Title:  OB protein-polymer chimeras

United States Patent:  6,620,413

Issued:  September 16, 2003

Inventors:  DeSauvage; Frederic (Foster City, CA); Levin; Nancy (Newbury Park, CA); Vandlen; Richard (Hillsborough, CA)

Assignee:  Genentech, Inc. (South San Francisco, CA)

Appl. No.:  775066

Filed:  December 27, 1996

Abstract

The present invention concerns long-half derivative of the obesity protein OB. The invention specifically concerns OB protein-immunoglobulin chimeras and polyethylene glycol (PEG)-OB derivatives, which have extended half-life as compared to the corresponding native OB proteins. The invention further relates to methods for treating obesity by using the long half-life derivatives of OB.

SUMMARY OF THE INVENTION

The present invention is based on the observation that the OB protein is significantly more effective at 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 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 is believed to be located in the brain.

In one aspect, the invention concerns long half-life derivatives of OB proteins, compositions containing them, and their administration for the treatment of conditions associated with the abnormal expression or function of the OB gene, such as obesity.

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 OB-immunoglobulin 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 association of two OB polypeptide-immunoglobulin heavy chain fusions (e.g., via covalent linkage by disulfide 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 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.

In another embodiment, the invention concerns a method of treating a condition associated with the abnormal expression or function of the OB gene by administering a therapeutically effective amount of a long half-life variant of an OB protein, such as an OB-immunoglobulin chimera. The invention specifically concerns a method of treating obesity.

In yet another embodiment, the invention concerns a composition for the treatment of a condition associated with the abnormal expression or function of a native OB gene, such as obesity, comprising an effective amount of a long half-life OB protein variant, such as an OB-immunoadhesin chimera.

DETAILED DESCRIPTION OF THE INVENTION

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 immunoglobulin (Ig) with domains of a cell-surface receptor that recognizes a specific ligand. This type of molecule is called an "immunoadhesin", because it combines "immune" and "adhesion" functions; other frequently used names are "Ig-chimera", "Ig- " or "Fc-fusion protein", or "receptor-globulin."

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 [1989]; Zettmeissl et al., DNA Cell Biol. USA 9 347-353 [1990]; Byrn et al., Nature 344, 667-670 [1990]); L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110, 2221-2229 [1990]; Watson et al., Nature 349, 164-167 [1991]); E-selectin [Mulligan et al., J. Immunol. 151, 6410-17 [1993]; Jacob et al., Biochemistry 34, 1210-1217 [1995]); P-selectin (Mulligan et al., supra; Hollenbaugh et al., Biochemistry 34, 5678-84 [1995]); ICAM-1 (Stauton et al., J. Exp. Med. 176, 1471-1476 [1992]; Martin et al., J. Virol. 67, 3561-68 [1993]; Roep 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]); L1 glycoprotein (Doherty et al., Neuron 14, 57-66 [1995]); TNF-R1 (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88, 10535-539 [1991]; Lesslauer et al., Eur. J. Immunol. 21, 2883-86 [1991]; Peppel 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. 1133-1144 (1991)]; NP receptors [Bennett et al., J. Biol. Chem. 266, 23060-23067 (1991)]; IgE receptor .alpha. [Ridgway and Gorman, J. Cell. Biol. 115, abstr. 1448 (1991)]; HGF receptor [Mark, M. R. et al., 1992, J. Biol. Chem. submitted]; IFN-.gamma.R .alpha.- and .beta.-chain [Marsters et al., Proc. Natl. Acad. Sci. USA 92, 5401-05 [1995]); trk-A, -B, and -C (Shelton et al., J. Neurosci. 15, 477-91 [1995]); IL-2 (Landolfi, J. Immunol. 146, 915-19 [1991]); IL-10 (Zheng et al., J. Immunol. 154, 5590-5600 [1995]).

The simplest and most straightforward immunoadhesin design combines the binding region(s) of the `adhesin` protein with the hinge and Fc regions of an immunoglobulin heavy chain. Ordinarily, when preparing the OB-immunoglobulin chimeras 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-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CH1 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 preferred embodiment, 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 114 [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 CH3 or CH1, 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-immunoglobulin chimeras are assembled as multimers, and particularly as homo-dimers or -tetramers (WO 91/08298). Generally, these assembled immunoglobulins will have known unit structures. A basic four chain structural unit is the form in which IgG; IgD, and IgE exist. A four unit is repeated in the higher molecular weight immunoglobulins; IgM generally exists as a pentamer of basic four units held together by disulfide bonds. IgA globulin, and occasionally IgG globulin, may also exist in multimeric form in serum. In the case of multimer, each four unit may be the same or different.

Various exemplary assembled OB-immunoglobulin chimeras within the scope herein are schematically diagrammed below:

(a) AC_L -AC_L ;

(b) AC_H -[AC_H, AC_L -AC_H, AC_L -V_H C_H, or V_L C_L -AC_H ];

(c) AC_L -AC_H -[AC_L -AC_H, AC_L -V_H C_H, V_L C_L -AC_H, or V_L C_L -V_H C_H ];

(d) AC_L -V_H C_H -[AC_H, or AC_L -V_H C_H, or V_L C_L -AC_H ];

(e) V_L C_L -AC_H -[AC_L -V_H C_H, or V_L C_L -AC_H ]; and

(f) [A-Y]_n -[V_L C_L -V_H C_H ]₂,

wherein

each A represents identical or different OB polypeptide amino acid sequences;

V_L is an immunoglobulin light chain variable domain;

V_H is an immunoglobulin heavy chain variable domain;

C_L is an immunoglobulin light chain constant domain;

C_H is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

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

Alternatively, the OB amino acid sequences can be inserted between immunoglobulin heavy 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. Similar constructs have been reported by Hoogenboom, H. R. et al., Mol. Immunol. 28, 1027-1037 (1991).

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

In a preferred embodiment, the immunoglobulin sequences used in the construction of the immunoadhesins of the present invention are from an IgG immunoglobulin heavy chain constant domain. For human immunoadhesins, the use of human IgG-1 and IgG-3 immunoglobulin sequences is preferred. A major 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-1. Possible IgG-based immunoadhesin structures are shown in FIGS. 3a-c. While IgG immunoadhesins are typically mono- or bivalent, other Ig subtypes like IgA and IgM may give rise to dimeric or pentameric structures, respectively, of the basic Ig homodimer unit. A typical IgM-based multimeric immunoadhesin is illustrated in FIG. 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-IgM (Traunecker et al., supra); ICAM-IgM (Martin et al., J. Virol. 67, 3561-68 [1993]); and CD2-IgM (Arulanandam et al., J. Exp. Med. 177, 1439-50 [1993]).

For OB-Ig immunoadhesins, which are designed for in vivo application, the pharmacokinetic properties and the effector functions specified by the Fc region are important as well. Although IgG-1, IgG-2 and IgG-4 all have in vivo half-lives of 21 days, their relative potencies at activating the complement system are different. IgG-4 does not activate complement, and IgG-2 is significantly weaker at complement activation than IgG-1. Moreover, unlike IgG-1, IgG-2 does not bind to Fc receptors on mononuclear cells or neutrophils. While IgG-3 is optimal for complement activation, its in vivo half-life is approximately one third of the other IgG isotypes. Another important consideration for immunoadhesins designed to be used as human therapeutics is the number of allotypic variants of the particular isotype. In general, IgG isotypes with fewer serologically-defined allotypes are preferred. For example, IgG-1 has only four serologically-defined allotypic sites, two of which (G1m and 2) are located in the Fc region; and one of these sites G1m1, is non-immunogenic. In contrast, there are 12 serologically-defined allotypes in IgG-3, all of which are in the Fc region; only three of these sites (G3m5, 11 and 21) have one allotype which is nonimmunogenic. Thus, the potential immunogenicity of a .gamma.3 immunoadhesin is greater than that of a .gamma.1 immunoadhesin.

In designing the OB-Ig immunoadhesins of the present invention regions that are not required for receptor binding, the structural integrity (e.g. 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 IgG1 hinge region.

OB-Ig immunoadhesins are 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 [1990]; Stamenkovic et al., Cell 66, 1133-1144 [1991]). The latter type of fusion requires the presence of Ig regulatory sequences for expression. cDNAs encoding IgG heavy-chain constant regions can be isolated based 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 .lambda.gtII 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 expression in mammalian cells pRK5-based vectors (Schall et 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 ClaI and HindIII is reversed. See U.S. Pat. No. 5,108,901 issued Apr. 28, 1992.). The exact junction can be created by removing the extra sequences between the designed junction codons using oligonucleotide-directed deletional mutagenesis (Zoller and Smith, Nucleic Acids Res. 10, 6487 [1982]; Capon et al., Nature 337, 525-531 [1989]). Synthetic oligonucleotides can be used, in which each half is complementary to 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.

Immunoadhesins can be expressed efficiently in a variety of host-cells, including myeloma cell lines, Chinese Hamster ovary (CHO) cells, monkey COS cells, human embryonic kidney 293 cells, and baculovirus infected insect cells. In these systems, the immunoadhesin polypeptides are assembled and secreted into the cell culture medium. Yeasts, e.g. Saccharomyces 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 al., 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 of heterologous proteins from the yeast into the culture media. According to Halaas et al., supra, expression of mouse and human OB genes in Saccharomyces cerevisiae transformed with 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 coding sequences of mouse and human OB-immunoglobulin chimeras can be subcloned into the PET15b expression vector (Novagen) and expressed in E. coli (BL21 (DE3)pIYsS) through use of the T7 E. coli RNA 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 et al., Gene 55, 189-96 [1987]).

The choice of host cell line for the expression of OB-Ig immunoadhesins depends mainly on the expression vector. Another consideration is the amount of protein that is required. Milligram quantities often can be produced by transient transfections. For example, the adenovirus EIA-transformed 293 human embryonic kidney cell line can be transfected transiently 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); Zettmeissl et 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 inhibitor methotrexate; clones are selected, in which the number of gene copies encoding the DHFR and immunoadhesin sequences is co-amplified. If the immunoadhesin contains 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 al., 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 et al., J. Immunol. 67, 3561-68 [1993]).

Immunoadhesins can 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 immunoadhesins that are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains [Lindmark et al., J. Immunol. Meth. 62, 1-13 (1983)]. Protein G is recommended for all mouse isotypes and for human .gamma.3 [Guss et 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 immunoadhesin to 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 .gamma.1 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 3.0), 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 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 [Al-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 also be a factor for immunoadhesins in 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 immunoadhesins produced 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 immunoglobulin regions 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. Pat. Nos. 5,225,538 (issued Jul. 6, 1993) and U.S. Pat. No. 5,455,165 (issued Oct. 30, 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, 104-115 (1995), and Peach and Linsley, Methods in Enzymology 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-immunoglobulin chimera, covalently bonded to a nonproteinaceous polymer. The nonproteinaceous polymer ordinarily is a hydrophilic synthetic polymer, i.e., a polymer not otherwise found in nature. However, polymers which exist in nature and are produced by recombinant or in vitro methods are useful, as are polymers which are isolated from native sources. Hydrophilic polyvinyl polymers fall within the scope of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularly useful are polyalkylene ethers such as polyethylene glycol (PEG); polyelkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; branched or unbranched polysaccharides which comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparon. The polymer prior to cross-linking need not be, but preferably is, water soluble, but the final conjugate must be water soluble. In addition, the polymer should not be highly immunogenic in the conjugate form, nor should it possess viscosity that is incompatible with intravenous infusion or injection if it is intended to be administered by such routes.

Preferably the polymer contains only a single group which is reactive. This helps to avoid cross-linking of protein molecules. However, it is within the scope herein to optimize reaction conditions to reduce cross-linking, or to purify the reaction products through gel filtration or chromatographic sieves to recover substantially homogenous derivatives.

The molecular weight of the polymer may desirably range from about 100 to 500,000, and preferably is from about 1,000 to 20,000. The molecular weight chosen will depend upon the nature of the polymer and the degree of substitution. In general, the greater the hydrophilicity of the polymer and the greater the degree of substitution, the lower the molecular weight that can be employed. Optimal molecular weights will be determined by routine experimentation.

The polymer generally is covalently linked to the OB protein or to the OB-immunoglobulin chimeras though a multifunctional crosslinking agent which reacts with the polymer and one or more amino acid or sugar residues of the OB protein or OB-immunoglobulin chimera to be linked. However, it is within the scope of the invention to directly crosslink the polymer by reacting a derivatized polymer with the hybrid, or via versa.

The covalent crosslinking site on the OB protein or OB-Ig includes the N-terminal amino group and epsilon amino groups found on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl or other hydrophilic groups. The polymer may be covalently bonded directly to the hybrid without the use of a multifunctional (ordinarily bifunctional) crosslinking agent. Covalent binding to amino groups is accomplished by known chemistries based upon cyanuric chloride, carbonyl diimidazole, aldehyde reactive groups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde; PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde, succinimidyl active esters, activated dithiocarbonate PEG, 2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate activated PEG.) Carboxyl groups are derivatized by coupling PEG-amine using carbodiimide.

Polymers are conjugated to oligosaccharide groups by oxidation using chemicals, e.g. metaperiodate, or enzymes, e.g. glucose or galactose oxidase, (either of which produces the aldehyde derivative of the carbohydrate), followed by reaction with hydrazide or amino derivatized polymers, in the same fashion as is described by Heitzmann et al., P.N.A.S., 71, 3537-41 (1974) or Bayer et al., Methods in Enzymology 62, 310 (1979), for the labeling of oligosaccharides with biotin or avidin. Further, other chemical or enzymatic methods which have been used heretofore to link oligosaccharides are particularly advantageous because, in general, there are fewer substitutions than amino acid sites for derivatization, and the oligosaccharide products thus will be more 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-terminus of the polypeptide linked, or which is reactive with the multifunctional cross-linking agent. In general, polymers bearing such reactive groups are known for the preparation of immobilized proteins. In order to use such chemistries here, one should employ a water soluble polymer otherwise derivatized in the same fashion as insoluble polymers heretofore employed for protein immobilization. Cyanogen bromide activation is a particularly useful procedure to employ in crosslinking polysaccharides.

"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 on the protein, whether all or a fragment of the protein is used, whether the protein is a fusion with a heterologous protein (e.g. an OB-immunoglobulin chimera), the molecular weight, hydrophilicity and other characteristics of the polymer, and the particular protein derivatization sites chosen. In general, the conjugate contains about from 1 to 10 polymer molecules, while any heterologous sequence may be substituted with an essentially unlimited number of polymer molecules so long as the desired activity is not significantly adversely affected. The optimal degree of cross-linking is easily determined by an experimental matrix in which the time, temperature and other reaction conditions are varied to change the degree of substitution, after which the ability of the conjugates to function in the desired fashion is determined.

The polymer, e.g. PEG, is cross-linked by a wide variety of methods known per se for the covalent modification of proteins with nonproteinaceous polymers 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 (>8.5), which can inactivate proteins. Moreover, since the "activated PEG" intermediate can react with water, a very large molar excess of "activated PEG" over protein is required. The high concentrations of PEG required for the carbonyl diimidazole chemistry also led to problems in purification, as both gel filtration chromatography and hydrophilic interaction chromatography are adversely affected. In addition, the high concentrations of "activated PEG" may precipitate protein, a problem that per se has been noted previously (Davis, U.S. Pat. No. 4,179,337). On the other hand, aldehyde chemistry (Royer, U.S. Pat. No. 4,002,531) is more efficient since it requires only a 40-fold molar excess of PEG and a 1-2 hr incubation. However, the manganese dioxide suggested by Royer for preparation of the PEG aldehyde is problematic "because of the pronounced tendency of PEG to form complexes with metal-based oxidizing agents" (Harris et al., J. Polym. Sci. Polym. Chem. Ed. 22, 341-52 [1984]). The use of a Moffatt oxidation, utilizing DMSO and acetic anhydride, obviates this problem. In addition, the sodium borohydride suggested by Royer must be used at high pH and has a significant tendency to reduce disulfide bonds. In contrast, sodium cyanoborohydride, which is effective at neutral pH and has very little tendency to reduce disulfide bonds is preferred.

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.

D. The Use of the OB-immunoglobulin Chimeras and Other Long half-life Derivatives

The OB-immunoglobulin chimeras and other long half-life OB derivatives of the present invention are useful 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-life OB derivatives, e.g. PEGylated OB, 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 7.4), and administered by intravenous or subcutaneous injection, or infusion.

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 (Remington'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 concentrations employed, 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, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or PEG.

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.

Therapeutic compositions herein generally are 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 (U.S. Pat. No. 3,773,919, EP 58,48 1), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et al., 1983, "Biopolymers" 22 (1): 547-556), poly (2-hydroxyethyl-methacrylate) (R. Langer, et al., 1981, "J. Biomed. Mater. Res." 15: 167-277 and R. Langer, 1982, Chem. Tech." 12: 98-105), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988A). Sustained release compositions also include liposomes. Liposomes containing a molecule within the scope of the present invention are prepared by methods known pert: 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 142641A; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324A. Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamelar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal NT-4 therapy.

An effective amount of a molecule of the present invention to be employed therapeutically will 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 1 .mu.g/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 progress of 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.

Claim 1 of 25 Claims

What is claimed is:

1. A derivative of an obesity (OB) protein having a longer plasma half-life and/or slower clearance and greater potency than a native OB protein, comprising an OB protein amino acid sequence having at least 85% amino acid sequence identity with a native OB protein represented by the amino acid sequence between the N-terminus and the cysteine (Cys) at position 167 of SEQ ID NO: 2, covalently bonded to a nonproteinaceous polymer, wherein said derivative is capable of reducing body weight and/or food intake in an individual.

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