NZ524612A - OB fusion protein compositions and methods - Google Patents

Abstract

Disclosed is a DNA sequence and uses thereof , where said DNA sequence encodes a fusion protein optionally having an N-terminal methionine , comprising an antibody constant region or portion thereof attached to the N-terminus of a human OB protein . Such nucleotide sequences and proteins encoded by them are useful in treatment of excess weight , diabetes , high blood lipid level , arterial sclerosis , arterial plaques , insufficient lean muscle mass , insufficient sensitivity to insulin , gall stone formation , and stroke .

Description

5246 I NEW ZEALAND PATENTS ACT 1953 No: Divided out of No. 514145 Date: Dated 11 December 1997 COMPLETE SPECIFICATION OB FUSION PROTEIN COMPOSITIONS AND METHODS We, AMGEN, INC., a Delaware corporation, of One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page 1 a) INTELLECTUAL PROPERTY OFFICE OF N.Z. " 7 MAR 2003 RBOEIVED -la (followed by 2)- OB FUSION PROTEIN COMPOSITIONS AND METHODS Field of the Invention 5 The present invention relates to Fc-OB fusion protein compositions and methods for preparation and use thereof.

Background Although the molecular basis for obesity is largely unknown, the identification of the "OB gene" and protein encoded ("OB protein" or *leptin") has shed some light on mechanisms the body uses to regulate body fat deposition. See. PCT publication, WO 96/05309 15 (12/22/96), Friedman et a!.; Zhang et al., Nature 372: 425-432 (1994); see also, the Correction at Nature 374: 479 (1995). The OB protein is active in vivo in both ob/ob mutant mice (mice obese due to a defect in the production of the OB gene product) as well as in normal, 20 wild type mice. The biological activity manifests itself in, among other things, weight loss. See generally. Barrinaga, "Obese" Protein Slims Mice, Science 269: 475-456 (1995). The OB protein, derivatives and use thereof as modulators for the 25 control of weight and adiposity of animals, including mammals and humans, has been disclosed in greater detail in PCT publication WO 96/05309 (12/22/96), hereby incorporated by reference, including figures.

The other biological effects of OB protein are 30 not well characterized. It is known, for instance, that in ob/ob mutant mice, administration of OB protein results in a decrease in serum insulin levels, and serum glucose levels. It is also known that administration of OB protein results in a decrease in body fat. This was 35 observed in both ob/ob mutant mice, as well as non-obese normal mice. Pelleymounter et al., Science 269: 540-543 (1995); Halaas et al., Science 231- 543-546 (1995). See also. Campfield et al., Science 269: 546-549 (1995) (Peripheral and central administration of microgram doses of OB protein reduced food intake and 5 body weight of ob/ob and diet-induced obese mice but not in db/db obese mice.) In none of these reports have toxicity's been observed, even at the highest doses.

Despite the promise of clinical application of the OB protein, the mode of action of the OB protein is 10 vivo is not clearly elucidated. Information on the OB receptor, shows high affinity binding of the OB protein detected in the rat hypothalamus, which indicates OB receptor location. Stephens et al., Nature 377: 530-532. The db/db mouse displays the identical phenotype 15 as the ob/ob mouse, i.e.. extreme obesity and Type II diabetes; this phenotype is thought to be due to a defective OB receptor, particularly since db/db mice fail to respond to OB protein administration. See Stephens et al., supra.

With the advances in recombinant DNA technologies, the availability of recombinant proteins for therapeutic use has engendered advances in protein formulation and chemical modification. One goal of such modification is protein protection and decreased 25 degradation. Fusion proteins and chemical attachment may effectively block a proteolytic enzyme from physical contact with the protein backbone itself, and thus prevent degradation. Additional advantages include, under certain circumstances, increasing the stability, 30 circulation time, and the biological activity of the therapeutic protein. A review article describing protein modification and fusion proteins is Francis, Focus on Growth Factors 2:4-10 (May 1992) (published by Mediscript, Mountview Court, Friern Barnet Lane, London N20, OLD, UK).

One such modification is the use of the Fc 5 region of immunoglobulins. Antibodies comprise two functionally independent parts, a variable domain known as "Fab", which binds antigen, and a constant domain, known as "Fc" which provides the link to effector functions such as complement or phagocytic cells. The 10 Fc portion of an immunoglobulin has a long plasma half-life, whereas the Fab is short-lived. Capon, et al., Nature H7.: 525-531 (1989) .

Therapeutic protein products have been constructed using the Fc domain to provide longer half-15 life or to incorporate functions such as Fc receptor binding, protein A binding, complement fixation and placental transfer which all reside in the Fc proteins of immunoglobulins. Id. For example, the Fc region of an IgGl antibody has been fused to the N-terminal end of 20 CD30-L, a molecule which binds CD30 receptors expressed on Hodgkin's Disease tumor cells, anaplastic lymphoma cells, T-cell leukemia cells and other malignant cell types. See. U.S. Patent No. 5,480,981. IL-10, an antiinflammatory and antir eject ion agent has been fused to 25 murine Fcy2a in order to increase the cytokine's short circulating half-life. Zheng, X. et al., The Journal of Immunology, 154: 5590-5600 (1995) . Studies have also evaluated the use of tumor necrosis factor receptor linked with .the Fc protein of human IgGl to treat 30 patients with septic shock. Fisher, C. et al., N. Engl. J. Med., 22A'- 1697-1702 (1996); Van Zee, K. et al.. The Journal of Immunology, 156: 2221-2230 (1996) . Fc has also been fused with CD4 receptor to produce a therapeutic protein for treatment of AIDS. See. Capon 35 eta 1., Nature, 117:525-531 (1989). In addition, the N-terminus of interleukin 2 has also been fused to the -4- (followed by 4a) Fc portion of IgGl or IgG3 to overcome the short half life of interleukin 2 and its systemic toxicity. See. Harvill ec al., Immunotechnology, 1: 95-105 (1995).

Due to the identification of the OB protein as a promising therapeutic protein, there exists a need to develop OB analog compositions for clinical application in conjunction with or in place of OB protein administration. Such development would include OB analog compositions where protein formulations and chemical modifications achieve decreased protein degradation, increased stability and circulation time. -4a- (followed by 4b) finmrnary r>f i-fya Invention In a first aspect, the present invention provides a DNA sequence encoding a fusion protein optionally having an N-terminal methionine, said fusion protein comprising an antibody constant region or portion thereof attached to the N-terminus of a human OB protein, wherein said OB protein is selected from the group consisting of: (a) the amino acid sequence 1-146 as set forth in SEQ ID NO: 6; (b) the amino acid sequence 1-146 as set forth in SEQ ID NO: 6 having a lysine residue at position 35 and an isoleucine residue at position 74; (c) the amino acid sequence of subparts (a) or (b) lacking a glutaminyl residue at position 28; and (d) the amino acid sequence of subparts (a), (b), or (c), wherein one or more amino acids selected from the group consisting of amino acids 32, 35, 50, 64, 68, 71, 74, 77, 89, 97, 100, 105, 106, 107, 108, 111, 118, 136, 138, 142 and 145 is substituted with the corresponding amino acid present in SEQ ID NO: 3 or a conserved amino acid.

In a further aspect, the present invention provides a process for producing a fusion protein optionally having an N-terminal methionine, said fusion protein comprising an antibody constant region or portion thereof attached to the N-terminus of a human OB protein, said process comprising expression of the DNA sequence of the invention in an isolated transformed host cell and isolating said fusion protein.

INTELLECTUAL PROPERTY OFFICE OF HI MAR 2004 -4b- (followed by 4c) In a yet further aspect, the present invention provides a process for producing a fusion protein optionally having an N-terminal methionine, said fusion protein comprising an antibody constant region or portion thereof attached to the N-terminus of a human OB protein, said process comprising expression of the DNA sequence of the invention in a transformed non-human host cell and isolating said fusion protein.

In a still further aspect, the present invention provides a vector comprising the DNA sequence of the invention.

In another aspect, the present invention provides an isolated host cell transformed or transfected with the DNA sequence of the invention.

In another aspect, the present invention provides a non-human host cell transformed or transfected with the DNA sequence of the invention.

In another aspect, the present invention provides the DNA sequence of the invention for use in treating obesity or increasing insulin sensitivity.

In another aspect, the present invention provides a method of treatment of a disorder selected from the group consisting of: excess weight; diabetes; high blood lipid level; arterial sclerosis; arterial plaque; insufficient lean tissue mass; insufficient sensitivity to insulin; and stroke; wherein the method consists of administering a therapeutically effective amount of the DNA sequence of the invention to a non-human animal.

INTELLECTUAL PROPERTY OFRCE OF HZ MAR 2m received -4c- (followed by 4d) In another aspect, the present invention provides a method of reducing or preventing gall stones formation; wherein the method consists of administering a therapeutically effective amount of the DNA sequence of the invention to a non-human animal.

In another aspect, the present invention provides a use of the DNA sequence of the invention in the preparation of a medicament for the treatment of a disorder selected from the group consisting of: excess weight; diabetes; high blood lipid level; arterial sclerosis; arterial plaque; insufficient lean tissue mass; insufficient sensitivity to insulin; and stroke; in a patient in need thereof.

In another aspect, the present invention provides a use of the DNA sequence of the invention in the preparation of a medicament for reducing or preventing gall stones formation in a patient in need thereof.

In another aspect, the present invention provides a fusion protein optionally having an N-terminal methionine, said fusion protein comprising an antibody constant region or portion thereof attached to the N-terminus of a human OB protein, when produced by a process of the invention.

INTELLECTUAL PROPERTY OFRCF OF N.Z MAR 2004 received -4d- (followed by 5) The present specification was divided out of New Zealand specification No. 514145.

NZ 514145 relates to Fc-OB fusion protein compositions, methods of preparation of such compositions and uses thereof. In particular, NZ 514145 relates to a genetic fusion protein comprising the Fc region or analogs of immunoglobulins fused to the N-terminal portion of the OB protein or analogs. The Fc-OB fusion protein is capable of dimerizing via the cysteine residues of the Fc region. Unexpectedly, genetic fusion modification with Fc at the N-terminus of the OB protein demonstrates advantages in stability, clearance rate and decreased degradation which are not seen in OB protein or with fusion of Fc to the C-terminus of the OB protein. Surprisingly and importantly, the N-terminus modification provides unexpected protein protection from degradation, increases circulation time and stability, when compared to the OB protein or Fc modification to the OB protein C-terminus. Such unexpected advantages from the Fc modification to OB protein would be advantageous to OB protein consumers, in that these changes contribute to lower doses required or less frequent dosing.

INTELLECTUAL PROPERTY OFFICE OF N.Z 1 5 MAR 2004 received NZ 514145 also relates to the genetic modification of proteins via fusion of the Fc region to the OB protein (or analogs thereof), as well as, specific modifications, preparations and methods of use 5 thereof.

NZ 514145 provides a Fc-OB fusion protein wherein Fc is genetically fused to the N-terminus of the OB protein (or analogs thereof) . In addition, the Fc portion may 10 also be linked to the N-terminus of the OB protein (or analogs thereof) via peptide or chemical linkers as known in the art. As noted above and described in more detail below, the Fc-OB fusion protein has unexpected protections from degradation and increased circulation 15 time and stability when compared to the OB protein or C-terminus OB-Fc fusion proteins. Additional aspects of NZ 514145, therefore, include not only Fc-OB fusion protein compositions, but also DNA sequences encoding such proteins, related vectors and host cells 20 containing such vectors, both useful for producing fusion proteins.

NZ 514145 also provides for preparing the Fc-OB fusion protein. Such methods include recombinant DNA techniques for 25 preparation of recombinant proteins. Furthermore, such aspects include methods of fermentation and purification as well.

Described but not claimed are methods for treating excess weight in an 30 individual or animals, including modulation of and/or fat deposition by the administration of Fc-OB fusion proteins. Due to the Fc-OB fusion protein characteristics, methods are contemplated which reduce the amount and/or frequency of dosage of OB protein by 35 using Fc-OB weight reducing agent.

INTELLECTUAL PROPERTY OFFICE OF N.Z MAR 2004 received Brief Description of the Drawings FIGURE 1 Recombinant murine metOB (double 15 stranded) DNA (SEQ. ID. NOs.: 1 and 2) and amino acid sequence (SEQ. ID. NO. 3) .

FIGURE 2 Recombinant human metOB analog (double stranded) DNA (SEQ. ID. NOs.: 4 and 5) and amino acid sequence (SEQ. ID. NO. 6) .

FIGURE 3 (A-C) Recombinant human metFc-OB (double stranded) DNA (SEQ. ID. NOs.: 7 and 8) and amino acid sequence (SEQ. ID. NO. 9) .

FIGURE 4 (A-C) Recombinant human metFc-OB variant (double stranded) DNA (SEQ. ID. NOs.: 10 and 11) 25 and amino acid sequence (SEQ. ID. NO. 12).

FIGURE 5 (A-C) Recombinant human metFc-OB" variant (double stranded) DNA (SEQ. ID. NOs.: 13 and 14) and amino acid sequence (SEQ. ID. NO. 15).

FIGURE 6 (A-C) Recombinant human metFc-OB 30 variant (double stranded) DNA (SEQ. ID. NOs.: 16 and 17) and amino acid sequence (SEQ. ID. NO. 18).

Detailed Description NZ 514145 relates to Fc-OB fusion protein compositions, methods of preparation of such compositions and uses thereof.

In particul^iNJELLECUJAL^PROPERTY MAR 2004 received NZ 514145 relates to the genetic or chemical fusion of the Fc region of immunoglobulins to the N-terminal portion of the OB protein. Unexpectedly, fusion of Fc at the N-terminus of the OB protein 5 demonstrates advantages which eure not seen in OB protein or with fusion of Fc at the C-terminus of the OB protein. Surprisingly, the N-terminally modified Fc-OB protein provides unexpected protein protection from degradation, increased circulation time and increased 10 stability. Accordingly, the Fc-OB fusion protein, and analogs or derivatives thereof, as well as, related methods of use and preparation, eure described in more detail below.

Compositions The Fc sequence of the recombinant human Fc-OB sequence set forth in SEQ. ID. NO. 9 (See Figure 3) may be selected from the human immunoglobulin IgG-1 heavy chain, see Ellison, J.W. et al., Nucleic Acids Res. lfl: 20 4071-4079 (1982) , or any other Fc sequence known in the art (e.g. other IgG classes including but not limited to IgG-2, IgG-3 and IgG-4, or other immunoglobulins). Variant, analogs or derivatives of the Fc portion may be constructed by, for example, making various 25 substitutions of residues or sequences.

Cysteine residues can be deleted or replaced with other amino acids to prevent formation of disulfide crosslinks of the Fc sequences. In particular amino acid at position 5 of SEQ. ID. NO. 9 is a cysteine 30 residue. The recombinant Fc-OB sequence of SEQ. ID.

NO. 9 is a 378 amino acid Fc-OB protein (not counting the methionine residue) . The first amino acid sequence for the recombinant Fc-OB protein of Figure 3 is referred to as +1 with the methionine at the -1 35 position.

INTELLECTUAL PROPERTY OFFICF OF N.Z MAR 2fflW received One may remove the cysteine residue at position 5 or substitute it with one or more amino acids. An alanine residue may be substituted for the cysteine residue at position 6 giving the variant amino 5 acid sequence of Figure 4 (SEQ. ID. NO. 12) . The recombinant Fc-OB protein of Figure 4 is a 378 amino acid Fc-OB protein (not counting the methionine residue) . The first amino acid sequence for the recombinant Fc-OB protein of Figure 4 is referred to as 10 +1 with the methionine at the -1 position.

Likewise, the cysteine at position 5 of SEQ. ID. NO. 9 could be substituted with a serine or other amino acid residue or deleted. A variant or analog may also be prepared by deletion of amino acids at positions 15 1, 2, 3, 4 and 5 as with the variant in SEQ. ID. NO. 15 (See Figure 5). Substitutions at these positions can also be made and cure with in the scope of this invention. The recombinant Fc-OB protein of Figure 5 is a 373 amino acid Fc-OB protein (not counting the 20 methionine residue) . The first amino acid sequence for the recombinant Fc-OB protein of Figure 5 is referred to as +1 with the methionine at the -1 position.

Modifications may also be made to introduce four amino acid substitutions to ablate the Fc receptor 25 binding site and the complement (Clq) binding site. --These variant modifications from SEQ. ID. NO. 15 would include leucine at position 15 substituted with glutamate, glutamate at position 98 substituted with alanine, and lysines at positions 100 and 102 30 substituted with alanines (see Figure 6 and SEQ. ID. NO. 18) . The recombinant Fc-OB protein of Figure 6 is a 373 amino acid Fc-OB protein (not counting the methionine residue) . The first amino acid sequence for the recombinant Fc-OB protein of Figure 6 is referred to as 35 +1 with the methionine at the -1 position.

Likewise, one or more tyrosine residues can be replaced by phenyalanine residues as well. In addition, other variant amino acid insertions, deletions and/or substitutions are also contemplated and are within the 5 scope of the present invention. Furthermore, alterations may be in the form of altered amino acids, such as peptidomimetics or D-amino acids. The Fc protein may be also linked to the OB proteins of the Fc-OB protein by "linker" moieties whether chemical or 10 amino acids of varying lengths. Such chemical linkers are well known in the art. Amino acid linker sequences can include but are not limited to: (a) ala, ala, ala; (b) ala, ala, ala, ala; (c) ala, ala, ala, ala, ala; (d) gly, gly; (e) giy» gly, gly; (f) gly, gly, gly, gly, gly; (g) gly, gly, gly, gly. gly, gly, gly; (h) gly-pro-gly; (i) gly, gly, pro, gly, gly; and (j) any combination of subparts (a) through (i).

The OB portion of the Fc-OB fusion protein may 25 be selected from the recombinant murine set forth in SEQ. ID. NO. 3 (See Figure 1), or the recombinant human protein as set forth in Zhang et al., Nature, supra, (herein incorporated by reference) or those lacking a glutaminyl residue at position 28. (See Zhang et al, 30 Nature, supra. at page 428.) One may also use the recombinant human OB protein analog as set forth in SEQ. ID. NO. 6 (See Figure 2), which contains: (1) an arginine in place of lysine at position 35; and (2) a leucine in place of isoleucine at position 74. (A 35 shorthand abbreviation for this analog is the recombinant human R->L^5, I->IP&) . The amino acid sequences for the recombinant human and recombinant murine proteins or analogs with or without the fused Fc portion at the N-terminus of the OB protein are set forth below with a methionyl residue at the -1 position; however, as with any of the present OB proteins and analogs, the methionyl residue may be absent.

The murine protein is substantially homologous to the human protein, particularly as a mature protein, and, further, particularly at the N-terminus. One may 10 prepare an analog of the recombinant human protein by altering (such as substituting amino acid residues), in the recombinant human sequence, the amino acids which diverge from the murine sequence. Because the recombinant human protein has biological activity in 15 mice, such an analog would likely be active in humans. For example, using a human protein having a lysine at residue 35 and an isoleucine at residue 74 according to the numbering of SEQ. ID. NO. 6, wherein the first amino acid is valine, and the amino acid at position 146 is 20 cysteine, one may substitute with another amino acid one or more of the amino acids at positions 32, 35, 50, 64, 68, 71, 74, 77, 89, 97, 100, 105, 106, 107, 108, 111, 118, 136, 138, 142, and 145. One may select the amino acid at the corresponding position of the murine 25 protein, (SEQ. ID. NO. 3), or another amino acid.

One may further prepare "consensus" molecules based on the rat OB protein sequence. Murakami et al., Biochem. Biophys. Res. Comm. 209: 944-952 (1995) herein incorporated by reference. Rat OB protein differs from 30 human OB protein at the following positions (using the numbering of SEQ. ID. NO. 6) : 4, 32, 33, 25., 5il, 68, II, 1A. 21, 78, £2, 21, 1M. 101, 102, 135, 1M, 1G1, 108. 111. 118. 13 6. 138 and 145. One may substitute with another amino acid one or more of the amino acids at 35 these divergent positions. The positions in bold print are those in which the murine OB protein as well as the rat OB protein are divergent from the human OB protein, and thus, are particularly suitable for alteration. At one or more of a positions, one may substitute an amino acid from the corresponding rat OB protein, or another 5 amino acid.

The positions from both rat and murine OB protein which diverge from the mature human OB protein are: 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, 10 and 145. An OB protein according to SEQ. ID. NO. 6 having one or more of the above amino acids replaced with another amino acid, such as the amino acid found in the corresponding rat or murine sequence, may also be effective.

In addition, the amino acids found in rhesus monkey OB protein which diverge from the mature human OB protein are (with identities noted in parentheses in one letter amino acid abbreviation): 8 (S), 35 (R) , 48 (V), 53(Q), 60(1), 66(1), 67(N), 68((L), 89(L), 100(L), 20 108 (E), 112 (D) , and 118 (L). Since the recombinant human OB protein is active in cynomolgus monkeys, a human OB protein according to SEQ. ID. NO. 6 (with lysine at position 35 and isoleucine at position 74) having one or more of the rhesus monkey divergent amino 25 acids replaced with another amino acid, such as the amino acids in parentheses, may be effective. It should be noted that certain rhesus divergent amino acids sure also those found in the above murine species (positions 35, 68, 89, 100 and 112) . Thus, one may prepare a 30 murine/rhesus/human consensus molecule having (using the numbering of SEQ. ID. NO. 6 having a lysine at position 35 and an isoleucine at position 74) having one or more of the amino acids at positions replaced by another amino acid: 4, 8, 32, 33, 15., 48, 50, 53, 60, 64, 66, 35 67, ££, 71, 74, 77, 78, £2, 97, !£>&, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145.

Other analogs may be prepared by deleting a part of the protein amino acid sequence. For example, the mature protein lacks a leader sequence (-22 to -1) . One may prepare the following truncated forms of human 5 OB protein molecules (using the numbering of SEQ. ID. NO. 6): (a) amino acids 98-146 (b) amino acids 1-32 (c) amino acids 40-116 (d) amino acids 1-99 and (connected to) 112-146 (e) amino acids 1-99 and (connected to) 112-146 having one or more of amino acids 100-111 placed between amino acids 99 and 112.

In addition, the truncated forms may also have altered one or more of the amino acids which are divergent (in the rat, murine, or rhesus OB protein) from human OB protein. Furthermore, any alterations may be in the form of altered amino acids, such as peptidomimetics or D-amino acids.

Therefore, nz 514145 encompasses a Fc-OB fusion protein wherein the OB protein is selected from: (a) the amino acid sequence 1-146 as set forth in SEQ. ID. NO. 3 (below) or SEQ. ID. NO. 6; (b) the amino acid sequence 1-146 as set forth in SEQ. ID. NO. 6 having a lysine residue at position 35 and an isoleucine residue at position 74; (c) the amino acid sequence of subpart (b) having a different amino acid substituted in one or more of the following positions (using the numbering according to SEQ. ID. NO. 6 and retaining the same numbering even in the absence of a glutaminyl residue at position 28): 4, 32,' 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111 i36, 138, 142, and 145; lltak&ECTUAL PROPERTY OFRCE OF KI.Z MAR im received (d) the amino acid sequence of subparts (a), (b) or (c) optionally lacking a glutaminyl residue at position 28; (e) the amino acid sequence of subparts (a), 5 (b), (c), or (d) having a methionyl residue at the N-terminus; (f) a truncated OB protein analog selected from among: (using the numbering of SEQ. ID. NO. 6) : (i) amino acids 98-146 10 (ii) amino acids 1-32 (iii) amino acids 40-116 (iv) amino acids 1-99 and 112-146 (v) amino acids 1-99 and 112-146 having one or more of eunino acids 100-111 placed between amino acids 99 and 112; and, (vi) the truncated OB analog of subpart (i) having one or more of amino acids 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, and 145 substituted with another amino acid; (vii) the truncated analog of subpart (ii) having one or more of amino acids 4, 8 and 32 substituted with another amino acid; (viii) the truncated analog of subpeirt (iii) having one or more of amino acids 50, 53, 60, 25 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111 and 112 replaced with another amino acid; (vix) the truncated analog of subpart (iv) having one or more of amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 112, 118, 136, 138, 142, and 145 replaced with another amino acid; and (x) the truncated analog of subpart (v) having one or more of amino acids 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, and 145 replaced with another amino acid; (xi) the truncated analog of any of 5 subparts (i)-(x) having an N-terminal methionyl residue; and (g) the OB protein or analog derivative of any of subparts (a) through (f) comprised of a chemical moiety connected to the protein moiety; 10 (h) a derivative of subpart (g) wherein said chemical moiety is a water soluble polymer moiety; (i) a derivative of subpart (h) wherein said water soluble polymer moiety is polyethylene glycol; (j) a derivative of subpart (h) wherein said 15 water soluble polymer moiety is a polyaminoacid moiety; (k) a derivative of subpart (h) through (j) wherein said moiety is attached at solely the N-terminus of said protein moiety; and (1) an OB protein, analog or derivative of 20 any of subparts (a) through (k) in a pharmaceutical^ acceptable carrier.

Derivatives The Fc-OB fusion proteins (herein the 25 term "protein" is used to include "peptide," Fc, OB .or analogs, such as those recited infra. unless otherwise indicated) are derivatized by the attachment of one or more chemical moieties to the Fc-OB fusion protein moiety. These chemically modified derivatives may be 30 further formulated for intraarterial, intraperitoneal, intramuscular subcutaneous, intravenous, oral, nasal, pulmonary, topical or other routes of administration as discussed below. Chemical modification of biologically active proteins has been found to provide additional 35 advantages under certain circumstances, such a!f"]NTELLECTUAL property increasing the stability and circulation time < f th§FFlCF OF NI.Z MAR 20M received therapeutic protein and decreasing immunogenicity. See. U.S. Patent No. 4,179,337, Davis et al., issued December 18, 1979. For a review, see Abuchowski et al., in Enzymes as Drugs. (J. S. Holcerberg and J. Roberts, 5 eds. pp. 367-383 (1981)); Francis et al., supra.

The chemical moieties suitable for such derivatization may be selected from among various water soluble polymers. The polymer selected should be water soluble so that the protein to which it is attached does 10 not precipitate in an aqueous environment, such as a physiological environment. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. One skilled in the art will be able to select the desired polymer based on such 15 considerations as whether the polymer/protein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations. For the present proteins and peptides, the effectiveness of the derivatization may be 20 ascertained by administering the derivative, in the desired form (i.e., by osmotic pump, or, more preferably, by injection or infusion, or, further formulated for oral, pulmonary or nasal delivery, for example), and observing biological effects as described 25 herein.

The water soluble polymer may be selected-from the group consisting of, for example, polyethylene glycol, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, 30 polyvinyl pyrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrolidone)polyethylene glycol, propylene glycol 35 homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohol. Polyethylene glycol propionaldenhyde may have advantages in manufacturing due to its stability in water. Also, succinate and styrene may also be used.

The OB or Fc proteins used to formulate the 5 Fc-OB fusion protein, may be prepared by attaching polyaminoacids or branch point amino acids to the Fc or OB protein (or analogs) moiety. For example, the polyaminoacid may be an additional carrier protein which, like the Fc fused to the OB protein or OB analog 10 of the present invention, serves to also increase the circulation half life of the protein in addition to the advantages achieved via the Fc-OB fusion protein above. For the present therapeutic or cosmetic purpose of the present invention, such polyaminoacids should be those 15 which have or do not create neutralizing antigenic response, or other adverse responses. Such polyaminoacids may be selected from the group consisting of serum album (such as human serum albumin) , an additional antibody or portion thereof (e.g. the Fc 20 region), or other polyaminoacids, e.g. lysines. As indicated below, the location of attachment of the polyaminoacid may be at the N-terminus of the Fc-OB protein moiety, or C-terminus, or other places in between, and also may be connected by a chemical 25 •linker* moiety to the Fc-OB protein.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 2 kDa and about 100 kDa (the term "about" indicating 30 that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of 35 sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

The number of polymer molecules so attached may vary, and one skilled in the art will be able to 5 ascertain the effect on function. One may mono-derivatize, or may provide for a di-, tri-, tetra-or some combination of derivatization, with the same or different chemical moieties (e.g., polymers, such as different weights of polyethylene glycols) . The 10 proportion of polymer molecules to protein (or peptide) molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted protein or polymer) will be determined 15 by factors such as the desired degree of derivatization (e.g., mono, di-, tri-, etc.), the molecular weight of the polymer selected, whether the polymer is branched or unbranched, and the reaction conditions.

The chemical moieties should be attached to 20 the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art. E.g.. EP 0 401 384 herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. 25 Hematol. 22.' 1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups cure those 30 to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residue. Those having a free carboxyl group may include aspartic acid residues, glutamic acid 35 residues, and the C-terminal amino acid residue.

Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecule(s) . Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group. Attachment at residues important for 5 receptor binding should be avoided if receptor binding is desired.

One may specifically desire N-terminally chemically modified Fc-OB fusion protein. Using polyethylene glycol as an illustration of the present 10 compositions, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be 15 performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally 20 pegylated material from a population of pegylated protein molecules. Selective N-terminal chemical modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the 25 N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved. For example, one may selectively 30 N-terminally pegylate the protein by performing the reaction at a pH which allows one to take advantage of the pKa differences between the e-amino group of the lysine residues and that of the a-amino group of the N-terminal residue of the protein. By such selective 35 derivatization, attachment of a water soluble polymer to a protein is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the protein and no significant modification of other reactive groups, such as the lysine side chain amino groups, occurs. Using reductive alkylation, the water 5 soluble polymer may be of the type described above, and should have a single reactive aldehyde for coupling to the protein. Polyethylene glycol propionaldehyde, containing a single reactive aldehyde, may be used.

An N-terminally monopegylated derivative is 10 preferred for ease in production of a therapeutic.

N-terminal pegylation ensures a homogenous product as characterization of the product is simplified relative to di-, tri- or other multi-pegylated products. The use of the above reductive alkylation process for 15 preparation of an N-terminal product is preferred for ease in commercial manufacturing.

Complexes The Fc-OB fusion protein, analog or derivative 20 thereof may be administered complexed to a binding composition. Such binding composition may have the effect of prolonging the circulation time even further them that achieved with the Fc-OB fusion protein, euialog or derivative. Such composition may be a protein 25 (or synonymously, peptide) . An example of a binding protein is OB protein receptor or portion thereof, such as a soluble portion thereof. Other binding proteins may be ascertained by examining OB protein or Fc-OB protein in serum, or by empirically screening for the 30 presence of binding. Binding proteins used will typically not interfere with the ability of OB protein, Fc-OB fusion proteins, or analogs or derivatives thereof, to bind to endogenous OB protein receptor and/or effect signal transduction.

Pharmaceutical Compositions Described but not claimed herein are methods of using pharmaceutical compositions of the Fc-OB fusion proteins and derivatives. Such pharmaceutical 5 compositions may be for administration for injection, or for oral, pulmonary, nasal, transdermal or other forms of administration. In general, comprehended by the invention are pharmaceutical compositions comprising effective amounts of protein or derivative products of 10 the invention together with pharmaceutical^ acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content {e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives 15 such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into 20 particulate preparations of polymeric compounds such as poly lactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the 25 physical state, stability, rate of in vivo release, and rate of in vivo cl ear cine e of the present proteins and derivatives. See, e.g.. Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated 30 by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations.

Contemplated for use herein are oral solid 35 dosage forms, which are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 199C intellectual property (Ma<S£RCe of hz htv MAR 2004 received Publisning Co. Easton PA 18042) at Chapter 89, which is herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid 5 encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 10 5,013,556). A description of possible solid dosage forms for the therapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated by reference. In general, the formulation will include the 15 Fc-OB fusion protein (or analog or derivative), and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.

Also specifically contemplated are oral dosage 20 forms of the above derivatized proteins. Fc-OB fusion protein may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the protein (or peptide) molecule 25 itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the protein and increase in circulation time in the body. Examples of such moieties 30 include: Polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, Soluble Polymer-Enzyme Adducts. In: "Enzymes as Drugs", 35 Hoceriberg and Roberts, eds., Wiley-Interscience, New York, NY, (1981), pp. 367-383; Newmark, et al., J. Appl.

Biochem. 4.: 185-189 (1982) . Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.

For the Fc-OB fusion protein, analog or derivative, the location of release may be the stomach, the small intestine (e.g., the duodenum, jejunum, or ileum) , or the large intestine. One skilled in the art has available formulations which will not dissolve in 10 the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleteriotis effects of the stomach environment, either by protection of the Fc-OB fusion protein, analog or derivative, or by release of 15 the biologically active material beyond the stomach environment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as 20 enteric coatings are cellulose acetate trimellitate (CAT) , hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These 25 coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. 30 Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or 35 tablet triturates, moist massing techniques can be used.

The therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration 5 could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, the protein (or derivative) may 10 be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the 15 therapeutic with an inert material. These diluents could include carbohydrates, especially taannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium 20 triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the fonnulation of the therapeutic into a solid dosage form. 25 Materials used as disintegrates include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, 30 acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as 35 agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl 5 cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropyimethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the 10 formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, 15 polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic 30 detergents might be used and could include benzalkonium chloride or benzethomium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated 35 castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the protein (or derivative) are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The drug could be incorporated into an inert 10 matrix which permits release by either diffusion or leaching mechanisms e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccahrides. Another form of a controlled release of this therapeutic is by a method 15 based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect. 20 Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan. The therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 25 groups. The first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene 30 glycols . The second group consists of the enteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by 35 compression coating.

Also contemplated herein is pulmonary delivery of the present protein (or derivatives thereof) . The protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung 5 epithelial lining to the blood stream. (Other reports of this include Adjei et al., Pharmaceutical Research 7^. 565-569 (1990); Adjei et al., International Journal of Pharmaceutics 63: 135-144 (1990)(leuprolide acetate); Braquet et al.. Journal of Cardiovascular 10 Pharmacology li (suppl. 5): s.143-146 (1989)(endothelin-1);Hubbard et al.. Annals of Internal Medicine '2.: 206-212 (1989) (al-antitrypsin) ; Smith et al.. J. Clin. Invest. M: 1145-1146 (1989) (a-l-proteinase); Oswein et al., "Aerosolization 15 of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, 1990 (recombinant human growth hormone); Debs et al., The Journal of Immunology 140: 3482-3488 (1988) (interferon-y and tumor necrosis factor a) and Platz et al., U.S. 20 Patent No. 5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the administration of the medicaments prepared according to the uses of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, 25 including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

Some specific examples of commercially available devices suitable for the practice of this 30 invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, 35 North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts.

All such devices require the use of formulations suitable for the dispensing of protein (or analog or derivative). Typically, each formulation is specific to the type of device employed and may involve 5 the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.

The protein (or derivative) should most advantageously be prepared in particulate form with an 10 average particle size of less than 10 (or microns), most preferably 0.5 to 5 pm, for most effective delivery to the distal lung.

Carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and 15 sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DO PC. Natural or synthetic surfactants may be used. Polyethylene glycol may be vised (even apart from its use in derivatizing the protein or analog) . Dextrans, such as 20 eyelodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or 25 microspheres, inclusion complexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise Fc-OB protein, analogs or derivatives thereof, 30 dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure) . The nebulizer formulation may also 35 contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided 5 powder containing the protein (or derivative) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a 10 hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafiuoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a 15 surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing protein (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, 20 mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal delivery of the protein (or analog or derivative) is also contemplated. Nasal delivery allows 25 the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport 30 across other mucus membranes is also contemplated.

Dosage One skilled in the art will be able to ascertain effective dosages by administration and 35 observing the desired therapeutic effect. Due to the N-terminus modification of the OB protein, the present invention provides unexpected protein protection from degradation, and increases circulation time and stability, when compared to OB protein or C-terminus modification of the OB protein. One skilled in the art, 5 therefore, will be able to ascertain from these changes that an effective dosage may require lower doses or less frequent dosing.

Preferably, the formulation of the molecule will be such that between about .10 jig/kg/day and 10 10 mg/kg/day will yield the desired therapeutic effect. The effective dosages may be determined using diagnostic tools over time. For example, a diagnostic for measuring the amount of OB protein or Fc-OB fusion protein in the blood (or plasma or serum) may first be 15 used to determine endogenous levels of protein. Such diagnostic tools may be in the form of an antibody assay, such as an antibody sandwich assay. The amount of endogenous OB protein is quantified initially, and a baseline is determined. The therapeutic dosages are 20 determined as the quantification of endogenous and exogenous OB protein or Fc-OB fusion protein (that is, protein, analog or derivative found within the body, either self-produced or administered) is continued over the course of therapy. The dosages may therefore vary 25 over the course of therapy, with a relatively high dosage being used initially, until therapeutic benefit is seen, and lower dosages used to maintain the therapeutic benefits.

Ideally, in situations where solely reduction 30 in blood lipid levels is desired, where maintenance of reduction of blood lipid levels is desired, or an increase in lean body mass is desired, the dosage will be insufficient to result in weight loss. Thus, during an initial course of therapy of em obese person, dosages 35 may be administered whereby weight loss and concomitant blood lipid level lowering or concomitemt fat tissue decrease/lean mass increase is achieved. Once sufficient weight loss is achieved, a dosage sufficient to prevent re-gaining weight, yet sufficient to maintain desired blood lipid levels or lean mass increase (or, prevention 5 of lean mass depletion) may be administered. These dosages can be determined empirically, as the effects of OB or Fc-OB protein are reversible, (e.g., Campfield et al., Science 269: 546-549 (1995) at 547). Thus, if a dosage resulting in weight loss is observed when weight 10 loss is not desired, one would administer a lower dose in order to achieve the desired blood lipid levels or increase in lean tissue mass, yet maintain the desired weight.

For increasing an individual's sensitivity to 15 insulin, similar dosage considerations may be taken into account. Lean mass increase without weight loss may be achieved sufficient to decrease the amount of insulin (or, potentially, amylin, thiazolidinediones, or other potential diabetes treating drugs) an individual would 20 be administered for the treatment of diabetes.

For increasing overall strength, there may be similar dosage considerations. Lean mass increase with concomitant increase in overall strength may be achieved with doses insufficient to result in weight loss. Other 25 benefits, such as an increase in red blood cells (and oxygenation in the blood) and a decrease in bone resorption or osteoporosis may also be achieved in the absence of weight loss. combinations The present methods may be used in conjunction with other medicaments, such as those useful for the treatment of diabetes (e.g., insulin, possibly, thiazolidinediones, amylin, or antagonists thereof), 35 cholesterol and blood pressure lowering medicaments (such as those which reduce blood lipid levels or other cardiovascular medicaments), and activity increasing medicaments {e.g., amphetamines). Appetite suppressants may also be used (such as those affecting the levels of serotonin or neuropeptide Y) . Such administration may be 5 simultaneous or may be in seriatim.

In addition, the present methods may be used in conjunction with surgical procedures, such as cosmetic surgeries designed to alter the overall appearance of a body (e.g., liposuction or laser 10 surgeries designed to reduce body mass). The health benefits of cardiac surgeries, such as bypass surgeries or other surgeries designed to relieve a deleterious condition caused by blockage of blood vessels by fatty deposits, such as arterial plaque, may be increased with 15 concomitant use of the present compositions and methods. Methods to eliminate gall stones, such as ultrasonic or laser methods, may also be used either prior to, during or after a course of the present therapeutic methods. Furthermore, the present methods may be used as an 20 adjunct to surgeries or therapies for broken bones, damaged muscle, or other therapies which would be improved by an increase in lean tissue mass.

The following examples axe offered to more fully illustrate the invention, but are not to be 25 construed as limiting the scope thereof.

EXAMPLE 1: Use of Murine FC-OB Protein Via Subcutaneous Injection This example demonstrates that injection 30 subcutaneously of murine Fc-OB protein results in weight loss in normal mice. Normal (non-obese) CD1 mice were administered murine Fc-OB protein via subcutaneous injections over a 22 day time period. A dosage of 10 mg protein/kg body weight/day resulted in a 14% ( + /- 1.1%) 35 loss from baseline weight by the 22nd day of injections. A dosage of PBS resulted in a 3.9% (+/- 3.3%) loss from baseline weight by the 22nd day of injections. The weight loss with the use of 10 mg protein/kg body weight/day of Fc-OB protein in obese CD1 mice resulted in a 10% (+/- 4.3%) loss from baseline weight anri a 5 dosage of PBS resulted in a 8.7% (+/- 1.3%) loss from baseline weight, both by the 22nd day of injections Presented below are the percent (%) differences from baseline weight in CD1 mice (8 weeks old) : Table I Weight Loss Upon Subcutaneous Injection Time (days) Vehicle (PBS) Lean/Recombinant Fc-OB Fusion Protein Obese/Recombinant Fc-OB Fusion Protein 1-2 -.44 +/1.1 -3.6 +/- .41 -1.03 +/- 1.36 3-4 -1.07 +/- .13 -6.8 +/- 1.5 -2.7 +/- 1.1 -6 -.13 +/- 1.1 -9.5 +/- 1.2 -4.9 +/- .95 7-8 -.92 + /- .29 -12.5 +/- 1.6 -7.7 +/- 2.9 9-10 1.6 +/- 1.3 -12.6 +/- 1.9 -8.2 +/- 2.9 11-12 -1.98 +/- 1 -13.6 +/- 1.96 -8.6 +/- 2.9 13-14 -5.2 +/- 1.3 -14.6 +/-1.7 -10.1+/- 3.6 -16 -8.6 */- 0.1 -14.5 +/- 2 -9.4 +/- 2.2 17-18 -8.5 ♦/- .64 -16.1 +/- 1.8 -9.6 +1-2.99 19-20 -4.1 +/- .99 -16 +/- 1.5 -10.4 +/- 3.3 21-22 -3.9 +/- 3.3 -14.1 +/- 1.1 -10 +/- 4.3 As can be seen, at the end of a 22 day subcutaneous regime, animals receiving the FC-OB protein lost over 14.1% of their body weight in lean and 10% of body weight in obese, as compared to animals only receiving the PBS vehicle and as compared to baseline.

Surprisingly, animals receiving Fc-OB protein up to 22 days continued to loose weight up until 28 days, 4 days after the last injection. Normal (non-obese) CD1 mice administered 10 mg protein/kg body weight/day of murine Fc-OB protein via subcutaneous injections stopped at day 22 resulted in a 21% loss from 5 baseline weight at day 28 as compared to 14% loss at day 22. Likewise, obese CD1 mice administered 10 mg protein/kg body weight/day of murine Fc-OB protein stopped at day 22 resulted in a 13% loss from baseline weight at day 28 compared to 10% loss at day 22. At day 10 34 weight loss was maintained at 10% loss in obese mice where lean mice recovered to 5% loss. Controls in each system from day 22 through day 34 averaged from 4% in obese mice and 7% gain in lean mice.

KXAMPT/E 7 • Use of Human FC-OB Protein Via Subcutaneous Injection in C57 Mice This example demonstrates that injection subcutaneously of human Fc-OB protein results in weight loss in normal mice. Normal (non-obese) C57 mice were 20 administered human Fc-OB protein via subcutaneous injections over a 7 day time period. A dosage of 10 mg protein/kg body weight/day resulted in a 12% (+/- 1.3%) loss from baseline weight by the 7th day of injections. A dosage of 1 mg protein/kg body weight/day resulted in 25 a 8.9% (+/- 1.5%) loss from baseline weight by the 7th day of injections. The weight loss with the use of TO mg protein/kg body weight/day of human OB protein in obese C57 mice resulted in a 1.1% (+/- .99%) loss from baseline weight and a dosage of 1 mg protein/kg body 30 weight/day resulted in a 2.5% (+/- 1.1%) loss from baseline weight, both by the 7th day of injections.

Results Presented below are the percent (%) 35 differences from baseline weight in C57 mice (8 weeks old) : Table 2 Weight Loss Upon Subcutaneous Injection Tine (days) Vehicle (PBS) Recombinant Fc-OB Fusion Protein Recombinant OB Protein 1-2 .258 +/- 1.3 -6.4 + /- 1.6 -2.1 + /- .91 3-4 2.2 +/- 1.1 -12.1 +/- 1.5 -.78 +/- .36 -6 4.5 +/- 2 -11.5 +/- 1.5 -1.7 +/- .6 7-8 7.0 +/- 2.1 -11.9 +/- 1.6 0.1 +/- 1.2 9-10 9.0 +/-1.9 -11.5 +/- 1.3 7.2 +/- 2.7 11-12 +/- 3.8 -9 +/-1.4 .9 +/- 2.9 13-14 12.5 +/- 4.4 -9.5 +/- 1.6 12.3 +/- 6.4 -16 11.1 +/- 1.0 -3.0 +/- 1.5 .3 +/- 3.3 17-18 17.2 +/- 3.6 8.0 +/- 1.3 13.3 +/- 3.4 As can be seen, at the end of a day 17 after a 7 day subcutaneous regime at 10 mg/kg/day, animals receiving the FC-OB protein recovered to 8% of their body weight. Animals receiving dosages of 1 mg/kg/day after a 7 day subcutaneous regime returned to 6.4% of 10 body weight after 12 days.

These studies also show that during recovery periods from day 7 to day 22, after the last injection at day 7, body weight recovery is slower in the FcOB treated C57 mice that with the OB treated mice. This 15 suggests that the Fc-OB protein is not cleared as quickly as OB protein thereby causing the extended weight loss effect.

EXAMPLE 3: Dose Response of CF7 Mice Treated with Fc-QB 20 Fusion Protein An additional study demonstrated that there was a dose response to continuous administration of Fc-OB protein. In this study, obese CF7 mice, weighing -40 g were administered recombinant human Fc-OB protein using methods similar to the above example. The results are set forth in Table 3, below, (with % body-weight lost as compared to baseline, measured as above) : Table 3 Dose Response With Continuous Administration Dose Time % Reduction in Body Weight 0.25 mg/kg/dav Dav 5 4 0.5 mg/kg/dav Dav 5 12 1 mg/kg/day Day 5 16 As can be seen, increasing the dose from 0.25 mg/kg/day to 1 mg/kg/day increased the weight lost from 4% to 16%. It is also noteworthy that at day 5, the 1 mg/kg/day dosage resulted in a 16% reduction in body weight. These studies also showed slow weight recovery 15 rates to 0% suggesting that the Fc-OB protein is not quickly cleared thereby causing the extended weight loss effect.

EXAMPLE 4-. Pharmacokinetics of recombinant human Fc-OB 20 in CD-I Mice and Docs This study demonstrated the pharmacokinetic properties of recombinant human met Fc-OB protein in CD-I mice and dogs. Following intravenous or subcutaneous dosing at 1 mg/kg/day, serum concentrations 25 of recombinant human met Fc-OB protein and human met OB protein were determined by an enzyme-linked immunosorbent assay (ELISA).

In both species, an increase in exposure, as quantified by higher peak serum concentrations and 30 larger areas under-the-serum-concentration-curve (AUC), was observed when compared to recombinant met-human OB protein. Fc-OB has lower systemic clearance than recombinant met-human OB protein. This is seen in the lower clearance and longer half-life of Fc-OB over OB protein. The increase in size causes not only an 5 increase in protein stability, but also a decrease in the efficiency of renal clearance. As a result, Fc-OB is cleared slower from the systemic circulation. The increases in peak time, peak serum concentrations and AUC for Fc-OB protein are consistent with lower 10 clearance. Fc-OB protein will yield substantially higher systemic exposure when compared to OB protein. Results are shown in Table 4 below: Tahlo A Pharmacokinetic Properties Species CD-I Mice CD-I Mice Beagle Dogs Route of Administration Intravenous Subcutaneous Subcutaneous OB protein Fc-OB protein OB protein Fc-OB protein OB protein Fc-OB protein Dose Level (mcr/kg) 1 1 1 1 0.5 0.5 Peak Time (h) 0.14 6 2.8 8 Peak Serum Concentrat ion (na/mL) 1520 7550 300 . 1120 AUC (ng-h/mL) 1470 366000 1230 132000 2200* 52500 Half-life (h) 0.491 21.4 0.388 2.13 22.9 Clearance (nsL/h/kg) 681 2.73 EXAMPLE 5: This example demonstrates that in normal mice 20 which are not obese and do not have elevated blood lipid levels, administration of human recombinant Fc-OB protein results in a lowering of cholesterol, glucose and triglyceride levels. In addition, this example demonstrates that these levels remain low over a three day recovery period.

Normal CDl mice were administered recombinant human Fc-OB protein via subcutaneous injections. Blood samples were taken 24 hours after day 23, the last day of injection. As discussed above, the animals lost weight at the dosages administered. As shown in Table 10 5, the mice had substantial reduction of serum cholesterol, glucose and triglycerides in a dose-dependent fashion when compared to controls: Dose Glucose Cholesterol Triglycerides PBS 232.6 +/- 15.1 67.8 +/- 3.6 52.6 +/- 3.7 1 mg/kg/dav 225.8 +/- 29.1 54 +/- 5.6 43 + /- 8.7 mg/kg/dav 193.2 +/- 21.4 53.4 + /- 5.7 38 +/- 11 1 mq/kg every 2 days 242.0 +/- 9.3 52.6 +/- 4.4 40.8 +/- 7.2 mg/kg every 2 days 197.4 +/- 27.9 51.4 +/- 5.9 29.8 +/- 6.3 1 mg/kg every 3 days 244.8 + /- 19.5 60.8 + /- 7.3 54 + /- 7.1 mg/kg every 3 days 188 +/- 31.2 52.2 +/- 6.9 26.2 +/- 10.7 These data demonstrate that the Fc-OB protein, or analogs or derivatives thereof, are effective blood lipid lowering agents.

EXAMPLE 6: A obese human patient is administered human Fc-OB protein, or analog or derivative for the purpose of weight reduction. The obese patient also has elevated levels of blood lipids, including elevated levels of cholesterol, above 200 ing/100 ml. The patient attains a satisfactory weight reduction over the course of Fc-OB therapy. A maintenance dose of Fc-OB protein or analog or derivative is administered to the non-obese patient to maintain lowered blood lipid levels, including lowered cholesterol levels, below 200 mg/100 ml. The dose administered is insufficient to result in further weight loss. Administration is chronic. Levels of circulating Fc-OB protein or analog or derivative may be monitored vising a diagnostic kit, such as an antibody assay against the OB protein (or other antigenic source if applicable).

EXAMPLE 7: A non-obese human patient undergoes coronary bypass surgery or other invasive treatment to alleviate advanced stages arterial plaque formation. After the surgery, the patient is administered a maintenance dose 20 of Fc-OB protein or analog or derivative in order to prevent the re-formation of arterial plaque. The dose administered is insufficient to result in weight loss. Administration is chronic. Levels of circulating Fc-OB protein or analog or derivative may be monitored using 25 a diagnostic kit, such as an antibody assay against the OB protein (or other antigenic source if applicable).

EXAMPLE 8: A non-obese human patient experiences 30 hypertension due to restricted blood flow from clogged arteries. The patient is administered a dose of Fc-OB protein, or analog or derivative thereof sufficient to reduce arterial plaque resulting in clogged arteries. Thereafter, the patient is monitored for further 35 arterial plaque formation, and hypertension. If the condition re-appears, the patient is re-administered an effective amount of Fc-OB protein, analog or derivative sufficient to restore blood flow, yet insufficient to result in weight loss. Levels of circulating Fc-OB protein or analog or derivative may be monitored using a diagnostic kit, such as an antibody assay against the Fc-OB protein (or other antigenic source if applicable) .

EXAMPLE 9: A human patient experiences gall stones.

Either the gall stones are not removed and the formation of additional gall stones is sought to be avoided, or the gall stones are removed but the gall bladder remains (as, for example, using laser or ultrasonic surgery) and the formation of additional gall stones is sought to be avoided. The patient is administered an effective amount of Fc-OB protein, analog or derivative thereof to result in prevention of accumulation of additional gall stones or re-accumulation of gall stones. Levels of circulating Fc-OB protein or analog or derivative may be monitored using a diagnostic kit, such as an antibody assay against the Fc-OB protein (or other antigenic source if applicable).

Em&hS 3,0: A diabetic human patient desires to use decreased dosages of insulin for treatment of diabetes. The patient is administered an effective amount of Fc-OB protein, analog or derivative thereof to result in an increase in lean tissue mass. The patient's sensitivity to insulin increases, and the dosage of insulin necessary to alleviate symptoms of diabetes is decreased, either in terms of a decrease in the units of insulin needed, or in terms of a decrease in the number of injections of insulin needed per day. Levels of circulating Fc-OB protein or analog or derivative may be monitored using a diagnostic kit, such as an antibody assay against the OB protein (or other antigenic source if applicable).

EXAMPLE 11: A non-obese human patient desires an increase in lean tissue mass for therapeutic purposes, such as recovery from illness which depleted lean tissue mass. The patient is administered an effective amount of Fc-OB 10 protein, analog or derivative thereof to result in the desired increase in lean tissue mass. Increase in lean tissue mass is monitored using DEXA scanning. Levels of circulating Fc-OB protein or analog or derivative may be monitored vising a diagnostic kit, such as an antibody 15 assay against the OB protein (or other antigenic source if applicable).

MATERIALS AMD METHODS Animals. Wild type CDl mice and (+/+) C57B16 mice were used for the above examples. The age of the mice at the initial time point was 8 weeks, and the animals were weight stabilized.

Feeding and Weight Measurement. Mice were given ground rodent chow (PMI Feeds, Inc.) in powdered food feeders (All en town Caging and Equipment) which allowed a more accurate and sensitive measurement than use of regular block chow. Weight was measured at the 30 same time each day (2:00 p.m.), for the desired period. Body weight on the day prior to the injection was defined as baseline weight. The mice used weighed 18-22 grams.

Housing. Mice were single-housed, and maintained under humane conditions.

Administration of Protein or Vehicle. Protein (as described below) or vehicle (phosphate buffered saline, pH 7.4) were administered by subcutaneous 5 injections or intravenously.

Controls. Control animals were those who were injected with the vehicle alone without either Fc-OB fusion protein or OB protein added to the vehicle.

Protein. Sequence ID. Nos. 1, 2 and 3 set forth murine recombinant OB DNA and protein (Figure 1), and Sequence ID. Nos. 4, 5 and 6 set forth an analog recombinant human OB DNA and protein (Figure 2) . As noted 15 above recombinant human OB protein as in SEQ. ID.. NO. 6 has a lysine residue at position 35 and an isoleucine residue at position 74. Furthermore, the recombinant human protein set forth in Zhang et al., Nature, supra, and PCT publication WO 96/05309 (12/22/96) (both 20 incorporated by reference including figures) , and the murine and human analog recombinant proteins of Figures 1 and 2 are illustrative of the OB protein which may be used in forming the Fc-OB fusion protein .of the present methods of treatment and manufacture of a medicament. Other OB or 25 Fc proteins or analogs or derivatives thereof may also be used to form the Fc-OB fusion protein.

Herein, the first amino acid of the amino acid sequence for recombinant OB protein is referred to as +1, and is valine, and the amino acid at position -1 is 30 methionine. The C-terminal amino acid is number 146 (cysteine) (see Figures 1 and 2) . The first amino acid sequence for recombinant human Fc-OB protein of Figure 3 is referred to as +1, and is glutamate, and the amino acid at position -1 is methionine. The C-terminal amino 35 acid is number 378 (cysteine) . The first amino acid sequence for the recombinant human Fc-OB protein variant of Figure 4 is referred to as +1, and is glutamate, and the amino acid at position -1 is methionine. The C-terminal amino acid is number 378 (cysteine) . The first amino acid sequence for the recombinant human 5 Fc-OB protein variant of Figure 5 is referred to as +1, and is aspartic acid, and the amino acid at position -1 is methionine. The C-terminal amino acid is number 373 (cysteine) . The first amino acid sequence for the recombinant human Fc-OB protein variant of Figure 6 is 10 referred to as +1, and is aspartic acid, apd the amino acid at position -1 is methionine. The C-terminal amino acid is number is 373 (cysteine).

Expression Vector and Host Strain 15 The plasmid expression vector used is pAMG21 (ATCC accession number 98113), which is a derivative of pCFM1656 (ATCC accession number 69576) and contains appropriate restriction sites for insertion of genes downstream from the lux PR promoter (see US Patent No. 20 5,169,318 for a description of the lux expression system) . The Fc-OB DNA, described below and shown in Figures 3-6, was created and ligated into the expression vector pAMG21 linearized with restriction endonucleases Ndel and BamHI and transformed into the £. coli host 25 strain, FM5. £. coli FM5 cells were derived at Amgen Inc., Thousand Oaks, CA from £. coli K-12 strain (Bachmann, et al., Bacterial. Rev. 4Q.: 116-167 (1976)) and contain the integrated lambda phage repressor gene, cl857 (Sussman et al., C. R. Acad. Sci. 254: 1517-1579 (1962) ) . Vector production, cell transformation, and colony selection were performed by standard methods, (e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2d Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.) Host cells 35 were grown in LB media.

Fc-OB DNA Construction The plasmid pFc-A3 (described below) served as the source of sequence for human immunoglobulin IgG-1 heavy chain from amino acid number 99 (Glu) to the 5 natural carboxyl terminus. The human IgG-1 sequence can be obtained from Genebank (P01857) .

The human OB sequence is disclosed above as well as Zhang et al., Nature, supra. and PCT publication WO 96/05309 both incorporated by reference including 10 drawings. The OB DNA was ligated into the expression vector pCFM1656 linearized with restriction endonucleases Xbal and BamHI using standard cloning procedures, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2d Edition, Cold Spring Harbor 15 Laboratory Press, Cold Spting Harbor, N.Y.. The plasmid pCFM1656 carrying the OB DNA sequence served as the source of sequence for the recombinant human OB gene.

The genetic fusing of these two sequences was carried out by the method of PCR overlap extension (Ho, 20 S.N., et al., Site Directed Mutagenesis Bv Overlap Extension Using The Polymerase Chain Reaction. Gene 77:51-59(1989)). The product of the PCR was cleaved with restriction endonuclease Ndel to create a 5 1 -cohesive end and with restriction endonuclease BamHI 25 to create a 3'-cohesive terminus. The vector, pAMG21, was similarly cleaved. A ligation was performed with the fusion fragment and the linearized vector. Ligated DNA was transformed by electroporation into the E. coli host strain. Clones surviving on kanamycin (50|ig/ml) 30 selection agar plates were checked for expression of Fc-OB-sized protein. Plasmid from individual clones was isolated and the sequence of the gene coding region verified.

When additional modifications of the Fc-OB 35 gene were desired, the PCR technique was used again to engineer the changes. Two sets of changes were performed at the N-terminus of the Fc portion of the fusion protein (SEQ. ID. No. 9) to create the variants SEQ. ID. NOS. 12 and 15. Another variant was constructed to introduce four, amino acid substitutions 5 to ablate the Fc-receptor binding site (leucine at position 15 substituted with glutamate), and the complement (Clq) binding site (glutamate at position 98 substituted with alanine, lysine at position 100 substituted with alanine, and lysine at position 102 10 substituted with alanine (See, Xin Xiao Zheng et. al, J. Immunol. 154 : 5590-5600 (1995)). The template for this construct was Seq. ID. No. 15 and the resulting variant was SEQ. ID. Nos. 18.

PFC-?V3 vector construction A plasmid, pFc-A3, containing the region encoding the Fc portion of human immunoglobulin IgG-1 heavy chain (See Ellison, J. W. et. al, Nucleic Acids Res. iQ.:4071-4079 (1982)), from the first amino acid 20 Glu-99 of the hinge domain to the carboxyl terminus plus a 5' -NotI fusion site and 3'-Sail and Xbal sites, was made by PCR amplification of the human spleen cDNA library. PCR reactions were in a final volume of 100 ml and employed 2 units of Vent DNA polymerase in 20 mM 25 Tris-HCl (pH 8.8), 10 mM KC1, 10 fflM (NH4)2S04, 2 mM MgS04, 0.1% Triton X-100 with 400 mM each dNTP and 1 ng of the cDNA library to be amplified together with 1 uM of each primer. Reactions were initiated by denaturation at 95 °C for 2 min, followed by 30 cycles of 95 °C for 30 30 s, 55 °C for 30 s, and 73 °C for 2 min. The 5'- primer incorporated a NotI site immediately 5' to the first residue (Glu-99) of the hinge domain of IgG-1. The 3'-primer incorporated Sail and Xbal sites. The 717 base pair PCR product was digested with NotI and Sail, 35 the resulting DNA fragment was isolated by electrophoresis through 1% agarose and purified and cloned into NotI, Sail-digested pBluescript II KS vector (Stratagene). The insert in the resulting plasmid, pFc-A3, was sequenced to confirm the fidelity of the PCR reaction.

Methods for Production The methods below for production have been used to produce biologically active recombinant methionyl murine or human analog OB protein and Fc-OB fusion proteins. Similar 10 methods may be used to prepare biologically active methionyl human OB protein.

Eerroefltation process A batch fermentation process was used. Media 15 compositions care set forth below.

A portion of the media consisting of primarily nitrogen sources was sterilized (by raising temperature to 120~123°C for 25-35 minutes) in the fermentation vessel. Upon cooling, carbon, magnesium, phosphate, and 20 trace metal sources were added aseptically. An overnight culture of the above recombinant murine protein-producing bacteria of 500 mL (grown in LB broth) was added to the fermentor. When the culture optical density (measured at 600 nm as an indicator for cell 25 density) reached 15-25 absorption units, an autoinducer solution (0.5 mg/mL homoserine lactone) was added (1 mL/L) to the culture to induce the recombinant gene expression. The fermentation process was allowed to continue for additional 10 to 16 hours, followed by 30 harvesting the broth by centrifugation.

Media Composition: Batch: 34 g/L Yeast extract 78 g/L Soy peptone 0.9 g/L Potassium chloride .0 g/L Hexaphos 1.7 g/L Citric acid 120 g/L Glycerol 0.5 g/L MgS04•7H20 0.2 mL/L Trace Metal Solution 0.5 mL/L P2000 Antifoam Trace Metal Solution: Ferric Chloride (FeCl3•6H2O): 27 g/L Zinc Chloride (ZnCl2 -4H20): 2 g/L Cobalt Chloride (C0CI2 * 6H2O): 2 g/L Sodium Molybdate (NaMoO4*2H20): 2 g/L Calcium Chloride (CaCl2 *2H20) : 1 g/L Cupric Sulfate (CuS04'5H20): 1.9 g/L Boric Acid (H3BO3): 0.5 g/L Manganese Chloride (MnCl2*4H20): 1-6 g/L Sodium Citrate dihydrate: 73.5 g/L Purification Process for Human Fc-OB Fusion Protein Purification for human Fc-OB fusion protein was accomplished by the steps below (unless otherwise noted, the following steps were performed at 4'C). Purification for murine and human OB protein is 30 disclosed in PCT publication WO 96/05309, supra, herein incorporated by reference. 1. Cell paste. E. coli cell paste was suspended in 5 times volumes of distilled water. The cells in the water were further broken by two passes through a microfluidizer. The broken cells were centrifuged at 4.2k rpm for 1 hour in a Beckman JB-6 centrifuge with a J5-4.2 rotor. 2. Inclusion body wash. The supernatant from 5 above was removed and the pellet was resuspended with five volumes of distilled water. The mixture was centrifuged as in step 1. 3. Solubilization. The pellet was solubilized with 10 volumes of 50 mM tris, pH 8.5, 8 M guanidine hydrochloride, 10 mM dithiothreitol and stirred for one hour at room temperature. The solution is made 40 mM cystamine dihydrochloride and stirred for one hour. 4. The solution from step 3 is added to 20 to 30 volumes of the following refold solution: 50 mM tris, pH 8.5, 0.8 M arginine, 2 M urea, and 4 mM cysteine. The refold is stirred for 16 hours at 8°C.

. Buffer exchange. The solution from step 4 is concentrated and diafiltered into 10 mM tris, pH 8.5. 6. Acid precipitation. The solution from step 5 is adjusted to pH 4.75 with 50% glacial acid and incubated for 30 minutes at room temperature. The solution is filtered. 7. Cation exchange chromatography. The solution from step 6 is adjusted to pH 7.0 and loaded onto a CM Sepharose Fast Flow column at 10' C. A twenty column volume gradient is done at 10 mM phosphate, pH 7.0, 0 to 0.1 M NaCl. 8. Anion exchange chromatography. The CM elution pool from step 7 is diluted 5 fold with 5 mM tris, pH 7.5 and loaded onto a Q Sepharose Fast Flow at 10*C. A 20 column volume gradient is done at 10 mM tris, pH 7.5, 0 to 0.2M NaCl. 9. Hydrophobic interaction chromatography. The Q sepharose pool is made 0.75M ammonium sulfate and loaded on a methyl Macroprep hydrophobic interaction column at room temperature. A 20 column volume gradient is done at 10 mM phosphate, pH 7.0, 0.75M to 0M ammonium sulfate.

. Buffer exchange. The pool from step 9 is concentrated as necessary and dialyzed against PBS buffer.

While the present invention has been described in terms of preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations which come within the scope of the invention as claimed.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Mann, Michael B.

Hecht, Randy I. (ii) TITLE OF INVENTION: OB FUSION PROTEIN COMPOSITIONS AND METHODS (iii) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Aragen Inc.

(B) STREET: 1840 DeHavilland Drive (C) CITY: Thousand Oaks (D) STATE: CA (E) COUNTRY: USA (F) ZIP: 91320-1789 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release *1.0, Version #1.30 (Vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US 08/770,973 (B) FILING DATE: 20-DEC-1996 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Knight, Matthew W.

(B) REGISTRATION NUMBER: 36.846 (C) REFERENCE/DOCKET NUMBER: A-416 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 491 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: misc_£eature (B) LOCATION: 41 (D) OTHER INFORMATION: /note= 'Met = ATG" (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: TCTAGATTTG AGTTTTAACT TTTAGAAGGA GGAATAACAT ATGGTACCGA TCCAGAAAGT 60 TCAGGACGAC ACCAAAACCT TAATTAAAAC GATCGTTACG CGTATCAACG ACATCAGTCA 120 CACCCAGTCG GTCTCCGCTA AACAGCGTGT TACCGGTCTG GACTTCATCC CGGGTCTGCA 180 CCCGATCCTA AGCTTGTCCA AAATGGACCA GACCCTGGCT GTATACCAGC AGGTGTTAAC 240 CTCCCTGCCG TCCCAGAACG TTCTTCAGAT CGCTAACGAC CTCGAGAACC TTCGCGACCT 300 GCTGCACCTG CTGGCATTCT CCAAATCCTG CTCCCTGCCG CAGACCTCAG GTCTTCAGAA 360 ACCGGAATCC CTGGACGGGG TCCTGGAAGC ATCCCTGTAC AGCACCGAAG TTGTTGCTCT 420 GTCCCGTCTG CAGGGTTCCC TTCAGGACAT CCTTCAGCAG CTGGACGTTT CTCCGGAATG 480 TTAATGGATC C 491 {2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 491 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDN'A (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AGATCTAAAC TCAAAATTGA AAATCTTCCT CCTTATTGTA TACCATGGCT AGGTCTTTCA 60 AGTCCTGCTG 7GGTTTTGGA ATTAATTTTG CTAGCAATGC GCATAGTTGC TGTAGTCAGT 120 GTGGGTCAGC CAGAGGCGAT TTGTCGCACA ATGGCCAGAC CTGAAGTAGG GCCCAGACGT 180 GGGCTAGGAT TCGAACAGGT TTTACCTGGT CTGGGACCGA CATATGGTCG TCCACAATTG 240 GAGGGACGGC AGGGTCTTGC AAGAAGTCTA GCGATTGCTG GAGCTCTTGG AAGCGCTGGA 300 CGACGTGGAC GACCGTAAGA GGTTTAGGAC GAGGGACGGC GTCTGGAGTC CAGAAGTCTT 360 TGGCCTTAGG GACCTGCCCC AGGACCTTCG TAGGGACATG TCGTGGCTTC AACAACGAGA 420 CAGGGCAGAC GTCCCAAGGG AAGTCCTGTA GGAAGTCGTC GACCTGCAAA GAGGCCTTAC 480 AATTACCTAG G 491 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 147 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "Met (ATG) starts at -1" (xi) SEQUENCE DESCRIi Met Val Pro lie Gin 1 5 Thr lie Val Thr Arg 20 Ala Lys Gin Arg Val 35 lie Leu Ser Leu Ser 50 Val Leu Thr Ser Leu 65 Leu Glu Asn Leu Arg ' 85 Cys Ser Leu Pro Gin 100 Gly Val Leu Glu Ala 115 Arg Leu Gin Gly Ser 130 TION: SEQ ID NO:3: Lys Val Gin Asp Asp Thr 10 lie Asn Asp lie Ser His 25 Thr Gly Leu Asp Phe lie 40 Lys Met Asp Glr. Thr Leu 55 Pro Ser Gin Asn Val Leu 70 75 Asp Leu Leu His Leu Leu 90 Thr Ser Gly Leu Gin Lys 105 Ser Leu Tyr Ser Thr Glu 120 Leu Gin Asp lie Leu Gin 135 Lys Thr Leu lie Lys 15 Thr Gin Ser Val Ser 30 Pro Gly Leu His Pro 45 Ala Val Tyr Gin Gin 60 Gin lie Ala Asn Asp 80 Ala ?he Ser Lys Ser 95 Pro Glu Ser Leu Asp 110 Val Val Ala Leu Ser 125 Gin Leu Asp Val Ser 140 Pro Glu Cys 145 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 454 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 4 (D) OTHER INFORMATION: /note= "Met = ATG" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CATATGGTAC CGATCCAGAA AGTTCAGGAC GACACCAAAA CCTTAATTAA AACGATCGTT ACGCGTATCA ACGACATCAG TCACACCCAG TCGGTGAGCT CTAAACAGCG TGTTACAGGC CTGGACTTCA TCCCGGGTCT GCACCCGATC CTGACCTTGT CCAAAATGGA CCAGACCCTG GCTGTATACC AGCAGATCTT AACCTCCATG CCGTCCCGTA ACGTTCTTCA GATCTCTAAC GACCTCGAGA ACCTTCGCGA CCTGCTGCAC GTGCTGGCAT TCTCCAAATC CTGCCACCTG CCATGGGCTT CAGGTCTTGA GACTCTGGAC TCTCTGGGCG GGGTCCTGGA AGCATCCGGT TACAGCACCG AAGTTGTTGC TCTGTCCCGT CTGCAGGGTT CCCTTCAGGA CATGCTTTGG CAGCTGGACC TGTCTCCGGG TTGTTAATGG ATCC (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 454 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GTATACCATG GCTAGGTCTT TCAAGTCCTG CTGTGGTTTT GGAATTAATT TTGCTAGCAA TGCGCATAGT TGCTGTAGTC AGTGTGGGTC AGCCACTCGA GATTTGTCGC ACAATGTCCG 60 120 * 180 240 300 360 420 454 GACCTGAAGT AGGGCCCAGA CGTGGGCTAG GACTGGAACA GGTTTTACCT GGTCTGGGAC 180 CGACATATGG TCGTCTAGAA TTGGAGGTAC GGCAGGGCAT TGCAAGAAGT CTAGAGATTG 240 CTGGAGCTCT TGGAAGCGCT GGACGACGTG CACGACCGTA AGAGGTTTAG GACGGTGGAC 300 GGTACCCGAA GTCCAGAACT CTGAGACCTG AGAGACCCGC CCCAGGACCT TCGTAGGCCA 360 ATGTCGTGGC TTCAACAACG AGACAGGGCA GACGTCCCAA GGGAAGTCCT GTACGAAACC 420 GTCGACCTGG ACAGAGGCCC AACAATTACC TAGG 454 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "Met (ATG) starts at -1" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys 15 10 15 Thr lie Val Thr Arg lie Asn Asp He Ser His Thr Gin Ser Val Ser 20 25 30 Ser Lys Gin Arg Val Thr Gly Leu Asp Phe lie Pro Giy Leu His Pro 35 40 45 lie Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin 50 55 60 lie Leu Thr Ser Met Pro Ser Arg Asn Val Leu Gin lie Ser Asn Asp 65 70 75 80 Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser 85 90 95 Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly 100 105 110 Gly Val Leu Glu Ala Ser Gly JTyr Ser Thr Glu Val Val Ala Leu Ser 115 120 125 Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser 130 135 140 Pro Gly Cys 145 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1150 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: i?.isc_feature (B) LOCATION: 4 (D) OTHER INFORMATION: /note= "Met = ATG" (xi) SEQUENCE DESCRIPTION: SSQ ID NO:7: CATATGGAAC CCAAATCTTG TGACAAAACT CACACATGCC CACCGTGCCC AGCACCTGAA 60 CTCCTGGGGG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC CCTCATGATC 120 TCCCGGACCC CTGAGGTCAC ATGCGTGGTG GTGGACGTGA GCCACGAAGA CCCTGAGGTC 180 AAGTTCAACT GGTACGTGGA CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG 240 GAGCAGTACA ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 300 CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC CCCCATCGAG" 360 AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC AGGTGTACAC CCTGCCCCCA 420 TCCCGGGATG AGCTGACCAA GAACCAGGTC AGCCTGACCT GCCTGGTCAA AGGCTTCTAT 480 CCCAGCGACA TCGCCGTGGA GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC 540 ACGCCTCCCG TGCTGGACTC CGACGGGTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC 600 AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCAC 660 AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA AAGTACCGAT CCAGAAAGTT 720 CAGGACGACA CCAAAACCTT AATTAAAACG ATCGTTACGC GTATCAACGA CATCAGTCAC 780 ACCCAGTCGG TGAGCTCTAA ACAGAAAGT? ACAGGCCTGG ACTTCATCCC GGGTCTGCAC 840 CCGATCCTGA CCTTGTCCAA AATGGACCAG ACCCTGGCTG TATACCAGCA GATCTTAACC 900 TCCATGCCGT CCCGTAACGT TATCCAGATC TCTAACGACC TCGAGAACCT TCGCGACCTG 960 CTGCACGTGC TGGCATTCTC CAAATCCTGC CACCTGCCAT GGGCTTCAGG TCTTGAGACT 1020 CTGGACTCTC TGGGCGGGGT CCTGGAAGCA TCCGGTTACA GCACCGAAGT TGTTGCTCTG 1080 TCCCGTCTGC AGGGTTCCCT TCAGGACATG CTTTGGCAGC TGGACCTGTC TCCGGGTTGT 1140 TAATGGATCC 1150 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1150 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GTATACCTTG GGTTTAGAAC ACTGTTTTGA GTGTGTACGG GTGGCACGGG TCGTGGACTT 60 GAGGACCCCC CTGGCAGTCA GAAGGAGAAG GGGGGTTTTG GGTTCCTGTG C-GAGTACTAG 120 AGGGCCTGGG GACTCCAGTG TACGCACCAC CACCTGCACT CGGTGCTTCT GGGACTCCAG 180 TTCAAGTTGA CCATGCACCT GCCGCACCTC CACGTATTAC GGTTCTGTTT CGGCGCCCTC 240 CTCGTCATGT TGTCGTGCAT GGCACACCAG TCGCAGGAGT GGCAGGACGT GGTCCTGACC - 300 GACTTACCGT TCCTCATGTT CACGTTCCAG AGGTTGTTTC GGGAGGGTCG GGGGTAGCTC 360 TTTTGGTAGA. GGTTTCGGTT TCCCGTCGGG GCTCTTGGTG TCCACATGTG GGACGGGGGT 420 AGGGCCCTAC TCGACTGGTT CTTGGTCCAG TCGGACTGGA CGGACCAGTT TCCGAAGATA 480 GGGTCGCTGT AGCGGCACCT CACCCTCTCG TTACCCGTCG GCCTCTTGTT GATGTTCTGG 540 TGCGGAGGGC ACGACCTGAG GCTGCCGAGG AAGAAGGAGA TGTCGTTCGA GTGGCACCTG 600 TTCTCGTCCA CCGTCGTCCC CTTGCAGAAG AGTACGAGGC ACTACGTACT CCGAGACGTG 660 TTGGTGATGT GCGTCTTCTC GGAGAGGGAC AGAGGCCCAT TTCATGGCTA GGTCTTTCAA 720 GTCCTGCTGT GGTTTTGGAA TTAATTTTGC TAGCAATGCG CATAGTTGCT GTAGTCAGTG 780 TGGGTCAGCC ACTCGAGATT TGTCTTTCAA TGTCCGGACC TGAAGTAGGG CCCAGACGTG 840 GGCTAGGACT GGAACAGGTT TTACCTGGTC TGGGACCGAC ATATGGTCGT CTAGAATTGG 900 AGGTACGGCA GGGCATTGCA ATAGGTCTAG AGATTGCTGG AGCTCTTGGA AGCGCTGGAC 960 GACGTGCACG ACCGTAAGAG GTTTAGGACG GTGGACGGTA CCCGAAGTCC AGAACTC7GA 1020 GACCTGAGAG ACCCGCCCCA GGACCTTCGT AGGCCAATGT CGTGGCTTCA ACAACGAGAC 1080 AGGGCAGACG TCCCAAGGGA AGTCCTGTAC GAAACCGTCG ACCTGGACAG AGGCCCAACA 1140 ATTACCTAGG 1150 (2) INFORMATION FOR SEQ NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 379 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "Met (ATG) starts at -1" (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 15 10 15 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25 30 Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val 35 40 45 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 50 55 60 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Fro Arg Glu Glu 65 70 75 80 Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 85 90 95 Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100 105 110 Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin 115 120 125 Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 130 135 140 Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 145 150 155 160 Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn 165 170 175 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Glv Ser Phe Phe Leu 180 185 190 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val 195 200 205 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin 210 215 220 Lys Ser Leu Ser Leu Ser Pro Gly Lys Val Pro lie Gin Lys Val Gin 225 230 235 240 Asp Asp Thr Lys Thr Leu lie Lys Thr lie Val Thr Arg He Asn Asp 245 250 255 lie Ser His Thr Gin Ser Val Ser Ser Lys Gin Lys Val Thr Gly Leu 260 265 270 Asp Phe lie Pro Gly Leu His Pro lie Leu Thr Leu Ser Lys Met Asp 275 280 285 Gin Thr Leu Ala Val Tyr Gin Gin lie Leu Thr Ser Met Pro Ser Arg 290 295 300 Asn Val lie Gin He Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu 305 . 310 315 320 His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly 325 330 335 Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr 340 345 350 Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gin Gly Ser Leu Gin Asp 355 360 365 Met Leu Trp Gin Leu Asp Leu Ser Pro Gly Cys 370 375 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1150 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double <D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: reisc_feature (B) LOCATION: 4 (D) OTHER INFORMATION: /note= "Met. = ATG" (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CATATGGAAC CAAAATCTGC TGACAAAACT CACACATGTC CACCTTGTCC AGCTCCGGAA 60 CTCCTGGGGG GTCCTTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC CCTCATGATC 120 TCCCGGACCC CTGAGGTCAC ATGCGTGGTG GTGGACGTGA GCCACGAAGA CCCTGAGGTC 180 AAGTTCAACT GGTACGTGGA CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG 240 GAGCAGTACA ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 300 CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC CCCCATCGAG 360 AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC AGGTGTACAC CCTGCCCCCA 420 TCCCGGGATG AGCTGACCAA GAACCAGGTC AGCCTGACCT GCCTGGTCAA AGGCTTCTAT 480 CCCAGCGACA TCGCCGTGGA GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC ~ 540 ACGCCTCCCG TGCTGGACTC CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC 600 AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCAC 660 AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA AAGTACCGAT CCAGAAAGTT 720 CAGGACGACA CCAAAACCTT AATTAAAACG ATCGTTACGC GTATCAACGA CATCAGTCAC 780 ACCCAGTCGG TGAGCTCTAA ACAGAAAGTT ACAGGCCTGG ACTTCATCCC GGGTCTGCAC 840 CCGATCCTGA CCTTGTCCAA AATGGACCAG ACCCTGGCTG TATACCAGCA GATCTTAACC 900 TCCATGCCGT CCCGTAACGT TATCCAGATC TCTAACGACC 7CGAGAACCT TCGCGACCTG 960 CTGCACGTGC TGGCATTCTC CAAATCCTGC CACCTGCCAT GGGCTTCAGG TCTTGAGACT 1020 CTGGACTCTC TGGGCGGGGT CCTGGAAGCA TCCGGTTACA GCACCGAAGT TGTTGCTCTG 1080 TCCCGTCTGC AGGGTTCCCT TCAGGACATG CTTTGGCAGC TGGACCTGTC TCCGGGTTGT 1140 TAATGGATCC 1150 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1150 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GTATACCTTG GTTTTAGACG ACTGTTTTGA GTGTGTACAG GTGGAACAGG TCGAGGCCTT 60 GAGGACCCCC CAGGAAGTCA GAAGGAGAAG GGGGGTTTTG GGTTCCTGTG GGAGTACTAG 120 AGGGCCTGGG GACTCCAGTG TACGCACCAC CACCTGCACT CGGTGCTTCT GGGACTCCAG 180 TTCAAGTTGA CCATGCACCT GCCGCACCTC CACGTATTAC GGTTCTGTTT CGGCGCCCTC 240 CTCGTCATGT TGTCGTGCAT GGCACACCAG TCGCAGGAGT GGCAGGACGT GGTCCTC-ACC 300 GACTTACCGT TCCTCATGTT CACGTTCCAG AGGTTGTTTC GGGAGGGTCG GGGGTAGCTC 360 TTTTGGTAGA GGTTTCGGTT TCCCGTCGGG GCTCTTGGTG TCCACATGTG GGACGGGGGT 420 AGGGCCCTAC TCGACTGGTT CTTGGTCCAG TCGGACTGGA CGGACCAGTT TCCGAAGATA"- 480 GGGTCGCTGT AGCGGCACCT CACCCTCTCG TTACCCGTCG GCCTCTTGTT GATGTTCTGG 540 TGCGGAGGGC ACGACCTGAG GCTGCCGAGG AAGAAGGAGA TGTCGTTCGA GTGGCACCTG 600 TTCTCGTCCA CCGTCGTCCC .CTTGCAGAAG AGTACGAGGC ACTACGTACT CCGAGACGTG 660 TTGGTGATGT GCGTCTTCTC GGAGAGGGAC AGAGGCCCAT TTCATGGCTA GGTCTTTCAA 720 GTCCTGCTGT GGTTTTGGAA TTAATTTTGC TAGCAATGCG CATAGTTGCT GTAGTCAGTG 780 TGGGTCAGCC ACTCGAGATT TGTCTTTCAA TGTCCGGACC TGAAGTAGGG CCCAGACGTG 840 GGCTAGGACT GGAACAGGTT TTACCTGGTC TGGGACCGAC ATATGGTCGT CTAGAATTGG 900 AGGTACGGCA GGGCATTGCA ATAGGTCTAG AGATTGCTGG AGCTCTTGGA AGCGCTGGAC 960 GACGTGCACG ACCGTAAGAG GTTTAGGACG GTGGACGGTA CCCGAAGTCC AGAACTCTGA 1020 GACCTGAGAG ACCCGCCCCA GGACCTTCGT AGGCCAATGT CGTGGCTTCA ACAACGAGAC 1080 AGGGCAGACG TCCCAAGGGA AGTCCTGTAC GAAACCGTCG ACCTGGACAG AGGCCCAACA 1140 ATTACCTAGG 1150 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 379 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: -r.known (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1 (D) OTHER INFORMATION: /r.ote= "Met (ATG) starts at -1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Met Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25 30 Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val 35 40 45 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 50 55 60 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 65 70 75 80 Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 85 90 95 Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100 105 110 Ala Leu Pro Ala Pro lie Glu Lys 115 120 Pro Arg Glu Pro Gin Val Tyr Thr 130 135 Thr Lys Asn Gin Val Ser Leu Thr 145 150 Ser Asp lie Ala Val Glu Trp Glu 165 Tyr Lys Thr Thr Pro Pro Val Leu 180 Tyr Ser Lys Leu Thr Val Asp Lys 195 200 Thr He Ser Lys Ala Lys Gly Gin 125 Leu Pro Pro Ser Arg Asp Glu Leu 140 Cys Leu Val Lys Gly Phe Tyr Pro 155 160 Ser Asn Gly Gin Pro Glu Asn Asn 170 175 Asp Ser Asp Gly Ser Phe Phe Leu 185 190 Ser Arg Trp Gin Gin Gly Asn Val 205 Phe Ser Cys Ser Val Met His Giu Ala Leu His Asn His Tyr Thr Gin 210 215 220 Lys Ser Leu Ser Leu Ser Pro Gly Lys Val Pro lie Gin Lys Val Gin 225 230 235 240 Asp Asp Thr Lys Thr Leu lie Lys Thr lie Val Thr Arg lie Asn Asp 245 250 255 lie Ser His Thr Gin Ser Val Ser Ser Lys Gin Lys Val Thr Giy Leu 260 265 270 Asp Phe He Pro Gly Leu His Pro lie Leu Thr Leu Ser Lys Met Asp 275 280 285 Gin Thr Leu Ala Val Tyr Gin Gin He Leu Thr Ser Met Pro Ser Arg 290 295 300 Asn Val He Gin He Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu 305 310 315 320 His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly 325 330 335 Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr 340 345 350 Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gin Gly Ser Leu Gin Asp 355 360 365 Met Leu Trp Gin Leu Asp Leu Ser Pro Giy Cys 370 375 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1135 base pairs (B) TYPE: nucleic acid (C> STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 4 (D) OTHER INFORMATION: /note= "Met = ATG" (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CATATGGACA AAACTCACAC ATGTCCACCT TGTCCAGCTC CGGAACTCC? GGGGGGTCCT 60 TCAGTCTTCC TCTTCCCCCC AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG 120 GTCACATGCG TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTAC 180 GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA GTACAACAGC 240 ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG ACTGGCTGAA TGGCAAGGAG 300 TACAAGTGCA AGGTCTCCAA CAAAGCCCTC CCAGCCCCCA TCGAGAAAAC CATCTCCAAA 360 GCCAAAGGGC AGCCCCGAGA ACCACAGGTG TACACCCTGC CCCCATCCCG GGATGAGCTG 420 ACCAAGAACC AGGTCAGCCT GACCTGCCTG GTCAAAGGCT 7CTATCCCAG CGACATCGCC 480 GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC TCCCGTGCTG 540 GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG TGGACAAGAG CAGGTGGCAG = 600 CAGGGGAACG TCTTCTCATG CTCCGTGATG CATGAGGCTC TGCACAACCA CTACACGCAG 660 AAGAGCCTCT CCCTGTCTCC GGGTAAAGTA CCGATCCAGA AAGTTCAGGA CGACACCAAA 720 ACCTTAATTA AAACGATCGT TACGCGTATC AACGACATCA GTCACACCCA GTCGGTGAGC 780 TCTAAACAGA AAGTTACAGG CCTGGACTTC ATCCCGGGTC TGCACCCGA7 CCTGACCTTG 840 TCCAAAATGG ACCAGACCCT GGCTGTATAC CAGCAGATCT TAACCTCCAT GCCGTCCCGT 900 AACGTTATCC AGATCTCTAA CGACCTCGAG AACCTTCGCG ACCTGCTGCA CGTGCTGGCA 960 TTCTCCAAAT CCTGCCACCT GCCATGGGCT TCAGGTCTTG AGACTCTGGA CTCTCTGGGC 1020 GGGGTCCTGG AAGCATCCGG TTACAGCACC GAAGTTGTTG CTCTGTCCCG TCTGCAGGGT 1080 TCCCTTCAGG ACATGCTTTG GCAGCTGGAC CTGTCTCCGG GTTGTTAATG GATCC 1135 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1135 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GTATACCTGT TTTGAGTGTG TACAGGTGGA ACAGGTCGAG GCCTTGAGGA CCCCCCAGGA 60 AGTCAGAAGG AGAAGGGGGG TTTTGGGTTC CTGTGGGAGT ACTAGAGGGC CTGGGGACTC 120 .

CAGTGTACGC ACCACCACCT GCACTCGGTG CTTCTGGGAC TCCAGTTCAA GTTGACCATG 180 CACCTGCCGC ACCTCCACGT ATTACGGTTC TGTTTCGGCG CCCTCCTCGT CATGTTGTCG 240 TGCATGGCAC ACCAGTCGCA GGAGTGGCAG GACGTGGTCC TGACCGACTT ACCGTTCCTC 300 ATGTTCACGT TCCAGAGGTT GTTTCGGGAG GGTCGGGGGT AGCTCTTTTG GTAGAGGTTT 360 CGGTTTCCCG TCGGGGCTCT TGGTGTCCAC ATGTGGGACG GGGGTAGGGC CCTACTCGAC 420 TGGTTCTTGG TCCAGTCGGA CTGGACGGAC CAGTTTCCGA AGATAGGGTC GCTGTAGCGG 480 CACCTCACCC TCTCGTTACC CGTCGGCCTC TTGTTGATGT TCTGGTGCGG AGGGCACGAC 540 CTGAGGCTGC CGAGGAAGAA GGAGATGTCG TTCGAGTGGC ACCTGTTCTC GTCCACCGTC 600 GTCCCCTTGC AGAAGAGTAC GAGGCACTAC GTACTCCGAG ACGTGTTGGT GATGTGCGTCr 660 TTCTCGGAGA GGGACAGAGG CCCATTTCAT GGCTAGGTCT TTCAAGTCCT GCTGTGGTTT 720 TGGAATTAAT TTTGCTAGCA ATGCGCATAG TTGCTGTAGT CAGTGTGGGT CAGCCAQTCG 780 AGATTTGTCT TTCAATGTCC GGACCTGAAG TAGGGCCCAG ACGTGGGCTA GGACTGGAAC 840 AGGTTTTACC TGGTCTGGGA CCGACATATG GTCGTCTAGA ATTGGAGGTA CGGCAGGGCA 900 TTGCAATAGG TCTAGAGATT GCTGGAGCTC TTGGAAGCGC TGGACGACGT GCACGACCGT 960 AAGAGGTTTA GGACGGTGGA CGGTACCCGA AGTCCAGAAC TCTGAGACCT GAGAGACCCG 1020 CCCCAGGACC TTCGTAGGCC AATGTCGTGG CTTCAACAAC GAGACAGGGC AGACGTCCCA 1080 AGGGAAGTCC TGTACGAAAC CGTCGACCTG GACAGAGGCC CAACAATTAC CTAGG 1135 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 374 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "Met (ATG) starts at -1" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 Ms Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn 85 90 95 ~- Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 lie G3ru Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr 225 230 235 240 Leu lie Lys Thr lie Val Thr Arg lie Asn Asp lie Ser His Thr Gin 245 250 255 Ser Val Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe lie Pro Gly 260 " 265 270 Leu His Pro lie Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val 275 280 285 Tyr Gin Gin lie Leu Thr Ser Met Pro Ser Arg Asn Val lie Gin lie 290 295 300 Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe 305 310 315 320 Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp 325 330 335 Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val 340 345 350 Ala Leu Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu 355 360 365 Asp Leu Ser Pro Gly Cys 370 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1135 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double <D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (ix) FEATURE: (A) NAME/KEY: nusc_feature (B) LOCATION: 4 (D) OTHER INFORMATION: /note= "Met = ATG" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CATATGGACA AAACTCACAC ATGCCCACCG TGCCCAGCTC CGGAACTCGA AGGTGGTCCG 60 TCAGTCTTCC TCTTCCCCCC AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG 120 GTCACATGCG TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTAC 180 GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA GTACAACAGC 240 ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG ACTGGCTGAA TGGCAAAGCT 300 TACGCATGCG CGGTCTCCAA CAAAGCCCTC CCAGCCCCCA TCGAGAAAAC CATCTCCAAA 360 GCCAAAGGGC AGCCCCGAGA ACCACAGGTG TACACCCTGC CCCCATCCCG GGATGAGCTG 420 ACCAAGAACC AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCC 480 GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC TCCCGTGCTG 540 GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG TGGACAAGAG CAGGTGGCAG 600 CAGGGGAACG TCTTCTCATG CTCCGTGATG CATGAGGCTC TGCACAACCA CTACACGCAG 660 AAGAGCCTCT CCCTGTCTCC GGGTAAAGTA CCGATCCAGA AAGTTCAGGA CGACACCAAA 720 ACCTTAATTA AAACGATCGT TACGCGTATC AACGACATCA GTCACACCCA GTCGGTGAGC 780 TCTAAACAGA AAGTTACAGG CCTGGACTTC ATCCCGGGTC TGCACCCGAT CCTGACCTTG 840 TCCAAAATGG ACCAGACCCT GGCTGTATAC CAGCAGATCT TAACCTCCAT GCCGTCCCGT 900 AACGTTATCC AGATCTCTAA CGACCTCGAG AACCTTCGCG ACCTGCTGCA CGTGCTGGCA " 960 TTCTCCAAAT CCTGCCACCT GCCATGGGCT TCAGGTCTTG AGACTCTGGA CTCTCTGGGC 1020 GGGGTCCTGG AAGCATCCGG TTACAGCACC GAAGTTGTTG CTCTGTCCCG TCTGCAGGGT 1080 TCCCTTCAGG ACATGCTTTG GCAGCTGGAC CTGTCTCCGG GTTGTTAATG GATCC 1135 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1135 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GTATACCTGT TTTGAGTGTG TACGGGTGGC ACGGGTCGAG GCCTTGAGCT TCCACCAGGC 60 AGTCAGAAGG AGAAGGGGGG TTTTGGGTTC CTGTGGGAGT ACTAGAGGGC CTGGGGACTC 120 CAGTGTACGC ACCACCACCT GCACTCGGTG CTTCTGGGAC TCCAGTTCAA GTTGACCATG 180 CACCTGCCGC ACCTCCACGT ATTACGGTTC TGTTTCGGCG CCCTCCTCGT CATGTTGTCG 240 TGCATGGCAC ACCAGTCGCA GGAGTGGCAG GACGTGGTCC TGACCGACTT ACCGTTTCGA 300 ATGCGTACGC GCCAGAGGTT GTTTCGGGAG GGTCGGGGGT AGCTCTTTTG GTAGAGGTTT 360 CGGTTTCCCG TCGGGGCTCT TGGTGTCCAC ATGTGGGACG GGGGTAGGGC CCTACTCGAC 420 TGGTTCTTGG TCCAGTCGGA CTGGACGGAC CAGTTTCCGA AGATAGGGTC GCTGTAGCGG 480 CACCTCACCC TCTCGTTACC CGTCGGCCTC TTGTTGATGT TCTGGTGCGG AGGGCACGAC 540 CTGAGGCTGC CGAGGAAGAA GGAGATGTCG TTCGAGTGGC ACCTGTTCTC GTCCACCGTC 600 GTCCCCTTGC AGAAGAGTAC GAGGCACTAC GTACTCCGAG ACGTGTTGGT GATGTGCGTC 660 TTCTCGGAGA GGGACAGAGG CCCATTTCAT GGCTAGGTCT TTCAAGTCCT GCTGTGGTTT 720 TGGAATTAAT TTTGCTAGCA ATGCGCATAG TTGCTGTAGT CAGTGTGGGT CAGCCACTCG 780 AGATTTGTCT TTCAATGTCC GGACCTGAAG TAGGGCCCAG ACGTGGGCTA GGACTGGAAC 840 AGGTTTTACC TGGTCTGGGA CCGACATATG GTCGTCTAGA ATTGGAGGTA CGGCAGGGCA 900 TTGCAATAGG TCTAGAGATT GCTGGAGCTC TTGGAAGCGC TGGACGACGT GCACGACCGT 960 AAGAGGTTTA GGACGGTGGA CGGTACCCGA AGTCCAGAAC TCTGAGACCT GAGAGACCCG 1020 CCCCAGGACC TTCGTAGGCC AATGTCGTGG CTTCAACAAC GAGACAGGGC AGACGTCCCA 10 80 AGGGAAGTCC TGTACGAAAC CGTCGACCTG GACAGAGGCC CAACAATTAC CTAGG 1135 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 374 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "Met (ATG) starts at -1" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Glu 15 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn 85 90 95 Gly Lys Ala Tyr Ala Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr 225 230 235 240 Leu lie Lys Thr lie Val Thr Arg lie Asn Asp lie Ser His Thr Gin 245 250 255 Ser Val Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe lie Pro Gly 260 265 270 Leu His Pro lie Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val 275 280 285 Tyr Gin Gin lie Leu Thr Ser Met Pro Ser Arg Asn Val lie Gin lie 290 295 300 Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe 305 310 315 320 Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp 325 330 335 Ser Leu Gly Gly Val Leu Glu Ala 340 Ala Leu Ser Arg Leu Gin Gly Ser 355 360 Ser Gly Tyr Ser Thr Glu Val Val 345 350 Leu Gin Asp Met Leu Trp Gin Leu 365 Asp Leu Ser Pro Gly Cys 370 INTELLECTUAL PROPERTY OFFICE OF N.z 1 5 MAR 2004 received

Claims (19)

WHAT WE CLAIM IS:

1. A DNA sequence encoding a fusion protein optionally having an N-terminal methionine, said fusion protein comprising an antibody constant region or portion thereof attached to the N-terminus of a human OB protein, wherein said OB protein is selected from the group consisting of: (a) the amino acid sequence 1-146 as set forth in SEQ ID NO: 6; (b) the amino acid sequence 1-14 6 as set forth in SEQ ID NO: 6 having a lysine residue at position 35 and an isoleucine residue at position 74; (c) the amino acid sequence of subparts (a) or (b) lacking a glutaminyl residue at position 28; and (d) the amino acid sequence of subparts (a), (b), or (c), wherein one or more amino acids selected from the group consisting of amino acids 32, 35, 50, 64, 68, 71, 74, 77, 89, 97, 100, 105, 106, 107, 108, 111, 118, 136, 138, 142 and 145 is substituted with the corresponding amino acid present in SEQ ID NO: 3 or a conserved amino acid.

2. A process for producing a fusion protein optionally having an N-terminal methionine, said fusion protein comprising an antibody constant region or portion thereof attached to the N-terminus of a human OB protein, said process comprising expression of the DNA sequence according to claim 1 -71 - in an isolated transformed host cell and isolating said fusion protein.

3. A process for producing a fusion protein optionally having an N-terminal methionine, said fusion protein comprising an antibody constant region or portion thereof attached to the N-terminus of a human OB protein, said process comprising expression of the DNA sequence according to claim 1 in a transformed non-human host cell and isolating said fusion protein.

4. A vector comprising the DNA sequence of claim 1.

5. An isolated host cell transformed or transfected with the DNA sequence of claim 1.

6. A non-human host cell transformed or transfected with the DNA sequence of claim 1.

7. The DNA sequence of claim 1 for use in treating obesity or increasing insulin sensitivity.

8. A method of treatment of a disorder selected from the group consisting of: excess weight; diabetes; high blood lipid level; arterial sclerosis; arterial plaque; insufficient lean tissue mass; insufficient sensitivity to insulin; and stroke; wherein the method consists of administering a therapeutically effective amount of the DNA sequence according to claim 1 to a non-human animal.

9. A method of reducing or preventing gall stones formation; wherein the method consists of administering a INTELLECTUAL PROPERTY OFRCE OF N.2 15 MAR 2004 received -72- therapeutically effective amount of the DNA sequence according to claim 1 to a non-human animal.

10. A use of the DNA sequence according to claim 1 in the preparation of a medicament for the treatment of a disorder selected from the group consisting of: excess weight; diabetes; high blood lipid level; arterial sclerosis; arterial plaque; insufficient lean tissue mass; insufficient sensitivity to insulin; and stroke; in a patient in need thereof.

11. A use of the DNA sequence according to claim 1 in the preparation of a medicament for reducing or preventing gall stones formation in a patient in need thereof.

12. A fusion protein optionally having an N-terminal methionine, said fusion protein comprising an antibody constant region or portion thereof attached to the N-terminus of a human OB protein, when produced by a process of claim 2.

13. A DNA sequence as claimed in claim 1 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

14. A process, as claimed in claim 2 or claim 3, for producing a fusion protein substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

15. A vector as claimed in claim 4 substantially as herein described with reference to any example thereof and with or / without reference to the accompanying drawings. 15 MAR 2004 -73-

16. A host cell as claimed in claim 5 or claim 6 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

17. A method as claimed in claim 8 or claim 9 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

18. A use as claimed in claim 10 or claim 11 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

19. A fusion protein as claimed in claim 12 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. INTELLECTUAL PROPERTY OFRCP OF M.Z 15 MAR 2004