Patent EA-004791-B1 (EA)
Title: Ob fusion protein and methods of uses thereof
Inventor: MANN MICHAEL BENJAMIN
IPC: C07K14/575
Filed: 1997-12-11
Abstract: 1. A protein having a formula selected from the group consisting of: R1 - R2 and R1 - L - R2, wherein R1 is a Fc protein or analog thereof, R2 is an OB protein or analog thereof, and L is a linker, wherein the Fc, analog or derivative thereof is selected from the group consisting of: (a) the Fc amino acid sequences as set forth in SEQ. ID. NOS.: 9, 12, 15 and 18; (b) the amino acid sequence of subpart (a) having a different amino acid substituted or deleted in one or more of the following positions (using the numbering according to SEQ. ID. NO. 9): (i) one or more cysteine residues replace by an alanine or serine residue; (ii) one or more tyrosine residues replaced by a phenylalanine residue; iii) the amino acid at position 5 replaced with an alanine; (iv) the amino acid at position 20 replaced with glutamate; (v) the amino acid at position 103 replaced with an alanine; (vi) the amino acid at position 105 replaced with an alanine; (vii) the amino acid at position 107 replaced with an alanine; (viii) the amino acids at positions 1, 2, 3, 4, or 5 deleted; (ix) one or more residues replaced or deleted to ablate the Fc receptor binding site; (x) one or more residues replaced or deleted to ablate the complement (C1q) binding site; and (xi) a combination of subparts i-x; (c) the amino acid sequence of subparts (a) or (b) having a methionyl residue at the N-terminus; (d) the Fc protein, analog or derivative of any of subparts (a) through (c) comprised of a chemical moiety connected to the protein moiety; (e) a derivative of subpart (d) wherein said chemical moiety is a water soluble polymer moiety; (f) a derivative of subpart (e) wherein said water soluble polymer moiety is polyethylene glycol; (g) A derivative of subpart (e) wherein said water soluble polymer moiety is a polyamino acid moiety; and (h) a derivative of subpart (e) wherein said water soluble polymer moiety is attached at solely the N-terminus of said protein moiety; OB protein, analog or derivative thereof is selected from the group consisting of: (a) the amino acid sequence 1-146 as set forth in SEQ. ID. NO. 3 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): 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145; (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), (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 having a lysine residue at position 35, and an isoleucine residue at position 74): (i) amino acids 98-146 (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 amino acids 100-111 sequentially placed between amino acids 99 and 112; and, (vi) the truncated OB analog of subpart (f)(i) having one or more of amino acids 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145 substituted with another amino acid; (vii) the truncated analog of subpart (f)(ii) having one or more of amino acids 4, 8 and 32 substituted with another amino acid; (viii) the truncated analog of subpart (f)(iii) having one or more of amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111 and 112 replaced with another amino acid; (ix) the truncated analog of subpart (f)(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; (x) the truncated analog of subpart (f)(v) 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, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145 replaced with another amino acid; (xi) the truncated analog of any of subparts (f)(i)-(x) having an N-terminal methionyl residue; (g) the OB protein or analog derivative thereof of any of subparts (a) through (f) comprised of a chemical moiety connected to the protein moiety; (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 water soluble polymer moiety is a polyamino acid moiety; and (k) a derivative of subpart (h) wherein said water soluble polymer moiety is attached at solely the N-terminus of said protein moiety, wherein the linker is selected from the group consisting of: (a) ala, ala, ala; (b) ala, ala, ala, ala; (c) ala, ala, ala, ala, ala; (d) gly, gly; (e) gly, 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; (j) chemical moiety; and (k) any combination of subparts (a) through (j). 2. A nucleic acid sequence encoding for a protein having the formula: R1 - R2 and R1 - L - R2, wherein R1 is a Fc protein or analog thereof, R2 is an OB protein or analog thereof, and L is a linker, wherein the Fc, analog or derivative thereof is selected from the group consisting of: (a) the Fc amino acid sequences as set forth in SEQ. ID. NOS.: 9, 12, 15 and 18; (b) the amino acid sequence of subpart (a) having a different amino acid substituted or deleted in one or more of the following positions (using the numbering according to SEQ. ID. NO. 9): (i) one or more cysteine residues replace by an alanine or serine residue; (ii) one or more tyrosine residues replaced by a phenylalanine residue; iii) the amino acid at position 5 replaced with an alanine; (iv) the amino acid at position 20 replaced with glutamate; (v) the amino acid at position 103 replaced with an alanine; (vi) the amino acid at position 105 replaced with an alanine; (vii) the amino acid at position 107 replaced with an alanine; (viii) the amino acids at positions 1, 2, 3, 4, or 5 deleted; (ix) one or more residues replaced or deleted to ablate the Fc receptor binding site; (x) one or more residues replaced or deleted to ablate the complement (C1q) binding site; and (xi) a combination of subparts i-x; (c) the amino acid sequence of subparts (a) or (b) having a methionyl residue at the N-terminus; (d) the Fc protein, analog or derivative of any of subparts (a) through (c) comprised of a chemical moiety connected to the protein moiety; (e) a derivative of subpart (d) wherein said chemical moiety is a water soluble polymer moiety; (f) a derivative of subpart (e) wherein said water soluble polymer moiety is polyethylene glycol; (g) A derivative of subpart (e) wherein said water soluble polymer moiety is a polyamino acid moiety; and (h) a derivative of subpart (e) wherein said water soluble polymer moiety is attached at solely the N-terminus of said protein moiety; OB protein, analog or derivative thereof is selected from the group consisting of: (a) the amino acid sequence 1-146 as set forth in SEQ. ID. NO. 3 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): 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145; (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), (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 having a lysine residue at position 35, and an isoleucine residue at position 74): (i) amino acids 98-146 (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 amino acids 100-111 sequentially placed between amino acids 99 and 112; and, (vi) the truncated OB analog of subpart (f)(i) having one or more of amino acids 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145 substituted with another amino acid; (vii) the truncated analog of subpart (f)(ii) having one or more of amino acids 4, 8 and 32 substituted with another amino acid; (viii) the truncated analog of subpart (f)(iii) having one or more of amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111 and 112 replaced with another amino acid; (ix) the truncated analog of subpart (f)(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; (x) the truncated analog of subpart (f)(v) 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, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145 replaced with another amino acid; (xi) the truncated analog of any of subparts (f)(i)-(x) having an N-terminal methionyl residue; (g) the OB protein or analog derivative thereof of any of subparts (a) through (f) comprised of a chemical moiety connected to the protein moiety; (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 water soluble polymer moiety is a polya