Incretin-insulin conjugates

An incretin-insulin conjugate comprising

• an incretin peptide;
• and
• an insulin agonist peptide, wherein the incretin peptide is linked to the insulin peptide via a linear chain spacer of 5 to 10 atoms, wherein the incretin peptide is selected from the group consisting of
• (i) the amino acid sequence:
• X1-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Z1 (SEQ ID NO:
• 839) with 1 to 3 amino acid modifications thereto, wherein
• X1 and/or X2 is a non-native (relative to SEQ ID NO:
• 701) amino acid that reduces susceptibility of the incretin peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),
• Z1 is selected from the group consisting of Asn-Thr-COOH, and Y—COOH, wherein Y is 1 to 2 amino acids, and further wherein
• (1) a lactam bridge connects the side chains of an amino acid at position i and an amino acid at position i+4, wherein i is 12, 16, 20 or 24 or
• (2) one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 of the incretin peptide is substituted with an α, α-disubstituted amino acid;
• and said incretin peptide has glucagon agonist activity;
• (ii) the amino acid sequence of SEQ ID NO:
• 701 modified to comprise at least one amino acid modification selected from the group consisting of:
• substitution of Asn at position 28 with a charged amino acid;
• substitution of Asn at position 28 with a charged amino acid selected from the group consisting of Asp, Glu, cysteic acid, and homocysteic acid;
• substitution at position 28 with Asn, Asp, or Glu;
• substitution at position 28 with Glu;
• substitution of Thr at position 29 with a charged amino acid;
• substitution of Thr at position 29 with a charged amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, and homocysteic acid;
• substitution at position 29 with Asp, Glu, or Lys;
• substitution at position 29 with Glu;
• insertion of 1-3 charged amino acids after position 29;
• insertion after position 29 of Glu or Lys;
• insertion after position 29 of Gly-Lys or Lys-Lys:
• or a combination thereof;
• and at least one amino acid modification selected from Group A or Group B, or a combination thereof;
• wherein Group A is an amino acid modification selected from the group consisting of substitution of Ser at position 16 with Thr or AIB;
• and
• wherein Group B is an amino acid modification selected from the group consisting of:
• substitution of His at position 1 with a non-native amino acid that reduces susceptibility of the incretin peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),
• substitution of Ser at position 2 with a non-native amino acid that reduces susceptibility of the incretin peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),
• substitution of Tyr at position 10 with Phe or Val;
• substitution of Lys at position 12 with Arg;
• substitution of Gln at position 20 with Ala or AIB;
• substitution of Asp at position 21 with Glu;
• substitution of Gln at position 24 with Ala or AIB;
• substitution of Met at position 27 with Leu or Nle;
• or a combination thereof;
• and wherein said incretin peptide has glucagon agonist activity;
• (iii) a incretin peptide of SEQ ID NO:
• 701, modified to comprise
• (a) an amino acid modification at position 1 that confers GIP agonist activity,
• (b) (1) a lactam bridge between the side chains of amino acids at positions i and i+4 or between the side chains of amino acids at positions j and j+3, wherein i is 12, 13, 16, 17, 20 or 24, and wherein i is 17, or
• (2) one, two, three, or all of the amino acids at positions 16, 20, 21, and 24 of the analog is substituted with an α,α-disubstituted amino acid,
• (c) amino acid modifications at one, two or all of positions 27, 28 and 29, and
• (d) 1-6 further amino acid modifications,
• wherein the EC50 of the analog for GIP receptor activation is about 10 nM or less;
• (iv) the sequence of SEQ ID NO:1 with
• (a) an amino acid at position 10 which is acylated or alkylated with a C4 to C30 fatty acid, and
• (b) an AIB at position 16;
• (v) an amino acid that differs from SEQ ID NO:
• 701 by no more than ten amino acid modifications, comprising one or more amino acid substitutions with AIB at positions 16, 20, 21, and/or 24, and an amino acid modification at position 1 and/or 2 that provides reduced susceptibility to cleavage by dipeptidyl peptidase IV, wherein said incretin peptide exhibits at least 20% of the activity of native GLP-1 at the GLP-1 receptor;
• (vi) an analog of any of claims (i)-(v), further comprising the amino acid sequence of GPSSGAPPPS (SEQ ID NO:
• 95) or XGPSSGAPPPS (SEQ ID NO:
• 96) C-terminal to the amino acid at position 29, wherein X is any amino acid, optionally glycine;
• (vii) amino acid of SEQ ID NO:
• 1 with the following amino acid modifications:
• Tyr at position 1, an AIB at position 2, Lys at position 10, wherein the Lys is covalently bound to a C16 fatty acyl group via a γ-Glu-γ-Glu dipeptide spacer, Ile at position 12, Lys at position 16, Gln at position 17, Ala at position 18, AIB at position 20, Glu at position 21, Asn at position 24, Leu at position 27, Ala at position 28, Gly at position 29, followed by the amino acid of SEQ ID NO:
• 95 C-terminal to the amino acid at position 29, and a C-terminal amide in place of the C-terminal alpha carboxylate;
• and
• (viii) X1X2EGTFTSDX10SIYLDKQAAX20EFVNWLLAGGPSSGAPPPS (SEQ ID NO:
• 2037) or X1X2EGTFTSDVSIYLDKQAAX20EFVNWLLAGGPSSGAPPPSX40 (SE ID NO:
• 2038), wherein
• X1 is His or Tyr;
• X2 is aminoisobutyric acid;
• X10 is Lys acylated with a C16 to C20 alkyl group optionally via a gamma Glu linker;
• X20 is aminoisobutyric acid;
• and
• X40 is Lys acylated with a C16 to C20 alkyl group optionally via a gamma Glu linker;
• wherein said insulin peptide comprises an A chain and a B chain wherein said A chain comprises a sequence GIVX4X4X5CCX8X9X10CX12LX14X15LX17X18YCX21-R13 (SEQ ID NO:
• 19), and said B chain comprises a sequence R22-X25LCGX29X30LVX33X34LYLVCGX41X42GFX45 (SEQ ID NO:
• 20), wherein
• X4 is glutamic acid or aspartic acid;
• X5 is glutamine or glutamic acid
• X8 is histidine, threonine or phenylalanine;
• X9 is serine, arginine, lysine, ornithine or alanine;
• X10 is isoleucine or serine;
• X12 is serine or aspartic acid;
• X14 is tyrosine, arginine, lysine, ornithine or alanine;
• X15 is glutamine, glutamic acid, arginine, alanine, lysine, ornithine or leucine;
• X17 is glutamic acid, aspartic acid, asparagine, lysine, ornithine or glutamine;
• X18 is methionine, asparagine, glutamine, aspartic acid, glutamic acid or threonine;
• X21 is selected from the group consisting of alanine, glycine, serine, valine, threonine, isoleucine, leucine, glutamine, glutamic acid, asparagine, aspartic acid, histidine, tryptophan, tyrosine, and methionine;
• X25 is histidine or threonine;
• X29 is selected from the group consisting of alanine, glycine and serine;
• X30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;
• X33 is selected from the group consisting of aspartic acid and glutamic acid;
• X34 is selected from the group consisting of alanine and threonine;
• X41 is selected from the group consisting of glutamic acid, aspartic acid or asparagine;
• X42 is selected from the group consisting of alanine, ornithine, lysine and arginine;
• X45 is tyrosine or phenylalanine;
• R22 is selected from the group consisting of AYRPSE (SEQ ID NO:
• 14), FVNQ (SEQ ID NO:
• 12), FVKQ (SEQ ID NO:
• 8), PGPE (SEQ ID NO:
• 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine, glutamine, glutamic acid and an N-terminal amine;
• and
• R13 is COOH or CONH2;
• wherein the spacer comprises:
• I. the structure of Formula I:
• wherein R1, R2, R3 and R4 are independently selected from the group consisting of H, and C1-C3 alkyl;
• Y is C, S, Se, or S═O,
• Z is C, Se, S, S(C1-C3)(C5-C6 aryl), or Z in combination with Y is —O—N═, or a 1,2,3 triazole;
• n is 0 or 1;
• and
• m is 1, 2 or 3, with the proviso that when Y is S═O, Z is C;
• II. the structure of Formula II:
• wherein R1, R2, R3 and R4 are independently selected from the group consisting of H and CH3, n is 0 or 1, and m is 1, 2 or 3;
• or
• III. the structure of Formula IV:
• wherein R3 is H or CH3;
• said spacer joining the carboxy terminus of the incretin peptide to the side chain of an amino acid at position B1, B2, B3, B22, B28 or B29 of the B chain, the N-terminal alpha amine of the B chain, or at the side chain of an amino acid at any position of a linking moiety that links the A chain and B chain of a single chain insulin analog.