AU1829101A - OB protein derivatives having prolonged half-life - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): GENENTECH, INC.

Invention Title: OB PROTEIN DERIVATIVES HAVING PROLONGED HALF-LIFE The following statement is a full description of this invention, including the best method of performing it known to me/us: 1A OB PROTEIN DERIVATIVES Field of the Invention The invention concerns long half-life derivatives of the OB protein. In particular, the invention concerns OB protein-immunoglobulin chimeras, and other long half-life derivatives of the OB protein, and compositions comprising and methods for administering them. The invention further relates to a method for treating obesity by administering a long half-life variant of the OB protein, such as, an OB proteinimmunoglobulin chimera.

Background of the Invention Obesity is the most common nutritional disorder which, according to recent epidemiologic studies, affects about one third of all Americans 20 years of age or older. Kuczmarski et al., J. Am. Med. Assoc. 272, 205-11 (1994). Obesity is responsible for a variety of serious health problems, including cardiovascular disorders, type II diabetes, insulin-resistance,hypertension, hypertriglyceridemia.dyslipoproteinemia,and some forms of cancer. Pi-Sunyer, Anns. Int. Med. 119, 655-60 (1993); Colfitz, Am. J. Clin. Nutr. 503S-507S(1992). A single-gene mutation (the obesity or "ob" mutation) has been shown to result in obesity and type 11 diabetes in mice. Friedman, Genomics 1, 1054-1062 (1991). Zhang et al., Nature 372, 425-431 (1994) have recently reported the cloning and sequencingof the mouse ob gene and its human homologue, and suggested that the ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot. Parabiosis experiments perfonned more than 20 years ago predicted that the geneticallyobese mouse containing two mutant copies of the ob gene (ob/ob mouse) does not produce 20 a satiety factor which regulates its food intake, while the diabetic (db/db) mouse produces but does not respond to a satiety factor. Coleman and Hummal, Am. J. Physiol. 217, 1298-1304(1969); Coleman, Diabetol 9, 294-98 (1973). Recent reports by three independent research teams have demonstrated that daily injections of •recombinantOB protein inhibit food intake and reduce body weight and fat in grossly obese ob/ob mice but not in db/db mice (Pelleymounter et al.. Science 269, 540-43 [1995]; Halaas etal., Science 269, 543-46 [1995]; 25 Campfield el al., Science 269, 546-49 [1995]), suggesting that the ob protein is such a satiety factor as proposed in early cross-circulationstudies. The results of these first studies leave many questions unanswered, and show a number of as yet unresolved discrepancies. For example, while modest effects of daily injections of the ob protein on food intake and body weight were reported in lean mice, there was a significant reduction in body fat as assessed by carcass composition in one (Halaas et al., supra) but not in another (Pelleymounter et al..

supra) of these reports, despite equivalentdecreases in body weight. Furthermore, Pelleymounter et al., supra observed that, for reasons unknown, ob/ob mice treated with a 0.1 mg/kg/day dose of the OB protein actually increased their body weight by 17.13 while the weight reduction in the obese mice that received a I mg/kg/day dose ofob was rather moderate. The receptor or receptors of the ob protein are as of yet unidentified.

While the existence of peripheral receptors cannot be ruled out at this time, the recent report that an increased 2 expression of the ob gene in adipose tissue of mice with hypothalamic lesions does not result in a lean phenotype suggests that the OB protein does not act directly on fat cells. Maffei et al., Proc. Natl. Acad. Sci. 92, 6957-60 (1995). Researchers suggest that at least one OB receptor is localised in the brain. The identification and expression cloning of a leptin receptor (OB-R) was reported by Tartaglia et al., Cell 83, 1263-71 (1995). Various isoforms of a leptin receptor are described by Cioffi et al., Nature 2, 585-89 (1996). A human hematopoetin receptor, which might be a receptor of the OB protein, is described in PCT application Publication No. WO 96/08510, published 21 March 1996. A receptor of the OB protein is disclosed in Tartaglia et al., Cell 83, 1263-71 (1995).

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Summary of the Invention The present invention is based on the observation that the OB protein is significantly more effective at S" reducing body weight and adipose tissue weight when delivered as a continuous subcutaneous infusion than when the same dose is delivered as a daily subcutaneous injection. The invention is further based on the unexpected finding that a chimeric protein, in which the OB polypeptide is fused to an immunoglobulin constant domain, is strikingly more potent in reducing the body weight and 30 adipose depots than native human OB, when both proteins are administered by subcutaneous injection once a day. The latter observation is particularly surprising, since the OB protein-immunoglobulin chimera due to its large molecular weight, is not expected to be able to cross the blood-brain barrier, and reach the OB receptor which has been believed to be located in the brain.

H:\cintae\Keep\speci\15200.97.doc 5/02/01 2a In one aspect, the invention concerns long halflife derivatives of an OB protein capable of reducing body weight and/or food intake in an individual treated. The invention further concerns compositions containing such derivatives, and their administration for reducing body weight and/or food intake.

In another aspect, the invention concerns chimeric polypeptides comprising an OB protein amino acid sequence capable of binding to a native OB receptor linked to an immunoglobulin sequence (briefly referred to as OBimmunoglobulin chimeras or immunoadhesins). In a specific embodiment, the chimeric polypeptides comprise a fusion of an OB amino acid sequence capable of binding a native OB receptor, to an immunoglobulin constant domain sequence.

The OB portion of the chimeras of the present invention preferably has sufficient amino acid sequences from a native OB protein to retain the ability to bind to and signal through a native OB receptor. Most preferably, the OB protein retains the ability to reduce body weight when administered to obese human or non-human subjects. The OB polypeptide is preferably human, and the fusion is preferably with an immunoglobulin heavy chain constant domain sequence. In a particular embodiment, the S* 25 association of two OB polypeptide-immunoglobulin heavy chain fusions via covalent linkage by disulfide S•bond(s)) results in a homodimeric immunoglobulin-like structure. An immunoglobulin light chain may further be associated with one or both of the OB-immunoglobulin 30 chimeras in the disulfide-bonded dimer to yield a homotrimeric or homotetrameric structure.

The invention further concerns nucleic acid encoding chimeric polypeptide chains of the present invention, expression vectors containing DNA encoding such molecules, transformed host cells, and methods for the production of the molecules by cultivating transformant host cells.

H:\cintae\Keep\speci\15200.97.doc 5/02/01 3 Although the long half-life derivatives of the present invention are particularly useful for reducing body weight and/or food intake, they can generally be used for the treatment of conditions associated with the abnormal expression or function of the OB gene and/or to elicit biological responses mediated by an OB receptor. Thus, the OB derivatives of the present invention may be used to treat bulimia, to reduce insulin levels, e.g. in Type I or II diabetic patients, and as mitogens of various cell types expressing an OB receptor. All these and related uses are within the scope of the present invention.

In another embodiment, the invention concerns the purification of an OB receptor by using an OB proteinimmunoglobulin chimera.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

Brief Description of the Figures S• Figure 1 top Lean female mice were treated with murine OB protein either as a continuous subcutaneous infusion or daily subcutaneous injections. The data shown are the mean body weight of each group, in grams, n 4 25 mice/point.

Figure 1 bottom The mean weight of the retroperitoneal fat pads are shown. Continuous subcutaneous infusions of the OB protein were also more effective than daily subcutaneous injections at reducing adipose tissue 30 weight.

Figure 2 top Obese female ob/ob mice were treated with human OB protein (hOB) or with a human OB-IgG-1 fusion protein (hOB-IgG-l). The data shown are the mean change in body weight for each treatment group from the first to the last day of the experiment, in grams, n 3 mice/bar except for the hOB 0.19 mg/kg/day by injection group, where n 4, and PBS injection group, where n 1.

:\Ema\Keep\Speci\15200.9 7 .doc 18/04/00 3a Figure 2 bottom The data shown were the mean food intake for each treatment group for the six 24 hour .periods of the experiment, in grams/mouse/day, n I/bar.

Figure 3 top and bottom Obese (ob/ob) female mice were treated with either hOB or the hOB-IgG-l fusion protein by daily subcutaneous injections for 7 days. The data are depicted as in Figure 2, with n 4 for all treatment groups.

Figure 4 top Obese female ob/ob mice were treated with human protein (hOB) or with PEG-hOB. The data shown are the mean change in body weight for each treatment group from the first to the least day of experiment, in grams, n 3-4 mice/bar except for the PBS injection group, where n 1. The materials were injected daily subcutaneously. The "PEG IX" and "PEG 2X" refer to the ratio of the PEG reagent to protein in the preparation of the molecule.

Figure 4 bottom The data shown were the mean food intake for each treatment group for the six 24 hour periods of the experiment, in grams/mouse/day, n 3-4/bar.

Figure 5 Obese (ob/ob) female mice were treated with either the hOB-IgG fusion protein, native hOB, or hCD4-IgG by daily subcutaneous injections for 7 days. n 6 for all treatment groups, except hOB at 3.8mg/kg/d, where 25 n 2. Again it was observed that the fusion protein was more effective than the native hOB protein at reducing body weight (top and middle panels) and food intake (bottom panel).

Figure 6 The nucleotide sequence (SEQ ID NO:1) and the amino acid sequence (SEQ ID NO:2) of the human OB-IgG-1 chimera of Example 1.

H:\E mma\Kep\Specis\15200 .97.doc 18/04/00 Detailed Description of the Invention A. Definitions The term "obesity" is used to designate a condition of being overweight associated with excessive bodily fat. The desirable weight for a certain individual depends on a number of factors including sex, height.

age, overall built, etc. The same factors will determine when an individual is considered obese. The determination of an optimum body weight for a given individual is well within the skill of an ordinary physician.

The phrase "long half-life" and grammatical variants thereof, as used in connection with OB derivatives, concerns OB derivatives having a longer plasma half-life and/or slower clearance than a corresponding native OB protein. The long half-life derivatives preferably will have a half-life at least about longer than a native OB protein; more preferably at least about 2-times longer than a native OB protein, more preferablyat least about 3-time longer than a native OB protein. The native OB protein preferably is that of the individual to be treated.

The terms "OB polypeptide" "OB protein" and their grammatical variants are used interchangeably and refer to "native" or "native sequence" OB proteins (also known as "leptins") and their functional derivatives. The OB polypeptides have the typical structural features of cytokines, i.e. polypeptides released by one cell population which act on another cell as intercellularmediators such as, for example, growth hormones, insulin-likegrowth factors, interleukins, insulin, glycoprotein hormones such as, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), tumor necrosis factor-a and -P (TNF-a and nerve growth factors, such as NGF-P, PDGF, transforminggrowth factors (TGFs) such as. TGF-a and TGF-P. insulinlike growth factor-1 and -2 (IGF-I and IGF-2), erythropoietin, osteoinductive factors, interferons (IFNs) such as, IFN-a, IFN-P and IFN-y, colony stimulating factors (CSFs) such as, M-CSF, GM-CSF, and G-CSF, interleukins (ILs) such as, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 and other polypeptide factors.

The terms "native" and "native sequence" OB polypeptide are used to refer to an OB polypeptide from 25 any animal species human, murine, rabbit, cat, cow, sheep, chicken, porcine, equine, etc.), as occurring in nature, including naturally-occurringalleles, deletion, substitution and/or insertion variants, as currently known or as might be identified in the future, provided that they retain the ability to bind to and, preferably, signal through the OB receptor. Thus, a native human OB polypeptide includes the amino acid sequence between the •N-terminusand the cysteine (Cys) at position 167 of the amino acid sequence shown in Figure 6 (see also SEQ.

30 ID. NO: 2 and Figure 6 of Zhang et al., supra), and naturally occurring variants of this protein, as currently known or might be identified in the future. Similarly, a "native" or "native sequence" murine OB polypeptide has the amino acid sequence shown in Figure 6 of Zhang et supra, and naturally occurring variants of that polypeptide, as currently known or might be identified in the future. The definition specifically includes variants with or without a glutamine at amino acid position 49, using the amino acid numbering of Zhang et a., 35 supra. The terms "native" and "native sequence" OB polypeptide include the native proteins with or without the initiatingN-terminal methionine (Met), and with or without the native signal sequence, either in monomeric or in dimeric form. The native human and murine OB polypeptidesknown in the art are 167 amino acids long, contain two conserved cysteines, and have the features of a secreted protein. The polypeptide is largely hydrophilic,and the predicted signal sequence cleavage site is at position 21, using the amino acid numbering of Zhang ci al., s ra. The overall sequence homology of the human and murine sequences is about 84%. The two proteins show a more extensive identity in the N-terminal region of the mature protein, with only four conservativeand three non-conservativesubstitutionsamong the residues between the signal sequence cleavage site and the conserved Cys at position 117. The molecular weight of OB proteins is about 16 kD in a monomeric form.

A "functionalderivative"ofa native polypeptide is a compound having a qualitativebiological property in common with the native polypeptide. A functionalderivative of an OB polypeptide is a compound that has a qualitative biological property in common with a native (human or non-human)OB polypeptide. "Functional derivatives" include, but are not limited to, fragmentsof native polypeptides from any animal species (including humans), and derivativesofnative (human and non-human) polypeptidesand their fragments, provided that they have a biological activity in common with a corresponding native polypeptide.

"Fragments" comprise regions within the sequence of a mature native OB polypeptide. Preferred fragments of OB polypeptides include the C-terminus of the mature protein, and may contain relatively short deletion(s) at the N-terminus and in other parts of the molecule not required for receptor binding and/or for structural integrity.

The term "derivative" is used to define amino acid sequence variants, and covalent modifications of a native polypeptide, whereas the term "variant" refers to amino acid sequence variants within this definition.

"Biological property" in the context of the definition of "functional derivatives" is defined as either I immunologicalcross-reactivity with at least one epitope of a native polypeptide a native OB polypeptide of any species), or 2) the possession of at least one adhesive, regulatory or effector function qualitatively in common with a native polypeptide.

Preferably, the functional derivatives are polypeptides which have at least about 65% amino acid sequence identity, more preferably about 75% amino acid sequence identity, even more preferably at least about 25 85% amino acid sequence identity, most preferably at least about 95% amino acid sequence identity with a onative polypeptide. In the context of the present invention, functional derivatives of native sequence human OB polypeptides preferably show at least 95% amino acid sequence identity with the native OB proteins, and are not immunogenic in the human.

Amino acid sequence identity or homology is defined herein as the percentage of amino acid residues S° 30 in the candidate sequence that are identical with the residues of a corresponding native polypeptide sequence, after aligning the sequences and introducinggaps, 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.

Immunologically cross-reactive as used herein means that the candidate (poly)peptide is capable of 35 competitively inhibiting the qualitative biological activity of a corresponding native polypeptide having this activity with polyclonal antibodies or antisera raised against the known active molecule. Such antibodies and antisera are prepared in conventional fashion by injecting an animal such as a goat or rabbit, for example, subcutaneously with the known native OB protein in complete Freud's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freud's.

The term "isolated OB polypeptide" and grammatical variants thereof refer to OB polypeptides (as hereinabovedefined) separated from contaminant polypeptides present in the human, other animal species, or in other source from which the polypeptide is isolated.

In general, the term "amino acid sequence variant" refers to molecules with some differences in their amino acid sequences as compared to a reference native sequence) polypeptide. The amino acid alterations may be substitutions,insertions, deletions or any desired combinations of such changes in a native amino acid sequence.

Substitutional variants are those that have at least one amino acid residue in a native sequence removed and a differentamino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

Insertional variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native amino acid sequence. Immediately adjacent to an amino acid means connected to either the a-carboxy or a-amino functional group of the amino acid.

Deletional variants are those with one or more amino acids in the native amino acid sequence removed.

Ordinarily,deletionalvariantswill have one or two amino acids deleted in a particular region of the molecule.

"Covalent derivatives" include modifications of a native polypeptide or a fragment thereof with an organic proteinaceousor non-proteinaceousderivatizing agent, and post-translational modifications. Covalent modificationsare traditionally introduced by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected sites or terminal residues, or by harnessing mechanisms of posttranslational 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 25 and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and *aspartyl residues. Alteratively,these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the OB-immunoglobulin chimeras of the present invention. Other posttranslational modifications include hydroxylationofproline and lysine, phosphorylation of hydroxyl groups of seryl, tyrosine or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side S 30 chains Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman Co., San Francisco, pp.

79-86 (1983)].

The terms "DNA sequence encoding", "DNA encoding"and "nucleic acid encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide chain. The DNA sequence thus 35 codes for the amino acid sequence.

The terms "replicable expression vector" and "expression 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. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independentlyof 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 moleculesof 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 ribosome binding site, and possibly, other as yet poorly understood 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 preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally.

"operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.

In the context of the present invention the expressions "cell", "cell line", and "cell culture" are used interchangeably,and all such designationsinclude progeny. Thus. the words "transformants" and "transformed (host) cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. 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 activity as screened for in 25 the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

Native immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy 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 30 chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one and (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues 35 are believed to form an interface between the light and heavy chain variable domains (Clothia et al., J. Mol.

Biol. 186, 651-663 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82, 4592-4596 [1985]).

Depending on the amino acid sequence of the constant region 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 furtherdivided into subclasses(isotypes).e.g. IgG- I. IgG-2, IgG-3. and IgG-4; IgA- and IgA-2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called a, delta, epsilon. y. and p, respectively.The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgA-l and IgA-2 are monomeric subclasses of IgA, which usually is in the form ofdimers or larger polymers. Immunocytes in the gut produce mainly polymeric IgA (also referred to poly-IgA including dimers and higher polymers). Such poly-lgA contains a disulfidelinked polypeptide called the "joining" or chain, and can be transported through the glandular epithelium together with the J-containing polymeric IgM (poly-lgM), comprising five subunits.

Hybridization is preferably performed under "stringent conditions" which means employing low ionic strength and high temperature for washing, for example, 0.015 sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50 0 C, or employing during hybridization a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.I% sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 0 C. Another example is use of 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate).

50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 pg/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.

B. OB protein-immunoglobulin chimeras (immunoadhesins) Immunoadhesins are chimeric antibody-like molecules that combine the functional domain(s) of a binding protein (usually a receptor, a cell-adhesion molecule or a ligand) with the an immunoglobulin sequence.

The most common example of this type of fusion protein combines the hinge and Fc regions of an gos., immunoglobulin (Ig) with domains of a cell-surface receptor that recognizes a specific ligand. This type of molecule is called an "immunoadhesin", because it combines "immune" and "adhesion" functions; other frequently used names are "Ig-chimera", or "Fc-fusion protein", or "receptor-globulin." o 25 To date, more than fifty immunoadhesins have been reported in the art. 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 I [1989]; Zettmeisslet DNA Cell Biol. USA 9, 347-353 [1990]; Bym et al., Nature 344, 667-670 [1990]); Lselectin (homing receptor) (Watson et al., J. Cell. Biol. 110, 2221-2229 [1990]; Watson et al., Nature 349, 164- 167 [1991]); E-selectin [Mulligan et a, J. Immunol. 151, 6410-17 [1993]; Jacob etal., Biochemistry 34, 1210- 1217 [1995]); P-selectin(Mulliganet al., supra; Hollenbaughet al., Biochemistry 34, 5678-84 [1995]); ICAM- *ge (Stauton et al., J. Exp. Med. 176, 1471-1476 [1992]; Martin et al., J. Virol. 67, 3561-68 [1993]; Roep et al, Lancet 343, 1590-93 [1994]); ICAM-2 (Damle et al., J. Immunol. 148, 665-71 [1992]); ICAM-3 (Holness et al., J. Biol. Chem. 270, 877-84 [1995]); LFA-3 (Kanner et al., J. Immunol. 148, 2-23-29 [1992]); LI 35 glycoprotein(Dohertyet al., Neuron 14, 57-66 [1995]); TNF-RI (Ashkenazi et al. Proc. Natl. Acad. Sci. USA 88, 10535-539[1991]; Lesslauer etal., Eur. J. Immunol. 21, 2883-86 [1991]; Peppel et al., 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 et al., J. Exp. Med. 173. 721-730 (1991)]; CTLA-4 [Lisley et al., J. Exp. Med. 174, 561-569 (1991)]; CD22 [Stamenkovic et al, Cell 66. 1 133- 144 (1991)]: NP receptors [Bennett et al.. J. Biol. Chem. 266. 23060-23067 (1991)]; IgE receptor a [Ridgway and Gorman. J. Cell. Biol. I 15. abstr. 1448 (1991)]; HGF receptor [Mark.

M.R. et 1992, J. Biol. Chem. submitted]; IFN-yR a- and P-chain [Marsters et al., Proc. Natl. Acad. Sci.

USA 92, 5401-05 [1995]); trk-A, and -C (Shelton et al., J. Neurosci. 5, 477-91 [1995]); IL-2 (Landolfi.

J. Immunol. 146, 915-19 [1991]); IL-10(Zheng e al., J. Immunol. 154. 5590-5600 [1995]).

The simplest and most straightforward immunoadhesin design combines the binding region(s) of the 'adhesin' protein with the hinge and Fc regions of an immunoglobulinheavy chain. Ordinarily, when preparing the OB-immunoglobulinchimeras of the present invention, nucleic acid encoding the desired OB polypeptide will be fused C-terminally to nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence, however N-terminal fusions are also possible. Typically, in such fusions the encoded chimeric polypeptide will retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminusof 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 OB-immunoglobulin chimeras.

In a preferredembodiment, the sequence of a native, mature OB polypeptide, is fused to the N-terminus of the C-terminal portion of an antibody (in particular the Fc domain), containing the effector functions of an immunoglobulin, e.g. IgG-1. It is possible to fuse the entire heavy chain constant region to the OB 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 I14 [Kobet et al., supra], or analogous sites of other immunoglobulins) is used in the fusion. In a particularly preferred embodiment, the OB polypeptide sequence is fused to the hinge region and CH2 and C-H3 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 OB-immunoglobulinchimeras are assembled as multimers, and particularly as homo-dimersor -tetramers(WO 91/08298). Generally, these assembled immunoglobulins will have known 30 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 molecularweight immunoglobulins;IgM generallyexists as a pentamer of basic four units held togetherby 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 OB-immunoglobulinchimeras within the scope herein are schematically diagrammed below:

ACL-ACL;

ACH-[ACH, ACL-ACH, ACL-VICH, or VLCL-ACH]; ACL-ACH-[ACL-ACH, ACL-VHCH, VLCL-ACI, or VLCL-VHCH]; ACL-VI(,C1-[ACII, or AC 1 -VIC, or ViCi-ACij]; VLC -AC 1 -[AC -V 1 Cj, or VLCL -ACH]; and CL-VIiC 2 wherein each A represents identical or different OB polypeptide amino acid sequences; VL is an immunoglobulin light chain variable domain;

V

H

i is an immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH. is an immunoglobulin heavy chain constant domain; n is an integer greater than I; Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show key features; they do not indicate joining or other domains of the immunoglobulins, nor are disulfide bonds shown. However, where such domains are required for binding activity, they shall be constructed as being present in the ordinary locations which they occupy in the immunoglobulin molecules.

Alternatively,the OB amino acid sequencescan be inserted between immunoglobulinheavy chain and light chain sequences such that an immunoglobulin comprising a chimeric heavy chain is obtained. In this embodiment, the OB polypeptide sequences are fused to the 3' end of an immunoglobulin heavy chain in each arm of an immunoglobulin, either between the hinge and the CH2 domain, or between the CH2 and CH3 domains. Similarconstructs have been reported by Hoogenboom, H. R. et Mol. Immunol. 28, 1027-1037 (1991).

Although the presence of an immunoglobulinlight chain is not required in the immunoadhesins of the present invention, an immunoglobulin light chain might be present either covalently associated to an OB protein-immunoglobulin heavy chain fusion polypeptide, or directly fused to the OB polypeptide. In the 25 former case, DNA encoding an immunoglobulin light chain is typically coexpressed with the DNA encoding the OB-immunoglobulinheavy chain fusion protein. Upon secretion, the hybrid heavy chain and the light chain will be covalently associated to provide an immunoglobulin-like structure comprising two disulfide-linked immunoglobulinheavy chain-light chain pairs. Method suitable for the preparation of such structures are, for example, disclosed in U.S. Patent No. 4,816,567 issued 28 March 1989.

30 In a preferred embodiment, the immunoglobulin sequences used in the construction of the immunoadhesinsof the present invention are from an IgG immunoglobulin heavy chain constant domain. For human immunoadhesins,the use of human IgG-1 and IgG-3 immunoglobulin sequences is preferred. A major advantage of using IgG-1 is that IgG-1 immunoadhesins can be purified efficiently on immobilized protein A.

In contrast, purification of IgG-3 requires protein G, a significantly less versatile medium. However, other structural and functional properties of immunoglobulins should be considered when choosing the Ig fusion partner for a particular immunoadhesin construction. For example, the IgG-3 hinge is longer and more flexible, so it can accommodate larger 'adhesin' domains that may not fold or function properly when fused to IgG-I.

Possible IgG-based immunoadhesin structures are shown in Fig. 3a-c. While IgG immunoadhesinsare typically mono- or bivalent, other Ig subtypes like IgA and IgM may give rise to dimeric or pentameric structures, respectively,of the basic Ig homodimer unit. A typical IgM-based multimeric immunoadhesin is illustrated in Figure 3d. Multimeric immunoadhesins are advantageous in that they can bind their respective targets with greater avidity than their IgG-based counterparts. Reported examples of such structures are CD4-lgM (Traunecker et spra); ICAM-IgM (Martin et J. Virol. 67, 3561-68 [1993]); and CD2-IgM (Arulanandam et al., J. Exp. Med. 177, 1439-50 [1993]).

For OB-lg immunoadhesins,which are designed for in vivo application,the pharmacokineticproperties and the effector functions specified by the Fc region are important as well. Although IgG- I, lgG-2 and IgG-4 all have in vivo half-livesof2 I 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- I.

Moreover, unlike IgG-I, IgG-2 does not bind to Fc receptors on mononuclearcells 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 immunoadhesinsdesigned 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-I has only four serologically-defined allotypic sites, two of which (G I m and 2) are located in the Fc region; and one of these sites G I m I, is non-immunogenic. In contrast, there are 12 serologically-defined allotypes in IgG-3. all of which are in the Fc region; only three of these sites I and 21)have one allotype which is nonimmunogenic. Thus, the potential immunogenicity of a y3 immunoadhesin is greater than that of a y I immunoadhesin.

In designing the OB-lg immunoadhesins of the present invention regions that are not required for receptor binding, the structural integrity proper folding) and/or biological activity of the molecule, may be deleted. In such structures, it is important to place the fusion junction at residues that are located between domains, to avoid misfolding. With respect to the parental immunoglobulin, a useful joining point is just upstream of the cysteines of the hinge that form the disulfide bonds between the two heavy chains. In a frequently used design, the codon for the C-terminal residue of the "adhesin" (OB) part of the molecule is placed directly upstream of the codons for the sequence DKTHTCPPCP of the IgG hinge region.

OB-Ig immunoadhesinsare most conveniently constructed by fusing the cDNA sequence encoding the OB portion in-frame to an Ig cDNA sequence. However, fusion to genomic Ig fragments can also be used (see, e.g. Gascoigne et al., Proc. Natl. Acad. Sci. USA 84, 2936-2940 [1987]; Aruffo et al., Cell 61, 1303-1313 30 [1990]; Stamenkovic et al., Cell 66, 1133-1144 [1991]). The latter type of fusion requires the presence of Ig oregulatory sequences for expression. cDNAs encoding IgG heavy-chain 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. Murine OB cDNA can, for example, be obtained by PCR from a mouse adipose tissue cDNA library (Clontech), using primers designed based on the sequence of Zhang et al. Human OB cDNA can be obtained in a similar manner. Alternatively, the mouse OB gene can be used as a probe to isolate human adipose tissue cDNA clones (Clontech), e.g. from a .gtll library, as described by Zhang et al. The cDNAs encoding the 'adhesin' and the Ig parts of the immunoadhesin are inserted in tandem into a plasmid vector that directs efficient expression in the chosen host cells. For

-II-

expression in mammalian cells pRK5-based vectors (Schall ec al. Cell 61. 361-370 [1990]), pRK7-vectors and CDM8-based vectors (Seed, Nature 329. 840 [1989])are preferred. (pRK7 is identical to pRK5 except that the order of the endonuclease restriction sites in the polylinker region between Clal and Hindlll is reversed. See U.S. Patent No. 5,108,901 issued 28 April 1992.). The exact junction can be created by removing the extra sequences between the designedjunction codons using oligonucleotide-directed deletional mutagenesis (Zoller and Smith, Nucleic Acids Res. 10. 6487 [1982]; Capon et al., Nature 337. 525-531 [1989]). Synthetic oligonucleotidescan be used, in which each half is complementaryto the sequence on either side of the desired junction; ideally, these are 36 to 48-mers. Alternatively, PCR technique can be used to join the two parts of the molecule in-frame with an appropriate vector.

Immunoadhesinscan be expressed efficiently in a variety of host cells, including myeloma cell lines, Chinese Hamsterovary (CHO) cells, monkey COS cells, human embryonic kidney 293 cells, and baculovirus infected insect cells. In these systems, the immunoadhesin polypeptidesare assembled and secreted into the cell culture medium. Yeasts, e.g. Saccharomvces cerevisiae. Pichia pastoris, etc., and bacterial cells, preferably E.

coli, can also be used as hosts. The OB-immunoglobulin chimeras can be expressed in yeast, for example, similarly to the process described for the expression of the OB proteins by Leiber et Crit. Res. Food Sci.

Nutr. 33 351 (1993); Friedman and Leibel, Cell 69, 217 (1992); and Beavis and Chait, Proc. Natl. Acad. Sci.

USA 87, 6873 (1990). Thus, the coding sequences can be subcloned into a yeast plasmid, such as the yeast expression plasmid pPIC.9 (Invitrogen). This vector directs secretion ofheterologous proteins from the yeast into the culture media. According to Halaas et al., supra, expression of mouse and human OB genes in Saccharomvces cerevisiae transformedwith this vector yields a secreted 16-kD protein, which is an unprocessed OB protein lacking the signal sequence. Expression of the mouse or human OB-immunoglobulin chimeras in E. coli can. for example, be performed on the analogy of the procedure described by Halaas et al., supra. The o coding sequences of mouse and human OB-immunoglobulin chimeras can be subcloned into the expression vector (Novagen) and expressed in E. coli (BL21 (DE3)plYsS) through use of the T7 E. coli RNA 25 polymerase system. Alternatively, the fusion protein can be expressed in E. coli by inserting the coding sequence in frame with the secretion sequence of the E. coli heat stable enterotoxin II, downstream of the E. coli alkaline phosphatase promoter (Chang e al., Gene 55, 189-96 [1987]).

The choice of host cell line for the expression of OB-lg immunoadhesins depends mainly on the expression vector. Another consideration is the amount of protein that is required. Milligram quantities often 30 can be produced by transient transfections. For example, the adenovirus EIA-transformed 293 human embryonic kidney cell line can be transfectedtransiently with pRK5- and pRK7-based vectors by a modification of the calcium phosphate method to allow efficient immunoadhesin expression. This method is illustrated in the examples. CDM8-based vectors can be used to transfect COS cells by the DEAE-dextran method (Aruffo et al, Cell 61, 1303-1313 (1990); Zettmeisslet al., DNA Cell Biol. (US) 9, 347-353 (1990)]. If larger amounts of protein are desired, the immunoadhesin can be expressed after stable transfection of a host cell line. For example, a pRK5- or pRK7-based vector can be introduced into Chinese hamster ovary (CHO) cells in the presence of an additional plasmid encoding dihydrofolate reductase (DHFR) and conferring resistance to G418.

Clones resistant to G418 can be selected in culture; these clones are grown in the presence of increasing levels of DHFR inhibitormethotrexate;clones are selected, in which the number of gene copies encoding the DHFR and immunoadhesinsequences is co-amplified. If the immunoadhesincontains a hydrophobic leader sequence at its N-terminus, it is likely to be processed and secreted by the transfected cells. The expression of immunoadhesins with more complex structures may require uniquely suited host cells; for example, components such as light chain or J chain may be provided by certain myeloma or hybridoma cell hosts [Gascoigne et al., 1987, supra; Martin et at., J. Virol. 67. 3561-3568 (1993)].

The expression of immunoadhesins with more complex oligomeric structures may require uniquely suited host cells; for example, components such as light chain or J chain may be provided by certain myeloma or hybridoma cell hosts (Gascoigne et al., supra; Martin el al., J. Immunol. 67, 3561-68 [1993]).

Immunoadhesinscan be conveniently purified by affinity chromatography. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain that is used in the chimera. Protein A can be used to purify immunoadhesinsthat are based on human y 1, y2, or y4 heavy chains [Lindmark et al., J. Immunol. Meth. 62, 1-13 (1983)]. Protein G is recommended for all mouse isotypes and for human y3 [Gusset al., EMBO J. 5, 15671575 (1986)]. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. The conditions for binding an immunoadhesinto the protein A or G affinity column are dictated entirely by the characteristics of the Fc domain; that is, its species and isotype. Generally, when the proper ligand is chosen, efficient binding occurs directly from unconditioned culture fluid. One distinguishing feature of immunoadhesins is that, for human y I molecules, the binding capacity for protein A is somewhat diminished relative to an antibody of the same Fc type. Bound immunoadhesin can be efficiently eluted either at acidic pH (at or above or in a neutral pH buffer containing a mildly chaotropic salt. This affinity chromatography step can result in an immunoadhesin preparation that is >95% pure.

Other methods known in the art can be used in place of, or in addition to, affinity chromatography on 25 protein A or G to purify immunoadhesins. Immunoadhesins behave similarly to antibodies in thiophilic gel chromatography [Hutchens and Porath, Anal. Biochem. 159, 217-226 (1986)] and immobilized metal chelate chromatography [AI-Mashikhi and Makai, J. Dairy Sci. 71, 1756-1763 (1988)]. In contrast to antibodies, however, their behavior on ion exchange columns is dictated not only by their isoelectric points, but also by a charge dipole that may exist in the molecules due to their chimeric nature. Microheterogeneity of charge can 30 also be a factor for immunoadhesinsin which the adhesin portion of the molecule is glycosylated and contains sialic acid. A specific purification protocol is described in the examples.

Results with the numerous immunoadhesinsproduced so far show that the fusion of the adhesin portion to an Fc region usually does not perturb the folding of the individual domains. Both the adhesin and the immunoglobulinregions appear to fold correctly, and the Fc portion retins many of the effector functions that are characteristic of antibodies, such as binding to Fc receptors.

Methods generally applicable for the construction, expression and purification of immunoadhesins are described, for example, in U.S. Patent Nos. 5,225,538 (issued 6 July 1993) and 5,455,165 (issued 30 October 1995), the disclosures of which are hereby expressly incorporated by reference. Immunoadhesin construction, expression, purification and various immunoadhesins designs are also described in the review articles by Ashkenazi and Chamow, Methods in Enzymology 8, 104-I 15 (1995), and Peach and Linsley. Methods in Enzvmologv 8, 116-123 (1995), the disclosures of which, along with the references cited therein, is hereby expressly incorporated by reference.

C. Other long half-life OB derivatives Other derivatives of the OB proteins, which possess a longer half-life than the native molecules comprise the OB protein or an OB-immunoglobulinchimera, covalently bonded to a nonproteinaceouspolymer.

The nonproteinaceouspolymer ordinarily is a hydrophilic synthetic polymer, 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 polyethyleneglycol (PEG); polyelkylenessuch 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-glucuronicacid, sialic acid, D-galacturonicacid, D-mannuronic acid polymannuronicacid, 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. hyaluronicacid; 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 o 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 crosslinking, 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 30 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 OB protein or to the OB-immunoglobulin chimeras though a multifunctionalcrosslinkingagent which reacts with the polymer and one or more amino acid or sugar residuesof the OB protein or OB-immunoglobulinchimera 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 via versa.

The covalent crosslinking site on the OB protein or OB-lg includes the N-terminal amino group and epsilon amino groups found on lysine residues, as well as other amino. imino. carboxvl, sulflydryl. hydroxyl or other hydrophilic groups. The polymer may be covalentlv 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 of4-hydroxybenzaldehyde, succinimidyl active esters, activated dithiocarbonate PEG, 2,4,5-trichlorophenylclorofonnate or P-nitrophenylcloroformate activated PEG.) Carboxyl groups are derivatized by coupling PEG-amine using carbodiimide.

Polymers are conjugated to oligosaccharide groups by oxidation using chemicals, e.g. metaperiodate, or enzymes, e.g. glucose or galactose oxidase, (either of which produces the aldehyde derivative of the carbohydrate), followed by reaction with hydrazide or amino derivatized polymers, in the same fashion as is describedby Heitzmanneta/., 71, 3537-41 (1974) or Bayer el al., Methods in Enzvmologv 62, 310 (1979), for the labeling of oligosaccharideswith 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 substitutionsthan amino acid sites for derivatization,and the oligosaccharide products thus will be more homogenous. The oligosaccharide substituents also are optionally modified by enzyme digestion to remove sugars, e.g. by neuraminidase digestion, prior to polymer derivatization.

The polymer will bear a group which is directly reactive with an amino acid side chain, or the N- or C-tenninus 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 S to use such chemistries here, one should employ a water soluble polymer otherwise derivatized in the same fashion as insoluble polymers heretofore employed for protein immobilization. Cyanogen bromide activation is a particularly useful procedure to employ in crosslinking polysaccharides.

25 "Water soluble" in reference to the starting polymer means that the polymer or its reactive intermediate used for conjugation is sufficiently water soluble to participate in a derivatization reaction.

"Water soluble" in reference to the polymer conjugate means that the conjugate is soluble in physiological fluids such as blood.

The degree of substitution with such a polymer will vary depending upon the number of reactive sites 30 on the protein, whether all or a fragment of the protein is used, whether the protein is a fusion with a heterologous protein an OB-immunoglobulin chimera), the molecular weight, hydrophilicity and other characteristicsofthe polymer, and the particular protein derivatization sites chosen. In general, the conjugate contains about from I to 10 polymer molecules, while any heterologous sequence may be substituted with an essentially unlimited number of polymer molecules so long as the desired activity is not significantly adversely 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 per se for the covalent modificationof proteins with nonproteinaceouspolymers such as PEG. Certain of these methods, however, are not preferred for the purposes herein. Cyanuronic chloride chemistry leads to many side reactions, including protein cross-linking. In addition, it may be particularly likely to lead to inactivation of proteins containing sulfhydryl groups. Carbonyl diimidazole chemistry (Beauchamp et al., Anal Biochem. 131, 25-33 [1983]) requires high pH 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 diimidazolechemistry 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 per se 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 J. Polvm.

Sci. Polvm. 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 cyanoborohydride, which is effective at neutral pH and has very little tendency to reduce disulfide bonds is preferred.

Functionalized PEG polymers to modify the OB protein or OB-Ig chimeras of the present invention are available from Shearwater Polymers, Inc. (Huntsville, AL). Such commercially available PEG derivatives include, but are not limited to, amino-PEG, PEG amino acid esters, PEG-hydrazide, PEG-thiol, PEG-succinate, carboxymethylated PEG, PEG-propionicacid, PEG amino acids, PEG succinimidylsuccinate, PEG succinimidyl propionate, succinimidyl ester of carboxymethylatedPEG, succinimidyl carbonate of PEG, succinimidyl esters of amino acid PEGs. PEG-oxycarbonylimidazole. PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl 25 ether, PEG-aldehyde, PEG vinylsulfone,PEG-maleimide,PEG-orthopyridyl-disulfide, heterofunctional PEGs, S* PEG vinyl derivatives, PEG silanes, and PEG phospholides. The reaction conditions for coupling these PEG derivatives will vary depending on the protein, the desired degree of PEGylation, and the PEG derivative •utilized. Some factors involved in the choice of PEG derivatives include: the desired point of attachment (lysine or cysteine), hydrolytic stability and reactivity of the derivatives, stability, toxicity and antigenicity of the linkeage,suitabilityfor analysis,etc. Specific instructions for the use of any particular derivative are available from the manufacturer.

o 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 conjugates may also be purified by ion-exchangechromatography. The chemistry of many of the electrophilicallyactivated PEG's results in a reduction of amino group charge of the PEGylated product. Thus, high resolution ion exchange chromatography can be used to separate the free and conjugated proteins, and to resolve species with different levels of PEGylation. In fact, the resolution of different species containing one or two PEG residues) is also possible due to the difference in the ionic properties of the unreacted amino acids.

D. The use of the OB-immunoglobulin chimeras and other long half-life derivatives The OB-immunoglobulinchimeras and other long half-life OB derivatives of the present invention are useful for weight reduction, and specifically, in the treatment of obesity and other disorders associated with the abnormal expression or function of the OB gene. Our studies indicate that the OB-immunoglobulin chimeras and other long half-lifeOB derivatives, e.g. PEGylatedOB, reduce the food intake and increase the energy use of animals treated, and are therefore very effective in reducing the weight of both obese and normal subjects.

For testing purposes, the molecules of the present invention may be dissolved in phosphate-buffered saline (PBS) (pH and administered by intravenous or subcutaneous injection, or infusion.

The long acting OB-derivatives of the present invention may further be used to treat other metabolic disorders such as diabetes and bulimia. The OB protein has been shown to reduce insulin levels in animals, and could be useful to reduce excessive levels of insulin in human patients. The reduction of insulin levels in obese or non-obese patients Type I or II diabetics) could restore or improve the insulin-sensitivity of such patients.

In addition, the long half-life OB-derivatives can be used for the treatment of kidney ailments, hypertension, and lung disfunctions, such as emphysema. The OB protein might also cause a mitogenic response in receptor-bearing tissues, acting as a growth factor for these cells.

Therapeutic formulations of the present invention are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers(Rernington's Pharmaceutical Sciences 16th edition. Osol, A. Ed. (1980)), in the form of lyophilized •formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate and other organic acids: antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, 25 such as serum albumin, gelatin or immunoglobulins;hydrophilicpolymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or PEG.

S 30 The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.

Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.

Therapeuticcompositionsherein generallyare placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods.e.g. injection or infusion by intravenous, intraperitoneal,etc. routes. Sustained released formulations are also foreseen. Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides Patent 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate Sidman et al., 1983, "Biopolymers" 22 547-556), poly (2-hydroxyethyl-methacrylate) Langer. et al.. 1981. Biomed.

Mater. Res." 15: 167-277 and R. Langer. 1982, Chem. Tech." 12: 98-105). ethylene vinyl acetate Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988A). Sustained release compositions also include liposomes. Liposomescontaininga molecule within the scope of the present invention are prepared by methods known per se: DE 3,218,121A; Epstein et al., 1985, "Proc. Natl. Acad. Sci. USA" 82: 3688-3692; Hwang et al., 1980, "Proc. Natl. Acad. Sci. USA" 77: 4030-4034; EP 52322A; EP 36676A; EP 88046A; EP 143949A; EP 142641 A; Japanese patent application 83- 18008; U.S. patents 4,485,045 and 4,544,545; and EP 102,324A.

Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamelartype in which the lipid content is greater than about 30 mol. cholesterol, the selected proportion being adjusted for the optimal therapy.

An effectiveamount of a moleculeof the present invention to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient.

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 I pg/kg to up to 100 mg/kg or more, depending on the factors mentioned above. Typically, the clinician will administer a molecule of the present invention until a dosage is reached that provides the required biological effect. The progressof this therapy is easily monitored by conventional assay techniques. If the purpose of the treatment is weight reduction, the therapy is normally continued until a desired body weight is reached.

25 Non-therapeuticuses of the OB protein-immunoglobulinfusions of the present invention include their use to identify and purify OB receptors. The identificationand expression cloning of an OB receptor, using an OB protein-immunoadhesin is described in a Reference Example hereinbelow.

The invention will be further illustrated by the following non-limiting examples.

Example I 30 Expression of OB- immunoadhesins Using protein engineering techniques, the human OB protein was expressed as a fusion with the hinge, CH2 and CH3 domains of IgG-1. DNA constructs encoding the chimera of the human OB protein and IgG-I Fc domains were made with the Fc region clones of human IgG-I. Human OB cDNA was obtained by PCR from human fat cell dscDNA (Clontech Buick-ClonecDNA product). The source of the IgG-1 cDNA was the plasmid pBSSK-CH2CH 3 The chimera contained the coding sequence of the full length OB protein (amino acids 1-167 in Figure 5) and human IgG-1 sequences beginning at aspartic acid 216 (taking amino acid 114 as the first residue of the heavy chain constant region (Kabat et al., Sequences of Proteins of Immunological -18- Interest 4th ed. [1987]), which is the first residue of the IgG-I hinge after the cysteine residue involved in heavy-light chain bonding, and ending with residues 441 to include the CH2 and CH3 Fc domains of IgG- I.

There was an insert of codons for three amino acids (GlyValThr)between the OB and IgG- I coding sequences.

If necessary, this short linker sequence can easily be deleted, for example by site directed deletion mutagenesis, to create an exact junction between the coding sequences of the OB protein and the IgG- hinge region. The coding sequence of the OB-IgG-I immunoadhesin was subcloned into the pRK5-based vector which containsa neomycineselectable marker, for transient expression in 293 cells using the calcium phosphate technique (Suva el al., Science 237, 893-896 [1987]). 293 cells were cultured in HAM's: Low Glucose DMEM medium (50:50), containing 10% FBS and 2 mM L-Gln. For purification ofOB-lgG-I chimeras, cells were changed to serum free production medium PS24 the day after transfection and media collected after three days.

The culture media was filtered.

The filtered 293 cell supernatant(400 ml)containing recombinant human OB-lgG-1 was made I mM in phenylmethylsulfonylfluorideand 2 pg/ml in aprotinin. This material was loaded at 4 °C onto a I x 4.5 cm Protein A agarose column (Pierce catalog 20365) equilibrated in 100 mM HEPES pH 8. The flow rate was 75 ml/h. Once the sample was loaded, the column was washed with equilibration buffer until the A 28 0 reached baseline. The OB-lgG- I protein was eluted with 3.5 M MgCI 2 2% glycerol (unbuffered) at a flow rate of ml/h. The eluate was collected with occasional mixing into 10 ml of 100 mM HEPES pH 8 to reduce the MgCI 2 concentration by approximately one-half and to raise the pH. The eluted protein was then dialyzed into phosphate buffered saline, concentrated, sterile filtered and stored either at 4°C or frozen at -70 The OB- IgG-I immunoadhesin prepared by this method is estimated by SDS-PAGE to be greater than 90% pure.

Example 2 Animal studies A. Materials and Methods OB protein Production Murine OB cDNA was obtained by PCR from an adipocyte cDNA library 25 using primers based on the sequence of Zhang et al., sulra. Mature OB protein (amino acids 22-167) was expressed in E. coli by inserting the OB coding sequence in frame with the secretion sequence of the E. coli heat-stable enterotoxin II, downstream of the E. coli alkaline phosphatase promoter. Chang et Gene 55, 189- 96(1987). After cell lysis, the insoluble fraction was solubilized in 8 M urea buffer pH 8.35 in the presence of mM DTT. Reduced OB protein was purified by size exclusion and reverse phase HPLC, then refolded in 30 the presence of glutathione. Refolded OB protein was purified by reverse phase HPLC and analyzed by SDS- PAGE and amino acid and mass spectrometry analyses.

Preparation of PEG-hOB The PEG derivatives of the human PB protein were prepared by reaction :of hOB purified by reverse phase chromatographywith a succinimidylderivativeof PEG propionic acid (SPA- PEG) having a nominal molecular weight of 10 kD, which had been obtained from Shearwater Polymers, Inc.

(Huntsville, AL). After purification of the hOB protein by reverse phase chromatography, an approximately 1-2 mg/ml solution of the protein in 0.1% trifluoroacetic acid and approximately 40% acetonitrile, was diluted with 1/3 to 1/2 volume of 0.2 M borate buffer and the pH adjusted to 8.5 with NaOH. SPA-PEG was added to -19the reaction mixture to make 1:1 and 1:2 molar ratios of protein to SPA-PEG and the mixture was allowed to incubate at room temperature for one hour. After reaction and purification by gel electrophoresis or ion exchange chromatography, the samples were extensively dialyzed against phosphate-buffered saline and sterilized by filtration through a 0.22 micron filter. Samples were stored at 4°C. Under these conditions, the PEG-hOB resulting from the 1:1 molar ratio protein to SPA-PEG reaction consisted primarily of molecules with one 10 kD PEG attached with minor amounts of the 2 PEG-containing species. The PEG-hOB from the 1:2 molar reaction consisted of approximately equal amounts of 2 and 3 PEGs attached to hOB. as determined by SDS gel electrophoresis. In both reactions, small amounts of unreacted protein was also detected. This unreacted protein can be efficiently removed by the gel filtration or ion exchange steps as needed. The PEG derivatives of the human OB protein can also be prepared essentially following the aldehyde chemistry described in EP 372,752 published June 13, 1990.

Animal Studies All manipulations involving animals were reviewed and approved by Genentech's Institutional Animal Care and Use Committee. Seven to eight week-old genetically obese C57Bl/6J-ob/ob (ob/ob) female mice were purchase from Jackson Labs (Bar Harbor, ME). Lean female mice of the same genetic background (C57BI/6) were purchased from Harlan Sprague Dawley (Hollister. CA). Mice were housed in groups 3 6 with adlibitum access to water and standard mouse chow (Purina 5010: Purina Mills, Richmond, IN) in a temperature-, humidity- and light-controlled (lights on at 06:00h, of at I8:00h) colony room.

Miniosmotic pumps (Alzet model 2002; Alza Corp., Palo Alto, CA) were filled with purified recombinantOB protein (100 pg/kg/day) in sterile phosphate-buffered saline (PBS) or PBS alone under sterile conditions following manufacturer's instructions and incubated overnight in sterile saline at room temperature prior to implantation into mice. Mice were anesthetized with ketamine/xylazine and miniosmotic pumps were implanted subcutaneously in the midscapular region. Daily subcutaneous injections of purified recombinant OB protein, hOB-lgG-I fusion protein or PBS were made into the midscapular region of conscious mice.

Injections were performed within one hour of lights out. The body weight of each mouse (to the nearest 0.1 25 gram) and the weight of the food contained in the food bin in each cage (to the nearest 0. gram) were recorded within one hour of lights out every one to two days. The data are depicted as the mean SEM. The number of animals is as described below and in the Figure legends.

B. Results with continuous subcutaneous infusion of OB protein Lean female mice were treated with murine OB protein either as a continuous subcutaneous infusion 30 or daily subcutaneous injections. The results are shown in Figure I. The upper chart shows that the OB protein is significantlymore effective in reducing body weight when delivered as a continuous infusion than when the same dose is delivered in the form of daily subcutaneous injections. The bottom chart shows the same difference in the ability of the OB protein to reduce adipose tissue weight.

C. Results with the OB-igG-1 chimera Obese female ob/ob mice were treated with human OB protein or with the human OB-lgG- chimera.

The data are shown in Figure 2. The data presented in the top chart demonstrate that the hOB-lgG-I fusion protein is more potent than the native hOB protein at reducing body weight, when both proteins are administered similarly by daily subcutaneous infusion. It is noted that the increase in potency would be even more expressed.

if the data were converted to molar amounts, as only about one third of the OB-lgG- I chimera comes from the OB protein. The data further confirm the previous observation that continuous subcutaneous infusion (pump) or the hOB protein is more effective than daily subcutaneous injections (inj) at reducing body weight.

The data shown at the bottom chart of Figure 2 show that the hOB-IgG- I fusion protein substantially reduced food intake. This result was unexpected as it was assumed that the fusion protein would be too large to cross the blood-brain barrier and exert its effect.

Obese (ob/ob) female mice were treated with either hOB or the hOB-lgG-1 chimera by daily subcutaneous injections for 7 days. The data shown in Figure 3 again demonstrate that the chimera is more effective than the native hOB protein at reducing body weight (top) and food intake (bottom).

In a further experiment, obese (ob/ob) female mice were treated with either the hOB-IgG- fusion protein, native hOB or hCD4-IgG-l (control) by daily subcutaneous injections for seven days. The results shown in Figure 5 affirm that the hOB-lgG-I fusion protein is more effective than the native hOB protein at reducing body weight (top and middle panels) and food intake (bottom panel).

D. Results with PEG-hOB Obese female ob/ob mice were treated with human OB protein or with PEG derivatives of human OB.

The data are shown in Figure 4. The data presented in the top chart demonstrate that PEG-hOB is more potent than the native hOB protein at reducing body weight, when both proteins are administered similarly by daily subcutaneous infusion.

The data shown at the bottom chart of Figure 4 show that the PEG-hOB proteins were substantially more effective in reducing food intake than unmodified native hOB.

Reference Example Identification and cloning of an OB receptor The OB protein-immunoadhesinof Example I was used to detect and expression clone an OB receptor.

First, to identify a receptor source, several cell lines were screened with I pg/ml OB-IgG-I fusion by flow cytometry. The detection system which consists of a biotin conjugated secondary antibody followed by streptavidin-phycoerythrinprovidesa dramatic amplification of the signal and allows the detection of cells expressing low numbers of receptors. Two cell lines, human embryonic kidney 293 30 and human lung A549 cells were found to bind OB-lgG-I but not an Flt-4 control immunoadhesin. Specific binding of OB-lgG- i to the cells was also demonstrated by the addition of excess of bacterially expressed human OB protein. Addition of 10 pg/ml of human OB completely blocks the binding of OB-lgG-1 to 293 cells.

To isolate a cDNA encoding the OB receptor, COSN cells were transiently transfected with pools of approximately 105 clones of an oligo dT primed 293 cell cDNA library in pRK5B. Transfected cells were enriched by panning on plates coated with an anti-human Fc antibody after incubation with OB-IgG-I. After three rounds of enrichment. I of 30 pools gave rise to OB-lgG-I mediated adherence of COSN cells to the binding plates which could be competed by human leptin. cDNA clones picked randomly from this third round were transfected in pools of 10-20. Individual clones were finally identified after breaking down one pool of that was scoring positive by panning.

Sequence analysis revealed a clone of approximately 5300 bp with an open reading frame encoding a protein of 896 amino acids. The sequence corresponded to a type I Iransmembrane protein with a 22 amino acid long signal peptide, 819 amino acid extracellulardomain, 21 am ino acid transmembranedomain and a short 34 amino acid intracellular domain. The sequence was found to essentially correspond to the human OB receptor identified and isolated by Tartaglia el al.. supra, and is identical with a human receptor sequence disclosed in copending application Serial No. 08/585,005 filed January 1 1, 1996.

While the invention has been illustrated by way of examples, the scope of 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.

All references cited throughout the specification, including the examples, and the references cited therein are hereby expressly incorporated by reference.

The entire disclosure in the complete specification of our Australian Patent Application No. 15200/97 is by this cross-reference incorporated into the present specification.

S e Page(s) 33 are claims pages they appear after the sequence listing SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Genentech, Inc.

De Sauvage, Frederic J.

Levin, Nancy Vandlen, Richard L.

(ii) TITLE OF INVENTION: OB Protein Derivatives (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Genentech, Inc.

STREET: 460 Point San Bruno Blvd CITY: South San Francisco STATE: California COUNTRY: USA ZIP: 94080 COMPUTER READABLE FORM: MEDIUM TYPE: 3.5 inch, 1.44 Mb floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: WinPatin (Genentech) (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 19-Dec-1996

CLASSIFICATION:

25 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/667184 FILING DATE: 20-JUN-1996 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/579494 S 30 FILING DATE: 27-DEC-1995 (viii) ATTORNEY/AGENT INFORMATION: NAME: Dreger, Ginger R.

REGISTRATION NUMBER: 33,055 REFERENCE/DOCKET NUMBER: 985P2PCT 35 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 415/225-3216 TELEFAX: 415/952-9881 TELEX: 910/371-7168 S(2) INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 7127 base pairs TYPE: Nucleic Acid STRANDEDNESS: Double TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TTCGAGCTCG CCCGACATTG TACGGGGTCA TTAGTTCATA TTACGGTAAA TGGCCCGCCT ACGTCAATAA TGACGTATGT TTGACGTCAA TGGGTGGAGT ATCAAGTGTA TCATATGCCA AAATGGCCCG CCTGGCATTA TACTTGGCAG TACATCTACG GGTTTTGGCA GTACATCAAT TTTCCAAGTC TCCACCCCAT AAATCAACGG GACTTTCCAA AAATGGGCGG TAGGCGTGTA TTAGTGAACC GTCAGATCGC CCATAGAAGA CACCGGGACC TTGGAACGCG GATTCCCCGT GTCTATAGGC CCACCCCCTT CATAACCTTA TGTATCATAC CATCCACTTT GCCTTTCTCT ACCTCGGTTC TATCGATATG CTTTGGCCCT ATCTTTTCTA AGATGACACC AAAACCCTCA TTTCACACAC GCAGTCAGTC 25 TTCATTCCTG GGCTCCACCC ACTGGCAGTC TACCAACAGA TCCAAATATC CAACGACCTG- GCCTTCTCTA AGAGCTGCCA

ATTATTGACT

GCCCATATAT

GGCTGACCGC

TCCCATAGTA

ATTTACGGTA

AGTACGCCCC

TGCCCAGTAC

TATTAGTCAT

GGGCGTGGAT

TGACGTCAAT

AATGTCGTAA

CGGTGGGAGG

CTGGAGACGC

GATCCAGCCT

GCCAAGAGTG

GGCTTCGTTA

ACATACGATT

CCACAGGTGT

CATTGGGGAA

TGTCCAAGCT

TCAAGACAAT

TCCTCCAAAC

CATCCTGACC

TCCTCACCAG

GAGAACCTCC

CTTGCCCTGG

AGTTATTAAT

GGAGTTCCGC

CCAACGACCC

ACGCCA-ATAG

AACTGCCCAC

CTATTGACGT

ATGACCTTAT

CGCTATTACC

AGCGGTTTGA

GGGAGTTTGT

CAACTCCGCC

TCTATATAAG

CATCCACGCT

CCGCGGCCGG

ACGTAAGTAC

GAACGCGGCT

TAGGTGACAC

CCACTCCCAG

CCCTGTGCGG

GTGCCCATCC

TGTCACCAGG

AGAAAGTCAC

TTATCCAAGA

TATGCCTTCC

GGGATCTTCT

GCCAGTGGCC

AGTAATCAAT

GTTACATAAC

CCGCCCATTG

GGACTTTCCA

TTGGCAGTAC

CAATGACGGT

GGGACTTTCC

ATGGTGATGC

CTCACGGGGA

TTTGGCACCA

CCATTGACGC

CAGAGCTCGT

GTTTTGACCT

GAACGGTGCA

CGCCTATAGA

ACAATTAATA

TATAGAATAA

GTCCAACTGC

ATTCTTGTGG

AAAAAGTCCA

ATCAATGACA

CGGTTTGGAC

TGGACCAGAC

AGAAACGTGA

TCACGTGCTG

TGGAGACCTT

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 GGACAGCCTG GGGGGTGTCC TGGAAGCTTC AGGCTACTCC ACAGAGGTGG 1350

TGGCCCTGAG

GACCTCAGCC

GTGCCCAGCA

CAAAACCCAA

GTGGTGGTGG

CGTGGACGGC

AGTACAACAG

GACTGGCTGA

CCCAGCCCCC

AACCACAGGT

CAGGTCAGCC

CGTGGAGTGG

CTCCCGTGCT

GTGGACAAGA

GCATGAGGCT

CGGGTAAATG

GTCGACCTGC

TATAATGGTT

20 ATTTTTTTCA

CTTATCATGT

TGAAATAACC

AGAACCAGCT

TCCCCAGCAG

CAGGTGTGGA

TGCATCTCAA

CCGCCCCTAA

AATTTTTTTT

CAGGCTGCAG

CTGGGTGCGG

CCTGAACTCC

GGACACCCTC

ACGTGAGCCA

GTGGAGGTGC

CACGTACCGT

ATGGCAAGGA

ATCGAGAAAA

GTACACCCTG

TGACCTGCCT

GAGAGCAATG

GGACTCCGAC

GCAGGTGGCA

CTGCACAACC

AGTGCGACGG

AGAAGCTTGG

ACAAATAAAG

CTGCATTCTA

CTGGATCGAT

TCTGAAAGAG

GTGGAATGTG

GCAGAAGTAT

AAGTCCCCAG

TTAGTCAGCA

CTCCGCCCAG

ATTTATGCAG

GGGTCTCTGC

GGTCACCGAC

TGGGGGGACC

ATGATCTCCC

CGAAGACCCT

ATAATGCCAA

GTGGTCAGCG

GTACAAGTGC

CCATCTCCAA

CCCCCATCCC

GGTCAAAGGC

GGCAGCCGGA

GGCTCCTTCT

GCAGGGGAAC

ACTACACGCA

CCCTAGAGTC

CCGCCATGGC

CAATAGCATC

GTTGTGGTTT

CGGGAATTAA

GAACTTGGTT

TGTCAGTTAG

GCAAAGCATG

GCTCCCCAGC

ACCATAGTCC

TTCCGCCCAT

AGGCCGAGGC

AGGACATGCT

AAAACTCACA

GTCAGTCTTC

GGACCCCTGA

GAGGTCAAGT

GACAAAGCCG

TCCTCACCGT

AAGGTCTCCA

AGCCAAAGGG

GGGAAGAGAT

TTCTATCCCA

GAACAACTAC

TCCTCTACAG

GTCTTCTCAT

GAAGAGCCTC

GACCTGCAGA

CCAACTTGTT

ACAAATTTCA

GTCCAA-ACTC

TTCGGCGCAG

AGGTACCTTC

GGTGTGGAAA

CATCTCAATT

AGGCAGAAGT

CGCCCCTAAC

TCTCCGCCCC

CGCCTCGGCC

GTGGCAGCTG

CATGCCCACC

CTCTTCCCCC

GGTCACATGC

TCAACTGGTA

CGGGAGGAGC

CCTGCACCAG

ACAAAGCCCT

CAGCCCCGAG

GACCAAGAAC

GCGACATCGC

AAGACCACGC

CAAGCTCACC

GCTCCGTGAT

TCCCTGTCTC

AGCTTCTAGA

TATTGCAGCT

CAAATAAAGC

ATCAATGTAT

CACCATGGCC

TGAGGCGGAA

GTCCCCAGGC

AGTCAGCAAC

ATGCAAAGCA

TCCGCCCATC

ATGGCTGACT

TCTGAGCTAT

1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700

TCCAGAAGTA

CTGTTAATTC

ACTTCGCATA

CAGCGACCCG

ACAGGATGAG

TTCTCCGGCC

AGACAATCGG

CGCCCGGTTC

GCAGGACGAG

GCGCAGCTGT

TTGGGCGAAG

CGAGAAAGTA

ATCCGGCTAC

GCACGTACTC

AGAGCATCAG

GCATGCCCGA

CCGAATATCA

CCGGCTGGGT

ATATTGCTGA

20 TACGGTATCG

TGACGAGTTC

GACGCCCAAC

AAAGGTTGGG

CAGCGCGGGG

GAGGCTAACT

TGACGGCAAT

TCATAAACGC

CGAGACCCCA

GTGAGGAGGC

GAACACGCAG

TTAAGGTGAC

CTTAACAGCG

GATCGTTTCG

GCTTGGGTGG

CTGCTCTGAT

TTTTTGTCA-A

GCAGCGCGGC

GCTCGACGTT

TGCCGGGGCA

TCCATCATGG

CTGCCCATTC

GGATGGAAGC

GGGCTCGCGC

CGGCGAGGAT

TGGTGGAA.AA

GTGGCGGACC

AGAGCTTGGC

CCGCTCCCGA

TTCTGAGCGG

CTGCCATCAC

CTTCGGAATC

ATCTCATGCT

GAAACACGGA

AAAAAGACAG

GGGGTTCGGT

TTGGGGCCAA

TTTTTTGGAG

ATGCAGTCGG

GCGTGTGGCC

TCAACAGCGT

CATGATTGAA

AGAGGCTATT

GCCGCCGTGT

GACCGACCTG

TATCGTGGCT

GTCACTGAAG

GGATCTCCTG

CTGATGCAAT

GACCACCAAG

CGGTCTTGTC

CAGCCGAACT

CTCGTCGTGA

TGGCCGCTTT

GCTATCAGGA

GGCGAATGGG

TTCGCAGCGC

GACTCTGGGG

GAGATTTCGA

GTTTTCCGGG

GGAGTTCTTC

AGGAGACAAT

AATAAAACGC

CCCAGGGCTG

TACGCCCGCG

GCCTAGGCTT

GGCGGCGCGG

TCGAACACCG

GCCGCAGATC

CAAGATGGAT

CGGCTATGAC

TCCGGCTGTC

TCCGGTGCCC

GGCCACGACG

CGGGAAGGGA

TCATCTCACC

GCGGCGGCTG

CGAAACATCG

GATCAGGATG

GTTCGCCAGG

CCCATGGCGA

TCTGGATTCA

CATAGCGTTG

CTGACCGCTT

ATCGCCTTCT

TTCGAAATGA

TTCCACCGCC

ACGCCGGCTG

GCCCACCCCG

ACCGGAAGGA

ACGGGTGTTG

GCACTCTGTC

TTTCTTCCTT

TTGCAAAAAG

TCCCAGGTCC

AGCGACCCTG

TGATCAAGAG

TGCACGCAGG

TGGGCACAAC

AGCGCAGGGG

TGAATGAACT

GGCGTTCCTT

CTGGCTGCTA

TTGCTCCTGC

CATACGCTTG

CATCGAGCGA

ATCTGGACGA

CTCAAGGCGC

TGCCTGCTTG

TCGACTGTGG

GCTACCCGTG

CCTCGTGCTT

ATCGCCTTCT

CCGACCAAGC

GCCTTCTATG

GATGATCCTC

GGAGATGGGG

ACCCGCGCTA

GGTCGTTTGT

GATACCCCAC

TTCCCCACCC

2750 2800 2850 2900 2950 3000 3050 3100 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900 3950 4000 4050 4100 CAACCCCCALA GTTCGGGTGA AGGCCCAGGG CTCGCAGCCA ACGTCGGGGC 4150

GGCAAGCCCG

ATGGGGAATG

GGGTCAGGTC

GCCTGGGCAT

TGGCTGCCAA

GCCGCCGGAC

GGTACGAGGA

CGGGACCCCG

AGTCATAAGT

TGACTGGGTT

CAACCATAGT

GGTTACGCGC

CTTTCGCTTT

CAAGCTCTAA

GCACCTCGAC

CATCGCCCTG

TTTAATAGTG

GGGCTATTCT

20 TAAAAAATGA

TTAACGTTTA

AGGTTAATGT

CGGGGAAATG

AAATATGTAT

ATTGAAAAAG

CCCTTTTTTG

GGTGAAAGTA

TCGAACTGGA

CCATAGCCAC

GTTTATGGTT

CACGACTGGA

GGACCGCATG

ACACCCCCGA

GAACTAAACC

GCGCTTTTGT

GCCAGGGCAC

GCGGCGACGA

GAAGGCTCTC

ACGCGCCCTG

AGCGTGACCG

CTTCCCTTCC

ATCGGGGGCT

CCCAAAAAAC

ATAGACGGTT

GACTCTTGTT

TTTGATTTAT

GCTGATTTAA

CAATTTTATG

CATGATAATA

TGCGCGGAAC

CCGCTCATGA

GAAGAGTATG

CGGCATTTTG

AAAGATGCTG

TCTCAACAGC

GGGCCCCGTG

CGTGGGGGTT

CTGAGCAGAC

TACTGGCGCG

CCCCCAAAAA

TGACTACGGC

TTTGTATTGG

CTGTCCTACG

TAGTCATGCC

AAGGGCATCG

TAGCGGCGCA

CTACACTTGC

TTTCTCGCCA

CCCTTTAGGG

TTGATTTGGG

TTTCGCCCTT

CCAAACTGGA

AAGGGATTTT

CAAAAATTTA

GTGCAGGCCT

ATGGTTTCTT

CCCTATTTGT

GACAATAACC

AGTATTCAAC

CCTTCCTGTT

AAGATCAGTT

GGTAAGATCC

GGTTAGGGAC

ATTCTTTTGG

AGACCCATGG

ACACGAACAC

CCACCGCGCG

ATCTCTGCCC

TCACCACGGC

AGTTGCATGA

CCGCGCCCAC

GTCGAGCGGC

TTAAGCGCGG

CAGCGCCCTA

CGTTCGCCGG

TTCCGATTTA

TGATGGTTCA

TGACGTTGGA

ACAACACTCA

GCCGATTTCG

ACGCGAATTT

CGTGATACGC

AGACGTCAGG

TTATTTTTCT

CTGATAAATG

ATTTCCGTGT

TTTGCTCACC

GGGTGCACGA

TTGAGAGTTT

GGGGTCCCCC

GCGTTGCGTG

TTTTTGGATG

CGGGCGTCTG

GATTTCTGGC

CTTCTTCGCT

CGAGTTTCCG

TAAAGAAGAC

CGGAAGGAGC

CGCATCAAAG

CGGGTGTGGT

GCGCCCGCTC

CTTTCCCCGT

GTGCTTTACG

CGTAGTGGGC

GTCCACGTTC

ACCCTATCTC

GCCTATTGGT

TAACAAAATA

CTATTTTTAT

TGGCACTTTT

AAATACATTC

CTTCAATAAT

CGCCCTTATT

CAGAAACGCT

GTGGGTTACA

TCGCCCCGAA

4200 4250 4300 4350 4400 4450 4500 4550 4600 4650 4700 4750 4800 4850 4900 4950 5000 5050 5100 5150 5200 5250 5300 5350 5400 5450 5500 a.

S

S

a..

GAACGTTTTC

ATTATCCCGT

ATTCTCAGAA

ACGGATGGCA

TGATAACACT

AGCTAACCGC

CGTTGGGAAC

CACGATGCCA

AACTACTTAC

GATAAAGTTG

TATTGCTGAT

CAGCACTGGG

ACGGGGAGTC

AGGTGCCTCA

ATATACTTTA

GTGAAGATCC

TTCGTTCCAC

GAGATCCTTT

CCGCTACCAG

20 TCCGAAGGTA

TAGTGTAGCC

ACATACCTCG

TAAGTCGTGT

CGCAGCGGTC

CGAACGACCT

CGCCACGCTT

GGGTCGGAAC

TATCTTTATA

CAATGATGAG

GATGACGCCG

TGACTTGGTT

TGACAGTAAG

GCGGCCAACT

TTTTTTGCAC

CGGAGCTGAA

GCAGCAATGG

TCTAGCTTCC

CAGGACCACT

AAATCTGGAG

GCCAGATGGT

AGGCAACTAT

CTGATTAAGC

GATTGATTTA

TTTTTGATAA

TGAGCGTCAG

TTTTCTGCGC

CGGTGGTTTG

ACTGGCTTCA

GTAGTTAGGC

CTCTGCTAAT

CTTACCGGGT

GGGCTGAACG

ACACCGAACT

CCCGAAGGGA

AGGAGAGCGC

GTCCTGTCGG

CACTTTTAAA

GGCAAGAGCA

GAGTACTCAC

AGAATTATGC

TACTTCTGAC

AACATGGGGG

TGAAGCCATA

CAACAACGTT

CGGCAACAAT

TCTGCGCTCG

CCGGTGAGCG

AAGCCCTCCC

GGATGAACGA

ATTGGTAACT

AAACTTCATT

TCTCATGACC

ACCCCGTAGA

GTAATCTGCT

TTTGCCGGAT

GCAGAGCGCA

CACCACTTCA

CCTGTTACCA

TGGACTCAAG

GGGGGTTCGT

GAGATACCTA

GAAAGGCGGA

ACGAGGGAGC

GTTTCGCCAC

GTTCTGCTAT

ACTCGGTCGC

CAGTCACAGA

AGTGCTGCCA

AACGATCGGA

ATCATGTAAC

CCAAACGACG

GCGCAAACTA

TAATAGACTG

GCCCTTCCGG

TGGGTCTCGC

GTATCGTAGT

AATAGACAGA

GTCAGACCAA

TTTAATTTAA

AAAATCCCTT

AAAGATCAAA

GCTTGCAAAC

CAAGAGCTAC

GATACCAAAT

AGAACTCTGT

GTGGCTGCTG

ACGATAGTTA

GCACACAGCC

CAGCGTGAGC

CAGGTATCCG

TTCCAGGGGG

CTCTGACTTG

GTGGCGCGGT

CGCATACACT

AAAGCATCTT

TAACCATGAG

GGACCGAAGG

TCGCCTTGAT

AGCGTGACAC

TTAACTGGCG

GATGGAGGCG

CTGGCTGGTT

GGTATCATTG

TATCTACACG

TCGCTGAGAT

GTTTACTCAT

AAGGATCTAG

AACGTGAGTT

GGATCTTCTT

AAAAAAACCA

CAACTCTTTT

ACTGTCCTTC

AGCACCGCCT

CCAGTGGCGA

CCGGATAAGG

CAGCTTGGAG

ATTGAGAAAG

GTAAGCGGCA

AAACGCCTGG

AGCGTCGATT

5550 5600 5650 5700 5750 5800 5850 5900 5950 6000 6050 6100 6150 6200 6250 6300 6350 6400 6450 6500 6550 6600 6650 6700 6750 6800 6850 6900 *4 4 4 4444** 4 4 4 44.* .4 4 4* 4.

444* 4 .4 4 S 4 4*44 4 4 TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCTGGC 6950 ACGACAGGTT TCCCGACTGG AAAGCGGGCA GTGAGCGCAA CGCAATTAAT 7000 GTGAGTTACC TCACTCATTA GGCACCCCAG GCTTTACACT TTATGCTTCC 7050 GGCTCGTATG TTGTGTGGAA TTGTGAGCGG ATAACAATTT CACACAGGAA 7100 ACAGCTATGA CCATGATTAC GAATTAA 7127 INFORMATION FOR SEQ ID NO:2: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 397 amino acids TYPE: Amino Acid CD) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: Met His Trp Gly Thr Leu Cys SEQ ID NO:2: Gly Phe Leu Phe Tyr Val 0 0000

S

S0 S

S*

0*

S

0

S

0@ 0 0e S Thr Lys Thr Leu Ser His Thr Gin Asp Phe Ile Pro Asp Gin Thr Leu Ser Arg Asn Val Asp Leu Leu His Trp Ala Ser Gly 30 Giu Ala Ser Gly Gin Gly Ser Leu Gly Cys Gly Val Ala Pro Giu Leu Gin Ile Ser Gly Ala 80 Ile 95 Val 110 Leu 125 Tyr 140 Gin 155 Thr 170 Leu Ala Val Pro Ile Lys Thr Ile Val Val Ser Ser Lys Leu His Pro Ile Val Tyr Gin Gin Gin Ile Ser Asn Leu Ala Phe Ser Glu Thr Leu Asp Ser Thr Giu Val Asp Met Leu Trp Asp Lys Thr His Gly Giy Pro Ser Leu Gin 25 Thr 40 Gin 55 Leu 70 Ile 85 Asp 100 Lys 115 Ser 130 Va1 145 Gin 160 Thr 175 Va1 Trp Leu Trp Pro Lys Vai Gin Asp Arg Ile Asn Asp Lys Val Thr Gly Thr Leu Ser Lys Leu Thr Ser Met Leu Giu Asn Leu Ser Cys His Leu Leu Gly Gly Val Ala Leu Ser Arg Leu Asp Leu Ser Cys Pro Pro Cys Phe Leu Phe Pro Tyr Asp Ile Leu Met Pro Arg 105 Pro 120 Leu 135 Leu 150 Pro 165 Pro 180 Pro 190 195 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 200 205 210 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 215 220 225 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 230 235 240 Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 245 250 255 Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys 260 265 270 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 275 280 285 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gln Val Tyr Thr 290 295 300 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu 305 310 315 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 320 325 330 Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 335 340 345 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 350 355 360 SThr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys 365 370 375 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 380 385 390 Leu Ser Leu Ser Pro Gly Lys 395 397

Claims (20)

1. A method of increasing the potency of an OB protein, comprising preparing a covalent derivative of said OB protein, selected from the group consisting of: an OB protein-immunoglobulin chimera, and a hydrophilic nonproteinaceous polymer covalently bonded to an OB-protein or an OB protein-immunoglobulin chimera. measuring the potency of said covalent derivative; and confirming that said covalent derivative has increased potency relative to the corresponding underivatized OB protein.

2. The method of claim 1, wherein said OB protein is a native OB protein.

3. The method of claim 2, wherein said OB protein is capable of binding to a native OB receptor. 20 4. The method of any one of claims 1 to 3, wherein said OB protein is fused to an immunoglobulin constant domain sequence. The method of claim 4, wherein said constant domain sequence is that of an immunoglobulin heavy chain.

6. The method of claim 5, wherein two OB protein-IgG S"heavy chain fusions are linked to each other by at least one disulfide bond to yield a homodimeric immunoglobulin- like structure.

7. The method of claim 6, wherein at least one of said OB protein-IgG heavy chain fusions is associated with an immunoglobulin light chain.

8. The method of claim 1, wherein said hydrophilic nonproteinaceous polymer is covalently bonded to an native OB protein.

9. The method of claim 8, wherein said hydrophilic nonproteinaceous polymer is polyethylene glycol (PEG). The method of any one of claims 1 to 9, wherein said H:\cintae\Keep\speci\15200.97.doc 5/02/01 32 OB protein is a native human OB protein.

11. The method of claim 1, wherein said covalent derivative is more potent in appetite suppression or in inducing weight loss than the corresponding OB protein.

12. The method of claim 1, wherein said covalent derivative is more potent in the treatment of obesity, bulimia, or type I or type II diabetes than the corresponding OB protein.

13. The method of claim 1, wherein said covalent derivative is more potent in the reduction of elevated insulin levels than the corresponding OB protein.

14. The method of any one of claims 1 to 7, wherein said OB protein-immunoglobulin chimera is prepared by culturing a host cell transformed with a nucleic acid encoding said chimera so as to express the nucleic acid, and isolating said chimera. The method of claim 14, wherein said host cells are cotransformed with nucleic acid encoding at least two OB protein-immunoglobulin chimeras. 20 16. The method of claim 1, further comprising the step of admixing said covalent derivative with a pharmaceutically acceptable carrier.

17. The method of claim 1 or claim 16, further comprising the step of administering said covalent derivative to an individual suffering from a condition associated with the abnormal expression or function of the OB gene.

18. The method of claim 1 or claim 16, further comprising the step of administering said covalent 30 derivative to an individual diagnosed with obesity, bulimia or type I or type II diabetes.

19. The method of claim 1 or claim 16, further comprising the step of administering said covalent .derivative to an individual in need of appetite suppression or of reduction of food intake. The method of claim 1 or claim 16, further comprising the step of administering said covalent derivative to an individual having elevated insulin levels.

21. Use of the said OB protein-immunoglobulin chimera H:\cintae\Keep\speci\15200.97.doc 5/02/01 33 or hydrophilic nonproteinaceous polymer covalently bonded to an OB protein or an OB protein-immunoglobulin chimera of claim 1, for the manufacture of a medicament for the treatment of an individual suffering from a condition associated with the abnormal expression or function of the OB gene.

22. Use according to claim 21, wherein the individual is suffering from obesity, bulimia or type I or type II diabetes.

23. Use according to claim 22, wherein the individual is in need of appetite suppression or of reduction of food intake.

24. Use according to claim 22, wherein the individual has elevated insulin levels.

25. A method according to claim 1, substantially as herein described with reference to the Examples.

26. Use according to claim 21, substantially as herein described with reference to the Examples. 20 Dated this 5th day of February 2001 GENENTECH, INC By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\cintae\Keep\speci\15200.97.doc 5/02/01