNA Cell Biol USA 9 347-353 (1990), Byrn et al , Nature 344 667-670 (1990)), L-seILP (homing receptor) (Watson et al , ] Cell Biol 1 10 2221 - 2229 (1990)), Watson et al , Nature 349 164-167 (1991)), E-seILP (Mulligan et al , J Immunol 151 6410- 17 (1993), Jacob et al , Biochemistry 34 1210-1217 (1995)), P-seILP (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 , J Virol 67 3561-68 (1993), Roep e/ α/ , Lancet 343 1590-93 (1994)), ICAM-2 (Damle e/ α/ , 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-R1 (Ashkenazi et al , Proc Natl Acad Sci USA 88 10535-539 (1991), Lesslauer et al , Eur J Immunol 21 2883-86 (1991 ), Peppel e/ α/ . J Exp Med 174 1483-1489 (1991)), TNF-R2 (Zack et al , Proc Natl 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 <?/ α/ , J Exp Med 173 721-730 (1991 )), CTLA-4 (Lisley et al , ] Exp Med 174 561-569 (1991 )). CD22 (Stamenkovic e/ α/ , Cell 66 1 133- 1 144 (1991)), NP receptors (Bennett et al , J Biol Chem 266 23060-23067 (1991 )), IgE receptor α (Ridgway and Gorman, J Cell Biol 1 15 abstr 1448 (1991 )), IFN-γR a- and β-chain (Marsters et al , Proc Natl Acad Sci USA 92 5401 -05 (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 et al . ] Immunol 154 5590-5600 (1995))
The simplest and most straightforward lmmunoadhesin design combines the binding regιon(s) of the 'adhesin' protein with the hinge and Fc regions of an immunoglobuhn heavy chain Ordinarily, when preparing the ILP-immunoglobuhn chimeras of the present invention, nucleic acid encoding the desired ILP 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 immunoglobuhn constant domain sequence, however fusion to the N-terminus of the immunoglobuhn 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 immunoglobuhn heavy chain Fusions are also made to the C-terminus of the Fc portion of a 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- lmmunoglobu n chimeras In a preferred embodiment, the sequence of a native, mature ILP polypeptide is fused 4o the N- terminus of the C-termmal portion of an antibody (in particular the Fc domain), containing the effector functions of an immunoglobuhn, 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 papain cleavage site (which defines IgG Fc chemically, residue 216. taking the first residue of heavy chain constant region to be 1 14, or analogous sites of other immunoglobulins) is used in the fusion In a particularK preferred embodiment, an ILP polypeptide chain is fused to the hinge region and CH2 and CH3 or CH 1 , hinge. CH2 and CH3 domains of an IgG-1, IgG-2, or IgG-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 In some embodiments, the ILP-immunoglobuhn 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, IgD, 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 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-immunoglobuhn chimeras within the scope of the invention are schematically diagrammed below (a) ACL-ACL, (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 ( [A-Y]n-[VLCL-VHCH]2, wherein each A represents identical or different novel ILP polypeptide amino acid sequences, Vj^ is an immunoglobuhn light chain variable domain, V is an immunoglobuhn heavy chain variable domain, CL is an immunoglobuhn light chain constant domain, Cj_j is an immunoglobuhn 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 are required for binding activity, they shall be construed as being present in the ordinary locations which the> occupy the immunoglobuhn molecules
Although the presence of an immunoglobuhn light chain is not required in the immunoadhesins of the present invention, an immunoglobuhn light chain might be present either covalently associated to an ILP- immunoglobuhn heavy chain fusion polypeptide. or directly fused to the ILP polypeptide In the former case. DNA encoding an immunoglobuhn light chain is typically coexpressed with the DNA encoding the ILP- immunoglobuhn heavy chain fusion protein Upon secretion, the hybrid heavy chain and the light chain will be covalently associated to provide an lmmunoglobu n-hke structure comprising two disulfϊde-Iinked immunoglobuhn 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 immunoglobuhn sequences used in the construction of the immunoadhesins of the present invention are from an IgG immunoglobuhn heavy chain constant domain For human immunoadhesins, the use of human IgG-1 and IgG-3 immunoglobuhn sequences is preferred A major 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 lmmunoadhesin 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 to IgG- 1 While IgG immunoadhesins are typically mono- or bivalent, other Ig subtypes like IgA and IgM may give rise to dimeπc or pentameπc 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 counteφarts Reported examples of such structures are CD4-IgM (Traunecker et al , supra). ICAM-IgM (Martin et al , J Virol 67, 3561-68 (1993)), and CD2-IgM (Arulanandam e/ α/ , J Exp Med 177, 1439-50 ( 1993))
For ILP-Ig 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 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 sites, two of which (Gl m and 2) are located in the Fc region, and one of these sites G lm l , is non- lmmunogenic 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, 1 1 and 21 ) have one allotype which is nonimmunogenic Thus, the potential lmmunogenicity of a γ3 lmmunoadhesin is greater than that of a γ l lmmunoadhesin
ILP-Ig immunoadhesins are most conveniently constructed by fusing the cDNA sequence encoding 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 e. αt . Cell 61 1303-1313 (1990), Stamenkovic et al , Cell 66 1 133-1 144 (1991)) The latter type of fusion requires the presence of Ig regulatory sequences for expression cDNAs encoding IgG heavy-cha constant regions can be isolated based 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 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); 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 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 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. 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. 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 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 groups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde. PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde, succmimidyl active esters, activated dithiocarbonate PEG, 2.4,5-tπchlorophenylcloroformate or P-nitrophenylcloroformate activated PEG ) Carboxyl groups are deπvatized by coupling PEG-amine using carbodnmide Polymers are conjugated to ohgosacchaπde groups by oxidation using chemicals, e g metapeπodate, 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 deπvatized polymers, 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 methods which have been used heretofore to link oligosaccharides are particularly advantageous because, in general, there are fewer substitutions than ammo acid sites for deπvatization, and the ohgosacchaπde products thus will be more homogeneous The oligosacchaπde substituents also are optionally modified by enzyme digestion to remove sugars, e g by neuraminidase digestion, prior to polymer deπvatization
The polymer will bear a group which is directly reactive with an amino acid side chain, or the N- or 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 deπvatized in the same fashion as insoluble polymers heretofore employed for protein immobilization Cyanogen bromide activation is a particularly useful procedure to employ in crosslinking polysacchaπdes "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 deπvatization reaction "Water soluble" in reference to the polymer conjugate means that the conjugate is soluble m physiological fluids such as blood
The degree of substitution with such a polymer will vary depending upon the number of reactive sites 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, hydrophihcity and other characteristics of the polymer, and the particular protein deπvatization sites chosen In general, the conjugate contains about from 1 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 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 m the art for the covalent modification of proteins with nonproteinaceous polymers such as PEG Certain of these methods, however, are not preferred for the puφoses 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 dnmidazole chemistry (Beauchamp et al . Anal Biochem 131.25-33 (1983)) requires high pH (>8 5), which can inactivate proteins Moreover, since the "activated PEG" 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 dnmidazole 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 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, 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 cyanoborohydπde, 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 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 entrappeα 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 disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A , Ed (1980)
G Antibody preparation
(l) 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 mtraperitoneal (lp) injections of the ILP and an adjuvant It may 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 thyroglobuhn, or soybean trypsin inhibitor using a bifunctional or deπvatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC^, or R'N=C=NR, where R and R' are different alkyl groups
Animals are immunized against the immunogenic conjugates or derivatives by combining approximately 1 mg or 1 μg of conjugate (for rabbits or mice, respectively) with three volumes of Freud's complete adjuvant and injecting the solution mtradermally 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 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 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
(n) Monoclonal antibodies
Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, I e . the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts 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 hybπdoma method first described by Kohler & Milstem, Nature 256 495 (1975), or may be made by recombinant DNA methods (Cabilly. et al , 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 ohgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) The hybπdoma 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 transfected into host cells such as simian COS cells Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobuhn 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 8 . 6851 (1984), or by covalently joining to the immunoglobuhn coding sequence 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 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, lmmunotox s may be constructed using a disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this puφose include lminothiolate and methyI-4-mercaptobutyπmιdate
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 - H, C, j2P, S, or
1
'•"I. a fluorescent or chemiluminescent compound, such as fluorescem isothiocyanate. rhodamine, or lucifeπn. biotin, radioactive isotopic labels, such as, e g , ' 5I, J^ P, 4 C, or1 H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase
Any method known m the art for separately conjugating the antibody to the detectable moiety ma> be employed, including those methods described by Hunter et al , Nature 144 945 (1962), David, et al Biochemistry 13 1014 (1974), Pain et al Immunol Meth 40 219 (1981 ), and Nygren J Histochem and Cytochem 30 407 (1982)
The antibodies of the present invention may be employed m any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (see, for example, Zola, Monoclonal Antibodies A Manual of Techniques, pp 147-158 (CRC Press, Inc , 1987)
(in) 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 "import" variable domain Humamzation 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 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 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 immunoglobuhn models are commonly available and are familiar to those skilled m the art Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobuhn sequences Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobuhn sequence l e the analysis of residues that influence the ability of the candidate immunoglobuhn 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 antιgen(s), is achieved In general, the CDR residues are directly and most substantially involved m influencing antigen binding (see, for example, WO 92/22653) 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 immunoglobuhn production For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J j) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production Transfer of the human germ-line immunoglobuhn gene array in such 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 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 Traditionally the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobuhn 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 immunoglobuhn heavy and light chains these hybπdomas (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, which is usually done by affinity chromatography steps, is rather cumbersome and the product yields are low Similai 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 According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobuhn constant domain sequences The fusion preferably is with an immunoglobuhn heavy chain constant domain, comprising at least part of the hinge, and second and third constant regions of an immunoglobuhn heavy chain (CH2 and CH3) It is preferred to have the first heavy chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions DNAs encoding the immunoglobuhn heavy chain fusions and, if desired, the immunoglobuhn 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 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 immunoglobuhn heavy chain with a first binding specificity in one arm, and a hybrid immunoglobuhn heavy chain-light chain pair (providing a second binding specificity) in the other arm It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobuhn chain combinations, as the presence of an immunoglobuhn 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 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 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- linking techniques
H Diagnostic Kits & Articles of Manufacture
Since the invention provides a diagnostic assay (l 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, 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 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 lyophihzed, 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 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 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 article of manufacture may further comprise a second container comprising a pharmaceutical ly-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 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 m Bodanszky et al , Peptide Synthesis. John Wiley & 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 Peptide Svntheha. Pierce Chemical Company, Rockford, IL (1984) Peptides may also be prepared by recombinant DNA technology, using a DNA sequence encoding the desired peptide 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 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 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 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, sequences from an ILP disclosed herein may be substituted or inserted into the ammo 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 novel insuhn-like polypeptide which is expressed, for example, in colon and uterus As a result of expression in these organs, the nucleic acid, dp, 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, 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 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 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 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 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 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 means of well established recombinant DNA techniques (cf Sambrook J et al (1989) Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory, New York) Useful nucleotide sequences for joining to p 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, 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 i/ -specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding ILP Such probes may also be used for the detection of similar insu n-like peptide encoding sequences The hybridization probes of the subject invention may be derived from the nucleotide sequences of SEQ ID NO 1 or its complement or from genomic sequences including promoters, enhancer elements and introns of naturally occurring dp Hybridization probes may be labeled by a variety of reporter groups including radionuchdes such as JΔP or J J 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 ohgonucleotides based upon the nucleotide sequences which encode ILP Such probes used in PCR may 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 dp include the cloning of nucleic acid sequences encoding ILP and ILP derivatives into vectors for the production of mRNA probes Such vectors 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 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 dp 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 dp 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 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 dp expression and the presence of disease The nucleotide sequence for tip 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
hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries 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 be found in genome issue of Science ( 1994) 265 1981 Correlation between the location of tip 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 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) G_ene Expression Technology. Methods and Enzvmology. 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 in which p 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 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 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 431 A 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 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 ammo acids, preferably at least 10 ammo acids They should mimic a portion of the am o 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 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 production of synthetic antibodies or other specific-binding molecules such as the screening of recombinant immunoglobuhn 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 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 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 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 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 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" m Toxicokmetics and New Drug Development. Yacobi et al (eds), Pergamon Press, NY, pp 42-96, each of which references are herein mcoφorated by reference m its entirety) An effective amount of ILP or ILP agonist or antagonist to be employed therapeutical ly 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 μg kg/day to 10 mg/kg/day Typically, the 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 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/i munogenicity, these and other characteristics may aid in defining an effective carrier 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 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 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 formulations (such as hposomally 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 μg/kg/day to 10 mg/kg/day, depending upon the route of administration 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 incoφorated 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 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-γ (rhIFN-γ), ιnterleukιn-2, and MN rgpl20 Johnson et al , Nat Med , 2 795-799 (1996), Yasuda, Biomed Ther , 22 1221-1223 (1993), Hora e/ α/ , Bιo Technol 8 755-758 (1990). Cleland, "Design and Production of Single Immunization Vaccines 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-coglycohc acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties The degradation products of PLGA, lactic and glyco c acids, can be cleared quickly within the human body Moreover, the degradabihty 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/glyco de polymer," in M Chasm and R Langer (Eds ). Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker New York. 1990), pp 1-41
There is a need m 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 μg/kg/day in mammals with a maximum body weight of 85 kg, the 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 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 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 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 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 Homology to Insulin Family of Proteins
The nucleic acid sequence of the relaxm molecule, a member of the insulin family of proteins, was 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 reiaxin nucleic acid sequence, and represent sequences within a gene of an insu n-like polypeptide (ILP) The EST corresponding to SEQ ID NO 15 was used to clone the full length ILP gene The full length ILP gene sequence was cloned using ohgonucleotide 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 ohgonucleotide primers were 5'CAC ATT CAG TCC TCA GCA AAA TGA A-3' (IN2328985 f, SEQ ID NO 1 1), 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, 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 is then hgated 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 bacteπophage particles and subsequent infection of £ 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 size of the genome and the size of the cloned fragments (see, Clark and Carbon. Cell 9, 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 deproteimzation 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 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 et al , 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 remove small and large fragments which would interfere with subsequent cloning Methods for size fractionation are w ell known in the art and are typically based on sucrose gradient fractionation or preparative gel electrophoresis The DNA is then hgated 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 library into E coli can be performed by any standard transformation techniques, including CaC^ 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 reverse transcπptase to synthesize complementary cDNA strands from the mRNA template In order to obtain double-stranded DNA suitable for hgation into a vector the dscDNA copy of the mRNA is methylated and equipped with suitable (usually £coRI) 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 ademne or cytosine residues within specific target sequences For example, £coRI methylates an ademne residue within the £coRI recognition sequence In the process of converting mRNA into double stranded cDNA in vitro, a first cDNA strand is synthesized by the reverse transcπptase 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 transcπptase that also has RNase H activity £ 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 molecules from the original poly(A)+ mRNA After converting the 5' and 3' ends into blunt ends, the dscDNA can be hgated to linkers/adaptors and subsequently hgated 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 21, 263-269 (1983), Okayama and Berg, Mol Cell Biol 2, 161-170 (1982). and Kato et al Gene 150, 243-250 ( 1994) Typical protocols for making cDNA libraries are also described in Ausubel et al supra, especialh in Units 5 2 1 , 5 5 2-5 5 7, 5 6 1 -5 6 8. and 5 8 1 -5 8 1 1
An optional method for converting mRNA into dscDNA is disclosed in copending patent application Serial No 08/872.861 filed 15 October 1996 herein incoφorated by reference in its entirety According to this method, reverse transcπptase-producing cells are transformed with vectors in which the 5' end of a mRNA molecule having a 5' ohgonucleotide cap is hgated to a s gle-stranded 5' overhang complementary to the ohgonucleotide cap, and the 3' end of the mRNA molecule is hgated to a single-stranded 3' overhang complementary to the 3' end of the mRNA molecule, so that the reverse transcπptase produced by the cell converts the mRNAs into dscDNAs to form a cDNA library As an alternative, a cDNA library may be prepared such that the library is enriched in signal sequences This library is enriched in am o 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 incoφorated by reference in its entirety 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 Briefly, the method involves transforming non-amylolytic yeast cells with exogenous DNA containing the coding sequence of a randomly selected, unidentified mammalian signal peptide hgated 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 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 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 ohgonucleotide primers are used 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 hgated to an adapter ohgonucleotide 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 hgated into the amylase expression vector described above which has been digested with enzvmes 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 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 1 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 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
Not\ primer-adapter (Life Tech , 2 μl, 0 5 μg/μl) was added to a sterile I 5 ml microcentπfuge tube to which was added poly A+ mRNA (7μl, 5μg) The reaction tube was heated to 70°C 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 μl), 0 1 M DTT (2 μl) and 10 mM dNTP Mix (Life Tech , 1 μl) were added and then heated to 37°C for 2 minutes to equilibrate the temperature SUPERSCRIPT II® reverse transcπptase (Life Tech , 5 μl) was then added, the reaction tube mixed well and incubated at 37°C for 1 hour, and terminated by placement on ice The final concentration of the reactants was the following 50 mM Tπs-HCl (pH 8 3), 75 mM KC1, 3 mM MgCl2, 10 mM DTT, 500 mM each dATP, dCTP, dGTP and dTTP, 50 mg/ml NotI primer-adapter. 5 mg (250 mg/ml) mRNA, 50,000 U/ml SUPERSCRIPT II® reverse transcπptase Second strand synthesis
While on ice, the following reagents were added to the reaction tube from the first strand synthesis, the reaction well mixed and allowed to react at 16°C for 2 hours, taking care not to allow the temperature to go above 16°C distilled water (93 ml) 5X Second strand buffer (30 ml), dNTP mix (3 ml), 10 U/ml E Coli DNA hgase (1 ml), 10 U/ml £ coli DNA polymerase I (4 ml), 2 U/ml £ 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 Tπs-HCl (pH 7 5), 100 mM KC1, 5 mM MgCl2, 10 mM (NH4)2S04, 0 15 mM b-NAD+, 250 mM each dATP, dCTP, dGTP, dTTP 1 2 mM DTT, 65 U/ml DNA hgase, 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 5M NaCl (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 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 SPEEDVAC™ drier 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 16°C for 16 hours distilled water (25 ml), 5X T4 DNA hgase buffer (10 ml), Sail adapters (10 ml), T4 DNA hgase (5 ml) The final composition of the reaction was the following 50 mM Tπs-HCl (pH 7 6), 10 mM MgCl2, 1 mM ATP, 5% (w/v) PEG 8000, 1 mM DTT, 200 mg/ml Sal I adapters. 100 U/ml T4 DNA hgase The reaction was extracted through phenol chloroform isoamyl alcohol (25 24 1 , 50 ml) the aqueous phase removed, collected and diluted into 5M NaCl (8 ml) and absolute ethanol (-20°C, 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 2 minutes at 14,000 x g Subsequently, the supernatant was removed and the resulting pellet dried in a SPEEDVAC™ 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 37°C for 2 hours DEPC -treated water (41 ml), Not 1 restriction buffer (REACT, Life Tech , 5 ml), Not 1 (4 ml) The final composition of this reaction was the following 50 mM Tπs-HCl (pH 8 0). 10 mM MgCl2. 100 mM NaCl, 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 1 kb, as determined by comparison with a molecular weight marker, were 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 m 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) Ligation of cDNA into pRK5B vector
The following reagents were added together and incubated at 16 °C for 16 hours 5X T4 hgase buffer (3 ml), pRK5B, Xhol, Notl 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 aqueous phase was removed, collected and diluted into 5M NaCl (10 ml) and absolute ethanol (-20CC, 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 hgation into bacteria The hgated 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 37CC for 30 minutes The transformants were then plated onto 20 standard 150 mm LB plates containing ampicilhn 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 dp The full length nucleic acid sequence of dp was obtained by screening a plasmid cDNA library
(prepared from uterine mRNA as described in Example 2) by colony hybridization using ohgonucleotides 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 ohgonucleotide sequences are indicated in Fig 1 by overhning or underlining of sense and antisense strands, respectively as 5'- C A C A TTC A G TC C TC A G C A A A A TG A A - 3 ' ( S EQ I D N O 1 1 ) , 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 ammo acid sequences are shown in Fig 1 Example 4 Homology 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 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 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 Dotmatπx homology plots were examined to distinguish regions of significant homology from chance matches
The dp nucleic acid sequence and the ILP amino acid sequence were homologous to but clearly 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 dp Fig 2 shows the comparison of ILP with other insulin and insuhn-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 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 sulin-hke polypeptides The most related of these molecules cluster together at the right hand side of the figure
The msuhn-hke molecules share several characteristics They are each secreted proteins, and each possesses a similar arrangement of six conserved cysteine residues Numerous additional ammo 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 108 (R, R, K, and K respectively)) are likely to have significant impact on function Example 5 ILP polypeptide structure
The mature insulin molecule, like other members of the insulin polypeptide family, is made up of two am o 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 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 α- ammo group of a protein chain with 2,4-dmιtro fluorobenzene (DNFB) to form yellow 2,4-dιnιtrophenyl derivatives, followed by acid hydrolysis and quantitation of the number of terminal ammo 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 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 lntrachain -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 mtrachain -S-S- cross-linkage between positions 6 and 1 1 Such -S-S- cross- linkages may be cleaved by oxidation with performic acid, which converts the two cystine half-residues into 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 1 1 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 mtrachain linkage between residues 13 and 18 of the A chain
Example 6 Use of the ILP Gene Sequence in Antisense Analysis
Knowledge of the correct, complete cDNA sequences of novel expressed genes encoding an msulin- like polypeptide will enable their use in antisense technology in the investigation of gene function Either ohgonucleotides, genomic or cDNA fragments comprising the antisense strand of dp 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 function of the gene can be ascertained by observing behavior at the cellular, tissue or orgamsmal level (e g lethality, loss of differentiated function, changes in moφhology, 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 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 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 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 incoφorated by reference in its entirety Typically, particular cells, such as retina, liver, pancreas, colon, uterus cells would be targeted for ILP transgene incoφoration with tissue- specific enhancers, which could result in altered cellular expression of the ILP Transgenic animals that 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 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 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 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 Cells A Practical Approach, E J Robertson, ed (IRL, Oxford, 1987), pp 1 13-152) 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 m 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 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 dp mutations Example 8 Expression of ILP
Expression of the ILP gene may be accomplished by subcloning the cDNA into an appropriate expression vector, transfectmg this vector into an appropriate expression host cell, and cultuπng the host cell The ILP may be intracellularly expressed or secreted into the peπplasm or culture medium of the host cell In this particular case, the cloning vector previously used for the generation of the tissue library also pro\ ides for direct expression of the included sequence in £ coli Upstream of the cloning site, this vector contains a promoter for β-galactosidase, followed by sequence containing the ammo-terminal Met and the subsequent 7 residues of β-galactosidase Immediately following these eight residues is an engineered bacteπophage promoter useful for artificial priming and transcription and a number of unique restriction sites, including £cøRI. 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 β-galactosidase, about 15 residues of "linker", and the peptide encoded within the cDNA Since cDNA clone inserts are generated by an essentially random process 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 ohgonucleotide linker inclusion The dp cDNA can be shuttled into other vectors known to be useful for expression of protein in specific hosts Ohgonucleotide amp mers containing cloning sites as w ell 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 enzy mes 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 segments with chemically synthesized ohgonucleotides Segments of the coding sequence from more than one gene can be hgated together and cloned in appropriate vectors to maximize expression of recombinant sequence 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 £ 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 marker such as the β-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 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 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 by nucleic acid sequence SEQ ID NO 1 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 Expression of ILP in £ coli
This example illustrates preparation of an unglycosylated form of ILP by recombinant expression in
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 expression vector A variety of expression vectors may be employed An example of a suitable vector is pBR322 (derived from £ coli, see Bolivar et al , Gene, 2 95 (1977)) which contains genes for ampicillin and tetracyclme resistance The vector is digested with restriction enzyme and dephosphorylated The PCR amplified sequences are then hgated into the vector The vector will preferably include sequences which encode for an antibiotic resistance gene, a tφ promoter, a polyhis leader (including, for example, the first six STII codons, polyhis sequence, and enterokmase cleavage site, or a lamB signal (USPN 5,324,820)), the ILP coding region, lambda transcπptional terminator, and an argU gene
The hgation mixture is then used to transform a selected £ coli strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic 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 then grown to a desired optical density, during which the expression promoter is turned on
After cultuπng the cells for several more hours, the cells can be harvested by centπfugation The cell pellet obtained bv the centπfugation 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 medium is processed by cleaving the signal sequence and isolating the protein as described
A method for isolating a recombinant polypeptide expressed in £ 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 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 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 dp DNA (DNA27865) is hgated into pRK5B with selected restriction enzyme sites such as Xbal to allow insertion of the dp DNA using hgation 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) are grown to confluence in tissue culture plates m medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics About 10 μg DNA27865-1091 DNA is mixed with about 1 μg DNA encoding the VA RNA gene (Thimmappaya et al , Cell, 31 543 (1982)) and dissolved in 500 μl of 1 mM Tπs-HCl, 0 1 mM EDTA, 0 227 M CaCl2 To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7 35), 280 mM NaCl, 1 5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25°C The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C The culture medium is aspirated off and 2 ml of 20% glycerol m 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 Approximately 24 hours after the transfections. the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml 35S-cysteme and 200 μO/mr-> S- methiomne 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 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-encod g 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 μg DN A27865- 1091 DNA is added The cells are first concentrated from the spinner flask by centπfugation 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-mtroduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0 1 μg/ml bovine transferπn After about four days, the conditioned media is centrifuged and filtered to remove cells and debris The sample containing expressed 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 ceils are lysed and the lysate is processed to recover the desired ILP
In another embodiment, ILP can be expressed in CHO cells The DN A27865- 1091 can be transfected into CHO cells using known reagents such as CaPO^ or DEAE-dextran As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 3 S-methionme 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 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 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 -chelate affinity chromatography
Expression of ILP in Yeast 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, 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 1 10, can then be transformed with the expression plasmids 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% tπchloroacetic 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 centπfugation and then concentrating the medium using selected cartridge filters 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 immunoglobuhn tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL 1393 (Novagen) Briefly, the ILP or the desired portion of the ILP (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may incoφorate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector Recombinant baculovirus is generated by co-transfectmg the above plasmid and BACULOGOLD M virus DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 171 1) using hpofectin (commercially available from GIBCO-BRL) After 4 - 5 days of incubation at 28°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 Press (1994)
Expressed poly-his tagged ILP can then be purified, for example, by Nr 9+ -chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 ceils 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 MgCl2, 0 1 mM EDTA, 10% glycerol, 0 1% NP-40, 0 4 M KC1), and sonicated twice for 20 seconds on ice The sonicates are cleared by centπfugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7 8) and filtered through a 0 45 μm filter A Nr -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 A2gQ with loading buffer, at which point fraction collection is started Next, the column is washed with a secondary wash buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 6 0), which elutes nonspecifically bound protein After reaching A2gQ baseline again, the column is developed with a 0 to 500 mM lmidazole gradient in the secondary wash buffer One mL fractions are collected and analyzed by SDS-PAGE and silver staining or western blot with Ni -NχA-conjugated to alkaline phosphatase (Qiagen) Fractions containing the eluted Hisj g-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 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 immunoglobuhn, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Coφ , Seattle Wash ) The inclusion of a cleavable linker sequence such as Factor XA or enterokmase (Invitrogen, San Diego Calif ) between the purification domain and the dp 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- 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 rabbit
For identifying mouse hybπdomas, the denatured protein can be radioiodmated and used to screen potential murine B-cell hybπdomas 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 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 Ohgopeptides 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-termmus and those 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 431 A 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 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 w ith antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG Hybπdomas may also be prepared and screened using standard techniques For example, hybπdomas 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 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 hybπdomas 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 hybπdomas are injected into pristine mice to produce ascites, and monoclonal antibody is purified from mouse ascitic fluid by affinity chromatography on Protein A Monoclonal antibodies with affinities of at least 10° M" 1, preferably lθ" M"' to 1010 M"' 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 incoφorated herein by reference, each in its entirety
Example 1 1 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 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 wide variety of labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature Suitable labels include radionuchdes 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 incoφorated by reference in their entirety Also, recombinant immunoglobulins may be produced as shown in U S Pat No 4,816,567, incoφorated 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 activated cell sorting (FACS) A two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-mterfeπng 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 158 121 1 ) Example 12 Purification Of ILP Using Specific Antibodies
Native or recombinant ILP may be purified by a variety of standard techniques m 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 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 immunoglobuhn is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology) The antibody is coupled to the resin, the resm is blocked, and the derivative resin is washed according to the manufacturer s 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 solubihzation of the whole cell or of a subcellular fraction obtained via differential centπfugation by the addition of detergent or bv 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 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 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-b ding molecules may be identified by interaction with radiolabeled ILP Radioactive labels may be incoφorated into ILP by various methods known in the art A preferred embodiment is the labeling of primary ammo groups in ILP with L I 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 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 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 ohgonucleotide probes for cloning the receptor gene
In an alternative method. mRNA is obtained from receptor-bearing cells and made into a cDNA 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 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 chromatography or density gradient centπfugation and are purified by methods known in the art The soluble receptors or binding proteιn(s) are subjected to N-termmal 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-Induced Cellular Response
The biological activity of ILP is measured, for example, by binding of an ILP of the invention to an 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)) m which activation of a receptor tyrosine kinase is monitored by immuno-capture of the activated receptor and quantitation of the level of ligand-mduced phosphorylation The assay may be adapted to monitor ILP-mduced 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 test may either be free m solution, affixed to a solid support, borne on a cell surface or located mtracellularly 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 m 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 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 thereof and assaying (I) 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 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. incoφorated 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, 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 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 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 9: 19-21 , incoφorated herein by reference in its entirety).
In one approach, the three-dimensional structure of the ILP, or of an ILP-inhibitor complex, is 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 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 incoφorated herein by reference in their entirety.
It is also possible to isolate a target-specific antibody, selected by functional assay, as described 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 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 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 incoφorated 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 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)
Material ATCC Pep 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 Puφose 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 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 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 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 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 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:

Claims

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 (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). 5. An isolated nucleic acid comprising DNA encoding an insulin-like polypeptide (ILP) having amino acid residues 1 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 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. 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- 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. 17. The host cell of claim 16 wherein the host cell is selected from the group consisting of a CHO cell, an £. coli cell, and a yeast cell.
18. A host cell comprising the expression vector of claim 1 1.
19. The host cell of claim 18 wherein the host cell is selected from the group consisting of a CHO cell, an £. coli cell, and a yeast cell. 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 £. coli cell, and a yeast cell.
22. A host cell comprising the expression vector of claim 13. 23 The host cell of claim 22 wherein the host cell is selected from the group consisting of a CHO cell, an £ coli cell, and a yeast cell
24 A method for producing an insu n-like polypeptide (ILP), said method comprising a) cultuπng the host cell of claim 14 under conditions suitable for expression of the ILP, and b) recovering the ILP from the culture
25 A method for producing an insu n-like polypeptide (ILP), said method comprising a) cultuπng 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 insu n-like polypeptide (ILP), said method comprising a) cultuπng 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 insulm- ke polypeptide (ILP), said method comprising a) cultuπng the host cell of claim 20 under conditions suitable for expression of the ILP, and b) recovering the ILP from the culture 28 A method for producing an insulm-hke polypeptide (ILP), said method comprising a) cultuπng 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 determming the presence of insulin-like polypeptide (ILP) mRNA in a sample, the method comprismg 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 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 insulm-hke polypeptide (ILP) in a sample, the method comprising a) contacting a detectable anti-ILP antibody with a sample suspected of containing ILP, and 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 comprismg an insulin-like polypeptide (ILP) fused to a heterologous ammo acid sequence
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 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) ofFig. 1 or a fragment thereof.
35. The chimeric molecule of claim 33 wherein the heterologous amino acid sequence is an epitope 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 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
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.
39. The antibody of claim 37 wherein the antibody is a monoclonal antibody.
EP98942285A 1997-09-24 1998-08-28 Insulin-like polypeptide and uses therefor Withdrawn EP1017816A1 (en)

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WO2000047776A2 (en) * 1999-02-12 2000-08-17 Zymogenetics, Inc. Insulin-family homolog localized to chromosome 1
KR20020093053A (en) 2000-04-21 2002-12-12 다케다 야쿠힌 고교 가부시키가이샤 Novel insulin/igf/relaxin family polypeptides and dnas thereof
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CN111269310B (en) * 2020-03-02 2023-03-31 郑州伊美诺生物技术有限公司 C-P tandem expression recombinant protein and gene thereof, preparation method, application and C peptide detection kit

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