FUSION PROTEINS

The present invention relates to fusion proteins for use in the treatment of diabetes. More particularly, the invention relates to fusion proteins comprising an insulin receptor agonist fused to a human IgG Fc region with a peptide linker, and the use of such proteins in the treatment of diabetes. The fusion proteins of the present invention have an extended time action profile and are useful for providing protracted basal glucose control and suppression of hepatic glucose output.

Diabetes mellitus is a chronic disorder characterized by hyperglycemia resulting from defects in insulin secretion, msulin action, or both. Type 1 diabetes mellitus is characterized by little or no insulin secretory capacity, and patients with type 1 diabetes mellitus require insulin for survival. Type 2 diabetes mellitus is characterized by elevated blood glucose levels resulting from impaired insulin secretion, insulin resistance, excessive hepatic glucose output, and/or contributions from all of the above. In at least one-third of patients with Type 2 diabetes mellitus, the disease progresses to an absolute requirement for insulin therapy.

In order to achieve normal glycemia, insulin replacement therapy is desired to parallel, as closely as possible, the pattern of endogenous insulin secretion in normal individuals. The daily physiological demand for msulin fluctuates and can be separated into two phases: (a) the mealtime phase requiring a pulse (bolus) of insulin to dispose of the meal-related blood glucose surge, and (b) the inter-meal phase requiring a sustained (basal) amount of insulin to regulate hepatic glucose output for maintaining optimal fasting blood glucose.

Because Type 1 diabetes patients produce little or no insulin, effective insulin therapy for Type 1 diabetics generally involves the use of two types of exogenously administered insulin: a rapid-acting, mealtime insulin provided by bolus injections, and a long-acting, basal insulin, administered once or twice daily to control blood glucose levels between meals. Treatment of patients with Type 2 diabetes typically begins with prescribed weight loss, exercise, and a diabetic diet, but when these measures fail to control elevated blood sugars, then oral medications and incretin-based therapy, such as administration of glucagon-like peptide-] (GLP-1) receptor agonists and/or dipeptidyl peptidase 4 (DPP-4) inhibitors that enable increased incretin levels, may be necessary. When these medications are still insufficient, treatment with insulin is considered. Type 2 diabetes patients whose disease has progressed to the point that insulin therapy is required are generally started on a single daily injection of a long-acting, basal insulin, although mealtime injections of rapid-acting insulins may be included, as necessary, in some cases.

Several types of basal insulins are currently available. Insulin glargine, sold under the tradename LANT US®, comprises a modified insulin structure in which the asparagine at position 21 in the insulin A-chain is replaced with glycine, and two arginines are added to the C-terminus of the B-chain. insulin detemir, sold under the tradename LEVEMIR®, comprises a modified insulin structure in which the threonine at position 30 of the B-chain has been deleted and the lysine at position 29 of the B-chain has been derivatized through the covalent linkage of a 14-carbon, myristovl fatty acid to the ε-amine group of lysine at B29. Insulin degludec, available in Europe and Japan under the tradename TRESIBA®, comprises a modified insulin structure in which the threonine at position 30 of the B-chain has been deleted, and the ε-amino group of the lysine at position 29 of the B-chain is covaiently derivatized with hexadecandioic acid via a γ-L-glutamic acid linker. Ail of these insulins are indicated for once-daily

administration.

Treatment regimens involving daily injections of existing insulin therapies can be complicated and painful to administer and can result in undesired side effects, such as hypoglycemia and weight gain. Therefore, many diabetic patients are unwilling or unable to comply, or are incapable of complying, with the insulin therapy necessary to maintain close control of blood glucose levels. Poor glycemic control increases a patient's risk for developing serious diabetes-related complications, including heart disease, stroke, nerve damage, lower limb amputation, vision loss, and kidney disease. Research is being conducted to identify insulin products with longer duration of action; thus, requiring fewer injections than currently available insulin products to improve acceptance and compliance.

CN1035091 18 describes proteins with a human insulin B-chain and human insulin A-chain joined by a 4 to 50 amino acid C-peptide connection sequence, and with the insulin A-chain attached directly, without an additional linker, to an immunoglobulin Fc fragment, and states that testing in mice shows that such proteins have an in vivo half-life as long as about 3 days. KR101324828 describes proinsulin analogs linked to immunoglobulin Fc regions through the use of non-peptidyl linkers, and states that such proteins provide increased serum half-life over existing therapies. The publication states that the non-peptidyl linkers represent an improvement over peptide linkers, asserting that fusion proteins using peptide linkers cannot increase the half-life of an active medication in the blood because peptide linkers are easily severed by enzymes in the body.

Despite the disclosures above, and/or in any other publications, the present inventors overcame multiple obstacles to discover fusion proteins comprising insulin receptor agonists, peptide linkers, and human IgG Fc regions that meet the ongoing need for a glucose-lowering product with increased duration of action, sufficient for less frequent dosing than currently available insulin products, including dosing as infrequently as once-weekly. For example, in order to achieve the desired prolonged time action profile, it was necessary to engineer fusion proteins with attenuated potency to avoid rapid receptor mediated clearance, a major route of insulin clearance, but that still have enough potency to provide sufficient glucose lowering. Further, in order to minimize renal clearance, the other major route of insulin clearance, and to regulate peripheral exposure through hydrodynamic size-limited paracellular diffusion, fusion proteins had to be engineered which were sufficiently large, and which would not have the insulin receptor agonist proteolytically cleaved from the human IgG Fc region after being administered, as such cleavage would result in faster than desired renal clearance of the insulin receptor agonist even if the human IgG Fc region remained in circulation. In addition, IgG Fc domains have evolved to self-associate to form stable dimers, and when such a dimer is formed from two fusion proteins, each comprising an insulin moiety fused with an IgG Fc region, the two insulin moieties are brought into close proximity to one another, enabling self-association, or dimerization, of the insulin moieties, mediated through the insulin B-chain self-association regions. Such insulin dimers are inactive, so fusion proteins with reduced self-association of the insulin receptor agonist moieties had to be engineered. Multiple additional challenges were overcome to create a fusion protein suitable for commercial manufacture and formulation as a therapeutic. The present invention provides fusion proteins which have prolonged duration of action compared to existing insulin treatments, allowing for less frequent injections than existing insulin products, including up to once weekly, thus reducing the complexity of and pain associated with existing treatment regimens involving more frequent injections. The fusion proteins of the present invention have a flat pharmacokinetic profile and restricted peripheral exposure, resulting in low day-to-day variability, and minimal incidence of hypoglycemia, including when provided in combination with an additional diabetes medication, such as an incretin-based therapy. The fusion proteins of the present invention may also provide prolonged duration of action without causing weight gain.

The present invention provides a fusion protein comprising:

a) an insulin receptor agonist having the general formula Z1-Z2-Z3, wherein t) Z1 is an insulin B-chain analog, comprising the amino acid

sequence:

X1 X2X3QHLCGSHLVEALX4Lv CGERGFX5YX5X7X8X9

wherein X1 is F, Q or A; X2 is V or G; X3 is N, K, D, G, Q, A or E; X4 is E, Y, Q, or H; X5 is H or F; X6 is G, T, S, H, V or is absent; X7 is G, E, P, K, D, S, H or is absent; X8 is G, E, K, P, Q, D, H or is absent; X9 is G, T, S, E, K, A or is absent, provided that the insulin B-chain analog includes at least one modification from the amino acid sequence of the B-chain of a molecule of human insulin at X4 , X5 , X6, X7 , X8, or X9 (SEQ ID NO: 1);

ti) Z- 2 is a first peptide linker comprising 5 to 10 amino acids, wherein at least 5 of said amino acids are G residues; and

iii) Z3 is an insulin A-chain analog comprising the amino acid

sequence:

G IVEQCC TS X1 CS LX2Q LEN Y C X3X4

X: is T or I; X2 is D, Y, Q or E; X3 is G, N, S or A; and X4 is any naturally occurring amino acid, or is absent, provided that if X3 is N, then X4 must be an amino acid other than G or N (SEQ ID NG:2);

b) a second peptide linker; and

c) a human IgG Fc region;

wherein the C-terminal residue of the insulin receptor agonist is directly fused to the N-terminal residue of the second peptide linker, and the C-terminal residue of the second peptide linker is directly fused to the N-terminal residue of the IgG Fc region.

The present invention also provides a fusion protein consisting of:

a) an insulin receptor agonist having the general formula Z1- Z2- Z3 , wherein t) Z1 is an insulin B-chain analog, having the amino acid sequence:

X1X2X3QHLCGSHLVEALX4LVCGERGFX5YX6X7X8X9

wherein X1 is F, Q or A; X2 is V or G; X3 is N, K, D, G, Q, A or E; X4 is E, Y, Q, or H; X5 is H or F; Xf, is G, T, S, FT, V or is absent; X7 is G, E, P, K, D, S, H or is absent; X8 is G, E, K, P, Q, D, H or is absent; X9 is G, T, S, E, K, A or is absent, provided that the insulin B-chain analog mcludes at least one modification from the amino acid sequence of the B-chain of a molecule of human insulin at X4 , X5 , X6, X7 , X8, or XQ (SEQ ID NO: 1);

ii) Z2 is a first peptide linker comprising 5 to 10 amino acids, wherein at least 5 of said amino acids are G residues; and

iii) Z3 is an insulin A -chain analog having the amino acid sequence: G IVEQCCTS X1 CSLX 2QLENYCX3X4

X·, is T or i; X2 is D, Y, Q or E; X3 is G, N, S or A; and X4 is any naturally occurring amino acid, or is absent, provided that if X3 is N, then X4 must be an amino acid other than G or N (SEQ ID NO:2);

b) a second peptide linker: and

c) a human IgG Fc region;

wherein the C-terminal residue of the insulin receptor agonist is directly fused to the N-terminal residue of the second peptide linker, and the C-terminal residue of the second peptide linker is directly fused to the N-terminal residue of the IgG Fc region.

The present invention also provides a pharmaceutical composition comprising a fusion protein of the present invention and at least one pharmaceutically acceptable excipient.

The present invention also provides a method of treating a patient with diabetes mellitus, obesity, dyslipidemia or metabolic syndrome, comprising administering to a patient in need thereof a therapeutically effective amount of a fusion protein of the present invention. The present invention also provides a method of treating or preventing a diabetes-related condition selected from the group consisting of heart disease, stroke, nephropathy, retinopathy, and kidney disease, comprising administering to a patient in need thereof a therapeutically effective amount of a fusion protein of the present invention.

The invention also provides a fusion protein of the present invention for use in therapy.

The present invention also provides the use of a fusion protein of the present invention in the manufacture of a medicament.

The present invention also provides polynucleotides encoding a fusion protein of the present invention.

The present invention also provides a process for producing a fusion protein of the present invention, said process comprising the steps of:

1. culturing a mammalian host cell comprising a polynucleotide encoding a fusion protein of the present invention under conditions such that said fusion protein is expressed; and

2. recovering from said host cell a fusion protein;

The present invention also provides a fusion protein produced by the process described above.

Figure 1. Figure 1 provides pharmacodynamic data for exemplary fusion proteins of the present invention in a streptozotocin (STZ)-treated rat diabetes model.

Figure 2. Figure 2 provides a schematic diagram of configurations of proteins described herein. It should be noted that the particular shapes (e.g., ovals, half-circles, etc.) used in the diagrams in Figure 2 are not intended to describe or characterize, and should not be used to construe, the meaning or structure of the individual components of the fusion proteins of the present invention.

In certain embodiments, the insulin B -chain analog includes at least one modification from the amino acid sequence of the B-chain of a molecule of human insulin at X4 or X5 of SEQ ID NO: 1 , and at least one modification from the amino acid sequence of the B-chain of a molecule of human insulin at X6, X7, X8, or X9 of SEQ ID NO: 1.

In certain embodiments, the insulin B-chain analog includes at least two modifications from the amino acid sequence of the B-chain of a molecule of human insulin at X6, X7, X8, or X9 of SEQ ID NO: 1.

In certain embodiments, the insulin B-chain analog includes modifications from the amino acid sequence of the B-chain of a molecule of human insulin at X6, X7, X8, and X9 of SEQ ID NO: 1.

In certain embodiments, the insulin B-chain analog has the amino acid sequence of SEQ ID NO: 1 wherein X6,- X9 are each G.

In certain embodiments, the insulin B-chain analog includes modifications from the amino acid sequence of the B-chain of a molecule of human insulin at X4 and X5 of SEQ ID NO: 1,

In certain embodiments, the insulin B-chain analog has the sequence of SEQ ID NO: 1, wherein: X, is F; X2 is V; X3 is N or D; X4 is E; X5 is H.

In certain embodiments, the insulin B-chain analog includes modification from the amino acid sequence of the B-chain of a molecule of human insulin at each of positions X4 , X5, X6, X7 , X8, and X9 of SEQ ID NO : 1.

In certain embodiments, the insulin B-chain analog comprises the sequence of SEQ ID NO: !, wherein: X1 is F; X2 is V; X3 is N or D; X4 is E; X5 is H; and X6-X9are each G.

In certain embodiments, the insulin A-chain analog includes at least one modification from the amino acid sequence of the human insulin A-chain at X- or X2 of SEQ ID NO:2.

In certain embodiments, the insulin A-chain analog includes modifications from the amino acid sequence of the human insulin A-chain at both X1 and X2 of SEQ ID

NO:2.

In certain embodiments, the insulin A-chain analog has the sequence of SEQ ID NO:2, wherein: X1 is I or T; X2 is D; X3 is G; and X4 is absent.

In certain embodiments, the insulin A-chain analog has the sequence of SEQ ID NO:2, wherein X3 is N and wherein X4 is an amino acid other than G, N, S, V L or P.

In certain embodiments, the insulin B -chain analog includes at least one modification from the amino acid sequence of the B-chain of a molecule of human insulin at X4 or X5 of SEQ ID NO: 1 , and at least one modification from the amino acid sequence of the B-chain of a molecule of human insulin at X6, X7 , X8, or X9 of SEQ ID NO: 1 ; and the insulin A-chain analog includes at least one modification from the amino acid sequence of the human insulin A-chain at X·, or X2 of SEQ ID NQ:2.

In certain embodiments, the insulin B-chain analog includes at least two modifications from the amino acid sequence of the B-chain of a molecule of human insulin at X6, X7, X8, or X9 of SEQ ID NO: 1 ; and the insulin A-chain analog includes at least one modification from the amino acid sequence of the human insulin A-chain at Xi or X2 of SEQ ID NO:2,

In certain embodiments, the insulin B-chain analog includes modifications from the amino acid sequence of the B-chain of a molecu le of human insulin at X6, X7, X8, and X9 of SEQ ID NO: 1 ; and the msulin A-chain analog includes at least one modification from the amino acid sequence of the human msulin A-chain at X: or X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog has the amino acid sequence of SEQ ID NO: 1 wherein X6- X9 are each G; and the insulin A-chain analog includes at least one modification from the amino acid sequence of the human insulin A-chain at X1 or X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog includes modifications from the amino acid sequence of the B-chain of a molecule of human insulin at X4 and X5 of SEQ ID NO: 1 ; and the insulin A-chain analog includes at least one modification from the amino acid sequence of the human insulin A-chain at X: or X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog comprises the sequence of SEQ ID NO: 1, wherein X: 1 is F; X2 is V; X3 is N or D; X4 is E; X5 is H; and the insulin A-chain analog includes at least one modification from the amino acid sequence of the human insulin A-chain at X1 or X2 of SEQ ID NO:2.

In certain embodiments, the insulin B -chain analog includes modification from the amino acid sequence of the B-chain of a molecule of human insulin at each of positions X4 , X5 , X6, X-7, X8, and X9 of SEQ ID NO: 1 ; and the insulin Λ-chain analog includes at least one modification from the amino acid sequence of the human insulin A-chain at X1 or X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog has the sequence of SEQ ID NO: 1 , wherein: X, is F; X2 is V; X3 is N or D; X4 is E; X5 is H; and X6-X9 are each G; and the msulin A-chain analog includes at least one modification from the amino acid sequence of the human insulin A-chain at X: or X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog includes at least one modification from the amino acid sequence of the B-chain of a molecule of human insulin at X4 or X5 of SEQ ID NO: 1, and at least one modification from the amino acid sequence of the B-chain of a molecule of human insulin at X6, X7 , X8, or X9 of SEQ ID NO: 1 ; and the insulin A-chain analog includes modifications from the amino acid sequence of the human insulin A-chain at both X1 and X2 of SEQ ID NO:2.

In certain embodiments, the tnsulm B-chain analog includes at least two modifications from the amino acid sequence of the B-chain of a molecule of human insulin at X6, X7, X8, or X9 of SEQ ID NO: 1 ; and the insulin A-chain analog includes modifications from the amino acid sequence of the human insulin A-chain at both X1 and X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog includes modifications from the amino acid sequence of the B-chain of a molecu le of human insulin at X6, X7, X8, and X9 of SEQ ID NO: 1 ; and the insulin A-chain analog includes modifications from the amino acid sequence of the human insulin A-chain at both X1 and X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog has the amino acid sequence of SEQ ID NO: 1 wherein X6-X9 are each G; and the insulin A-chain analog includes modifications from the amino acid sequence of the human tnsulm A-chain at both X1 and

X2 of SEQ ID NO:

In certain embodiments, the insulin B-chatn analog includes modifications from the amino acid sequence of the B-chain of a molecule of human insulin at X4 and X5 of

SEQ ID NO: l; and the insulin A-chain analog includes modifications from the amino acid sequence of the human insulin A-chain at both X1 and X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog comprises the sequence of

SEQ ID NO: 1, wherein: X1 is F: X2 is V; X3 is N or D; X4 is E; X5 is H; and the insulin

A-chain analog includes modifications from the amino acid sequence of the human

insulin A-chain at both X: and X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog includes modification from the amino acid sequence of the B-chain of a molecule of human insulin at each of positions X4, X5, X6, X7 , X8, and X9 of SEQ ID NO: 1 ; and the insulin A-chain analog includes modifications from the amino acid sequence of the human insulin A-chain at both and X1 X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog has the sequence of SEQ ID

NO: 1 , wherein: X1 is F; X2 is V; X3 is N or D; X4 is E; X5 is H; and X6-X9 are each G;

and the insulin A-chain analog includes modifications from the amino acid sequence of the human insulin A-chain at both X1 and X2 of SEQ ID NO:2.

In certain embodiments, the insulin B-chain analog includes at least one

modification from the amino acid sequence of the B-chain of a molecule of human insulin at X4 or X5 of SEQ ID NO: 1 , and at least one modification from the amino acid sequence of the B-chain of a molecule of human insulin at X6, X7, X8, or X9 of S EQ ID NO: 1 ; and the insulin A-chain analog has the sequence of SEQ ID NO:2, wherein: X1 is I or T; X2 is D; X3 is G; and X4 is absent.

In certain embodiments, the insulin B-chain analog includes at least two

modifications from the amino acid sequence of the B-chain of a molecule of human

insulin at X6, X7, X8, or X9 of SEQ ID NO: 1 ; and the insulin A-chain analog has the

sequence of SEQ ID NO:2, wherein: X1 is I or T; X2 is D; X3 is G; and X4 is absent.

In certain embodiments, the insulin B-chain analog includes modifications from the amino acid sequence of the B-chatn of a molecule of human insulin at X6, X7, X8, and X9 of SEQ ID NO: 1 ; and the insulin A-chain analog has the sequence of SEQ ID NO:2, wherein: X1 is I or T; X2 is D; X3 is G; and X4 is absent.

In certain embodiments, the insulin B-chain analog has the amino acid sequence of SEQ ID NO: 1 wherein X6-X9 are each G, wherein: X. is I or T; X2 is D; X3 is G; and X4 is absent.

In certain embodiments, the insulin B-chain analog includes modifications from the amino acid sequence of the B-chain of a molecule of human insulin at X4 and X5 of SEQ ID NO: 1 ; and the insulin A-chain analog has the sequence of SEQ ID NO:2, wherein: X1 is I or T; X2 is D; X3 is G; and X4 is absent.

In certain embodiments, the insulin B-chain analog comprises the sequence of

SEQ ID NO: 1, wherein: X1 is F; X2 is V; X3 is N or D; X4 is E; X5 is H; and the insulin A-chain analog has the sequence of SEQ ID NO:2, wherein: X1 is I or T; X2 is D; X3 is G; and X4 is absent.

In certain embodiments, the insulin B-chain analog includes modification from the amino acid sequence of the B-chain of a molecule of human insulin at each of positions X4 , X5 , X6, X7 , X8, and X9 of SEQ ID NO: 1 ; and the insulin A-chain analog has the sequence of SEQ ID NO:2, wherein: X1 is I or T: X2 is D; X3 is G; and X4 is absent.

In certain embodiments, the insulin B-chain analog has the sequence of SEQ ID NO: 1 , wherein: X: is F; X2 is V; X3 is N or D; X4 is E; X5 is II; and X6-X9 are each G; and the insulin A-chain analog has the sequence of SEQ ID NO:2, wherein: X1 is I or T; X2 is D; X3 is G; and X4 is absent.

In certain embodiments, the first peptide linker (Z2) comprises the amino acid sequence: X1 GX2GGGG, wherein X 1is G or is absent; and X2is G, S or is absent (SEQ ID NO:3).

In certain embodiments, the insulin receptor agonist, i.e., Z1Z2-Z3 in the fusion protein described above, comprises the amino acid sequence:

X1X2X3QHLCGSHLVEALX4LVCGERGFX5YX6X7X8X9X10G X1 1GGGGGIVEQCCTS X12CSL X13QLENYC X1 4 X1 5

wherein X1 is F, Q or A; X2 is V or G; X3 is N, K, D, G, Q, A or E; X4 is E, Y, Q, or H; X5 is H or F; X6 is G, T, S, H, V or is absent; X7 is G, E, P, K, D, S, H or is absent; X8 is G, E, K, P, Q, D, H or is absent; X9 is G, T, S, E, K, A or is absent; X10 is G or is absent; X11 is G, S or is absent; X12 is T or I; X13 is D, Y, Q or E; X14 is G, N, S or A; and X15 is any naturally occurring amino acid, or is absent, provided that at least one of X4 , X5, X6, X7, X8, or X9 must be a different amino acid than that found, respectively, at position B16, B25, B27, B28, B29 or B30 of the B-chain of a molecule of human insu lin, and further provided that if X14 is N, then X15 must be an amino acid other than G or N (SEQ ID NO:4).

In certain preferred embodiments, the insulin receptor agonist, i.e., Z1-Z2-Z3 in the fusion protein described above has the following amino acid sequence:

FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCG

(SEQ ID NO:5).

In certain embodiments, the second peptide linker is a peptide having between 10 and 25 amino acids, wherein at least 50% of said amino acids are G residues. In certain embodiments, the second peptide linker is a peptide comprising the amino acid sequence [GGGGX]n, wherein X is Q, E or S; and wherein n is 2-5. In certain embodiments, the second peptide linker comprises the amino acid sequence:

GGGG X1 GGGGX 2GGGGX3GGGGX4X5X6

wherein X1 is Q or E; X2 is Q or E; X3 is Q or E; X4 is G, E, Q or is absent; X5 is G or absent; and X6 is G or is absent (SEQ ID NO: 6).

In certain preferred embodiments, the second peptide linker has the amino acid sequence:

GGGGQGGGGQGGGGQGGGGG ( SEQ I D NO : 7 ) .

In certain embodiments, the human IgG Fc region comprises fragments from one heavy chain of an IgG antibody. A schematic diagram of a fusion protein comprising such an IgG region is provided in diagram (A) in Figure 2. In other embodiments, the human IgG Fc region comprises fragments from two heavy chains of an IgG antibody. A schematic diagram of a fusion protein comprising such an IgG region is provided in diagram (B) in Figure 2.

In certain embodiments, the human IgG Fc region is an Fc region from an IgGl , IgG2 or IgG4 antibody.

In certain embodiments, the human IgG Fc region is an Fc region from an IgGl antibody comprising the following amino acid sequence:

CPPCPAPELLGGPS VX, LX 2PPKPKDTLMISRTPEVTCX3VX4DVSHEDPEVKFNWY

VDGVEVHNAKTKPREEQYXjSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGX6

wherein X1 is F, Q or E; X2 is F, Q or E; X3 is V or T; X4 is V or T; X5 is N, D or Q; and X6 is K or is absent. (SEQ ID NO: 8)

In certain embodiments, the IgG Fc region comprises the amino acid sequence of SEQ ID NO: 8 and further comprises some or all of the amino acids that would be found in a wild-type IgGl Fc sequence to the N-terminal side of the C residue at position 1 in SEQ ID NO:8.

Preferably, the human IgG Fc region is from either an IgG2 or IgG4 antibody. In certain embodiments, the human IgG Fc region is an Fc region from an IgG4 antibody comprising the following amino acid sequence:

PCPPCPAPEAAGGPSV X1 LX2PPKPKDTLMISRTPEVTCX3VX4DVSQEDPEVQFNW

Y^DGVEVIMAKTKPREEQFXsSTYRWSVLTYLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLYSX6LTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGX7

wherein X1 is F, Q or E; X2 F, Q or E; X3 is V or T; X4 is V or T; X5 is N, D or Q; X6 is R, or K; X7 is K or is absent. (SEQ ID NO: 9)

In certain embodiments, the IgG Fc region comprises the amino acid sequence of SEQ ID NO: 9, and further comprises some or all of the amino acids that would be found in a wild type IgG4 Fc sequence to the N-terminal side of the C residue at position 1 in SEQ ID NO: 9. In certain embodiments, the human IgG Fc region comprises the amino acid sequence of SEQ ID NO:9, wherein X1 is F; X2 is F; X3 is V; X4 is V; X5 is N; X6 is R; and X7 is absent.

In certain embodiments, the human IgG Fc region is an Fc region from an XgG2 antibody having the following amino acid sequence:

wherein X1 is F, Q or E; X2 is F, Q or E; X3 is V or T; X4 is V or T; X5 is N, D or Q; and X6 is K or absent, (SEQ ID NO: 10)

In certain embodiments, the IgG Fc region comprises the amino acid sequence of

SEQ ID NO: 10, and further comprises some or all of the amino acids that would be found in a wild type IgG2 Fc sequence to the N -terminal side of the E residue at position 1 in SEQ ID NO: 10. In certain embodiments, the human IgG Fc region comprises the amino acid sequence of SEQ ID NO: 10, wherein X1 is F; X2 is F; X3 is V; X4 is V; X5 is N; and X6 is absent.

In certain embodiments, the fusion protein comprises the amino acid sequence:

wherein X1 is F, Q or A; X2 is V or G; X3 is N, K, D, G, Q, A or E; X4 is E, Y, Q, or H; X5 is H or F; X6 is G, T, S, H, V or is absent; X7 is G, E, P, K, D, S, H or is absent; X8 is G, E, K, P, Q, D, H or is absent; X9 is G, T, S, E, K, A or is absent, provided that at least one of X4, X5, X6, X7, X¾ or X9 is an amino acid other than that which is present, respectively, at position Β16, B25, B27, B28, B29 or B30 of a human insulin B-chain: X10 is G or is absent; X11 is G, S or is absent; X12 is T or I; X13 is D, Y, Q or E; X14 is G, N, S or A; X15 is any naturally occurring amino acid, or is absent, provided that if X14 is N, then X15 must be an amino acid other than G or N; X16 is Q or E; X17 is Q or E; X18 is Q or E; X19

is G, E, Q or is absent; X20 is G or absent; X21 is G or is absent; X22 is E or P; X23 is E or P; X24 is A or V; X25 is G or is absent; X26 is Q or H; X27 is Y or F; X28 is L or V; X29 is S or A; X30 is S or P; X31 is A or T; X32 is Q or R; X33 is V or M; X34 is R or K; X35 is E or Q; and X36 is L or P (SEQ ID NO: 11).

In certain embodiments, the present invention provides a fusion protein selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.

In a referred embodiment, the fusion rotein has the amino acid se uence:

In certain embodiments, the fusion proteins of the present invention are present in the form of a dimer. A schematic diagram of such a dimer is provided in diagram (C) in Figure 2. In certain embodiments, the dimer is a homodimer, wherein the amino acid sequences of the two fusion proteins that make the dimer are the same. In certain embodiments, the dimer is a heterodimer, wherein the amino acid sequences of the two fusion proteins that make up the dimer are different.

In certain embodiments, the pharmaceutical composition of the present invention comprises a fusion protein of the present invention, a buffering agent, a surfactant, and an isotonicitv agent. In certain embodiments, the buffering agent is citric acid and/or citrate, the surfactant is polysorbate 80, and the isotonicity agent is mannitol. In certain embodiments, the pH of the composition ranges from about 5.5 to about 8.0. In certain

embodiments, the pH ranges from about 6,0 to about 7.4. In certain embodiments, the pH ranges from about 6.0 to 6.75.

in certain embodiments, the pharmaceutical composition further comprises an additional active ingredient. In certain embodiments, the additional active ingredient is an incretin-based therapy. In certain preferred embodiments, the incretin-based therapy is a GLP-1R agonist. Preferably, the GLP-1R agonist is dulaglutide.

In certain embodiments, the method of the present invention comprises administration of a therapeutically effective amount of a fusion protein once daily. In preferred embodiments, a therapeutically effective amount of the fusion protein is administered once weekly. In certain embodiments, a therapeutically effective amount of the fusion protein is administered once monthly. In certain embodiments, the present invention provides a method of treating a patient with diabetes mellitus while reducing the risk of hypoglycemia and/or weight gain, comprising administering to the patient a therapeutically effective amount of a fusion protein of the present invention.

The present invention also provides a method of treating a patient with diabetes mellitus, obesity, dyslipidemia, and/or metabolic syndrome, comprising administering a therapeutically effective amount of a fusion protein of the present invention in combination with an additional active ingredient. The fusion protein and additional active ingredient in such embodiments may be administered simultaneously, sequentially or in a single combined formulation. In certain embodiments, the additional active ingredient is an incretin-based therapy. In certain preferred embodiments, the incretin-based therapy is a GLP-1R agonist. Preferably, the GLP- 1 R agonist is dulaglutide. In certain embodiments, the combination is admmistered once daily. In certain preferred embodiments, the combination is administered once weekly. In certain embodiments, the combination is administered once monthly.

In certain embodiments, the present invention provides a fusion protein of the present invention for use in treatment of diabetes mellitus, obesity, dyslipidemia or metabolic syndrome. In certain embodiments, the present invention provides a fusion protein of the present invention for use in treating or preventing a diabetes-related condition selected from the group consisting of heart disease, stroke, nephropathy,

retinopathy, and kidney disease. In certain embodiments, the present invention provides a fusion protein of the present invention for use in treating a patient with diabetes mellitus while reducing the risk of hypoglycemia and/or weight gain, comprising administering to the patient a fusion protein of the present invention. In certain embodiments, the fusion protein of the present invention is provided for use in simultaneous, separate or sequential combination with another active ingredient. In certain embodiments, the additional active ingredient is an incretin-based therapy. In certain preferred embodiments, the incretin-based therapy is a GLP-1R agonist.

In certain embodiments, the present invention provides the use of a fusion protein of the present invention in the manufacture of a medicament for the treatment of diabetes mellitus, obesity, dyslipidemia or metabolic syndrome. In certain embodiments, the present invention provides the use of a fusion protein of the present invention in the manufacture of a medicament for the treatment or prevention of a diabetes-related condition selected from the group consisting of heart disease, stroke, nephropathy, retinopathy, and kidney disease. In certain embodiments, the present invention provides the use of a fusion protein of the present invention in the manufacture of a medicament for treating dia betes mellitus while reducing the risk of hypoglycemia and/or weight gain, comprising administering to the patient a fusion protein of the present invention. In certain embodiments, the present invention provides the use of a fusion protein of the present invention in the manufacture of a medicament for the treatment of diabetes mellitus, obesity, dyslipidemia or metabolic syndrome, wherein the medicament is to be administered simultaneously, separately or sequentilaly in combination with another active ingredient.

When used herein, the term "insulin receptor agonist" refers to a protein that binds to and activates the insulin receptor, resulting in a lowering of blood glucose levels and/or suppression of hepatic glucose output, characteristics which can be tested and measured using known techniques, such as those shown in the studies described below.

The insulin receptor agonist portion of the fusion proteins of the present in vention includes an analog of an insulin B-chain and an analog of an insulin A-chain. When used herein, the terms "insulin A-chain" and "insulin B-chain" refer to the A and B chains of the human insulin molecule (CAS No. 1 1061-68-0), whose native wild type sequences are well-known. The human msulin A-chain consists of 21 amino acids, referred to in the art as A1-A21 , having the following sequence:

GI VEQCCTS I CSLYQLENYCN (SEQ ID NO; ! 3).

The human insulin B-chain consists of 30 amino acids, referred to in the art as Bi- B30, having the following sequence:

FWQHLCGS HLVEALYLVCGERGFFYTPKT (SEQ ID NO: 14).

In a molecule of human insulin, the A- and B-chains are joined by two disulfide bonds, CysA7-CysB7 and CysA20-CysB19. The A-chain has an intra-chain disulfide bond at CysA6-CysAl 1.

To achieve the desired extended time action profile, the insulin receptor agonist of the fusion protein of the present invention must remain in circulation, and be capable of interacting with the msulin receptor, over an extended period of time. In order for the fusion proteins of the present invention to remain in circulation for the desired period of time, elimination of the fusion proteins must be attenuated. The two primary routes of insulin elimination are renal clearance and insulin receptor-mediated clearance. See. Igiesias P, et al. Diabetes Obes. Metab. 2008; 10:811-823. To minimize renal clearance, a molecule with hydrodynamic size of at least about the size of human serum albumin is needed, and such a hydrodynamic size is provided in the fusions proteins of the present invention by the human IgG Fc region. As for receptor-mediated clearance, the fusion protein cannot be so potent at the insulin receptor that it results in more rapid receptor-mediated clearance than desired, but the fusion protein must be potent enough, however, to provide sufficient glucose control at doses that are commercially feasible. Thus, the potency of the fusion protein must be carefully balanced, and the structure of the fusion protein of the present invention allows it to achieve such a balance.

Insulin molecules have a tendency to self-associate into dimers and hexamers. Numerous roles have been proposed for the evolutionarily-conserved, self-association tendencies of msulin, including: (1) chemical and thermal stabilization of the molecule during intracellular vacuole storage; (2) protection of the monomeric insulin from fibrillation in vivo; (3) substitution for chaperone-assisted stabilization and folding during intracellular expression; and/or (4) essential for secretory trafficking. The active form of insulin, however, is the monomer.

The tendency of insulin molecules to self-associate, and the inactivity of such self-associated molecules, is relevant to the present invention, because human IgG Fc regions also tend to self-associate to form dimers, typically associated covalently through disulfide bonds in the hinge region, and such dimers are formed from the human IgG Fc region s in the fusion proteins of the present invention. As a result of the dimerization of the human IgG Fc regions, the two insulin receptor agonist "arms" are in close proximity to one another, and thereby exist at relatively high local concentration. In the case of human insulin, such close proximity would tend to favor self-association, or dimerization, of the insulin moieties, affecting the activity of the molecules. A schematic diagram of an Fc fusion protein dirtier with insulin moiety arms that have become self-associated, or dimerized, is provided in diagram (D) of Figure 2. The tendency of insulin molecules to self-associate could also lead to self-association, or dimerization, of insulin moieties from more than one Fc fusion protein dimer; a schematic diagram of a dimer of two Fc fusion protein dimers with self-associated, or dimerized, insulin moieties is provided in diagram (E) of Figure 2, Further, the insulin moieties in more than two Fc fusion protein dimers could also self-associate in such a fashion to form, for example, a trimer comprised of three dimers, or even higher order aggregates comprised of more than three dimers.

The insulin receptor agonist portion of the fusion protein of the present invention, however, has a reduced tendency to self-associate or dimerize, and thus fusion protein dimers comprised of fusion proteins of the present invention tend to favor the structure depicted in diagram (C) of Figure 2, as opposed to the structures depicted in diagrams (D) and (E) of Figure 2. Thus, while the present invention provides a dimer of two fusion proteins, the insulin receptor agonist "arm" of each fusion protein in the dimer maintains a predominately monomeric state, as depicted for example in diagram (C) of Figure 1 , and is thus more capable of interacting with the insulin receptor.

In the fusion proteins of the present invention, the analog of the insulin B-chain in the insulin receptor agonist includes one or more modifications to the amino acid sequence of the human insulin B-chain. In particular, in order to reduce the propensity of the insulin receptor agonist portions to self-associate, or dimerize, the insulin B-chain analog includes one or more modifications from the B-chain of a molecule of human insulin at positions B1 6, B25 or B27-30, which are represented in SEQ ID NO: 1 as positions X4, X5 and X6-9, respectively. For example: X4 (which corresponds with B16 in the B-chain of a human insulin molecule) may be modified to E, Q or H; X5 (which corresponds with B25 in the B-chain of a human insulin molecule) may be modified to H; X6 (which corresponds with B27 in the B-chain of a human insulin molecule) may be deleted or modified to G, S, H or V; X7 (which corresponds with B28 in the B-chain of a human insulin molecule) may be deleted or modified to G, E, K, D, S or H; X8 (which corresponds with B29 in the B-chain of a human insulin molecule) may be deleted or modified to G, E, P, Q, D or H; and X9 (which corresponds with B30 in the B-chain of a human insulin molecule) may be deleted or modified to G, S, E or K. In addition to reducing the propensity of the insulin receptor agonist portions to self-associate, modifications to positions B16 and B25 on the insulin B-chain analog - X4 and X5 of SEQ ID NO: 1 , respectively - may also be made to adjust potency, improve expression, improve chemical and/or physical stability, improve the ease with which the fusion proteins can be formulated with other commonly used excipients and/or to eliminate deamidation. The insulin B-chain analog may also include additional modifications for these reasons. Referring to the variables in SEQ ID NO: 1 , such additional modifications include the following: modification of X1 (which corresponds with B1 in the B-chain of a human insulin molecule) to Q or A; modification of X2 (which corresponds with B2 in the B-chain of a human insulin molecule) to G; and/or modification of X3 (which corresponds with B3 in the B-chain of a human insulin molecule) to K, D or G.

in certain preferred embodiments, the insulin B-chain analog includes more than one modification to the amino acid sequence of the human insulin B-chain at positions X4, X5 and X6-y of SEQ ID NO : 1 . In a preferred embodiment, X4 is E and X5 is H. In certain embodiments, the amino acid sequence of X6-X9 of SEQ ID NO: 1 is selected from the group consisting of: GGES, GGGS, GGDS, GGEG, GGGG, SSES, SSGS, GGEE, GGGE, GGEK, GGGK, TPGS, TGGS, HGES, GHES, GGHS, GGEH, HGGS, GHGS, GGGH, GGDD, VGES, ΤΈΕΤ, TKPT, GGGG, TGGG, TPGG, EPKT, TDKT, TPGS,

EGGS, EGES, EEES, EPES, EPEP and GGDD. in preferred embodiments, the sequence of these four amino acids is GGG S, GGGG or TΈΕΤ. In particularly preferred embodiments, X4 is E, X5 is H and X6-9 is GGGG.

It should be noted that, while X6-X9 of SEQ ID NO: 1 are described above as comprising the C-terminal end of the insulin B-chain analog (Z1), these amino acids are not critical to the activity of the fusion proteins at the insulin receptor, and thus may alternatively be considered an extension of the first peptide linker (Z2). For example, in the context of SEQ ID NO:4, X6-X9 may be considered either part of the insulin B-chain analog or the first peptide linker in that insulin receptor agonist.

In the fusion proteins of the present invention, the analog of the insulin A-chain in the insulin receptor agonist portion may include one or more modifications to the amino acid sequence of the human insulin A-chain intended to improve chemical and physical stability, adjust potency, and/or enhance expression. Referring to the variables in SEQ ID NO:2, these modifications include the following: modification of X1 (which corresponds with Aioin the A-chain of a human insulin molecule) to T; modification of X2 (which corresponds with A14in the A-chain of a human insulin molecule) to D, Q or E; and/or modification of X3 (which corresponds with A21 in the A-chain of a human insulin molecule) to G, S or A. In a preferred embodiment, X1 is T, X2 is D, and X3 is G.

WE CLAIM:

1. A fusion protein comprising:

a) an insulin receptor agonist having the general formula Z1-Z2-Z3, wherein:

i) Z1 is an insulin B-chain analog, comprising the amino acid

sequence:

X1 X2X3QHLCGSHLVEALX4LVCGERGFX5YX6X7X8X9

wherein X. is F, Q or A; X2 is V or G; X3 is N, K, D, G, Q, A or E; X4 is E, Y, Q, or H; X5 is H or F; X6 is G, T, S, H, V or is absent; X7 is G, E, P, K, D, S, H or is absent: X8 is G, E, K, P, Q, D, H or is absent; X9 is G, T, S, E, K, A or is absent, provided that the insulin B-chain analog includes at least one modification from the amino acid sequence of the B-chain of a molecule of human insulin at X4, X5, X6, X7 , X8, or X9 (SEQ ID NO: l);

ii) Z2 is a first peptide linker comprising 5 to 10 amino acids, wherein at least 5 of said amino acids are G residues; and

iii) Z3 is an insulin A-chain analog comprising the amino acid

sequence:

GIVEQCCT S X1 CSLX 2QLENYCX3X4

wherein X. is T or I; X2 is D, Y, Q or E; X3 is G, N, S or A; and X4 is any naturally occurring amino acid, or is absent, provided that if X3 is N, then X4 must be an amino acid other than G or N (SEQ ID NO: 2);

b) a second peptide linker; and

c) a human IgG Fc region;

wherem the C-terminal residue of the insulin receptor agonist is directly fused to the N-terminal residue of the second peptide linker, and the C-terminal residue of the second peptide linker is directly fused to the N-terminal residue of the human IgG Fc region.

2. The fusion protein of claim 1 , wherein:

the insulin B-chain analog includes at least one modification from the amino acid sequence of the human insulin B-chain at X4 or X5 of SEQ ID NO: 1 ; and

and the insulin A-chain analog includes at least one modification from the amino acid sequence of the human insulin A-cihna at X1 or X2 of SEQ ID NO:2.

3. The fusion protein of either of claims 1 or 2, wherein:

the insulin B-chain analog comprises the sequence of SEQ ID NO: 1 , wherein: X1 is F; X2 is V; X3 is N or D; X4 is E; X5 is H; and

the insulin A-chain analog comprises the sequence of SEQ ID NO:2, wherein: X1 is I or T; X2 is D; X3 is G; and X4 is absent.

4. The fusion protein of any of claims 1-3, wherein the insulin B-chain analog comprises the sequence of SEQ ID NO: 1, wherein X6,-X9 are each G.

5. The fusion protein of any of claims 1-4, wherein the first peptide linker comprises the following amino acid sequence:

X1 GX2GGGG

wherein X1 is G or is absent; and X2 is G, S or is absent (SEQ ID NQ:3).

6. The fusion protem of claim 5, wherein X1 and X2 of SEQ ID NO:3 are G and S, respectively.

7. The fusion protem of any of claims 1 -6, wherein the msulin receptor agonist has the following amino acid sequence:

FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYC

G (SEQ ID NO:5).

8. The fusion protein of any of claims 1 -7, wherein the second peptide linker is a peptide having between 10 and 25 amino acids, wherein at least 50% of said amino acids are G residues.

9. The fusion protein of any of claims 1 -8, wherein the second peptide linker comprises a peptide having the sequence [GGGGX]n

wherein X is Q, E or S; and wherein n is 2-5.

10. The fusion protem of any of claims 1-9, wherein the second peptide linker comprises the following amino acid sequence:

GGGGX1GGGGX 2GGGGX3GGGGX4 X5X6

X1 is Q or E

X2 is Q or E

X3 is Q or E

X4 is G, E, Q or is absent

X5 is G or absent; and

X6 is G or is absent

(SEQ ID NO:6).

11. The fusion protein of any of claims 1-10, wherein the second peptide linker has the following amino acid sequence:

GGGGQGGGGQGGGGQGGGGG (SEQ ID NO:7).

12. The fusion protein of any of claims 1-11, wherein the human IgG Fc region is an Fc region from an IgGl , IgG2 or IgG4.

13. The fusion protein of any of claims 1-12, wherein the human IgG Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10.

14. A fusion protein having the amino acid sequence of SEQ ID NO: 12.

15. A homodimer of two fusion proteins of any of claims 1-14,

16. A pharmaceutical composition comprising either a fusion protein of any of claims 1- 14 or a homodimer of claim 15, and at least one excipient.

17. The pharmaceutical composition of claim 16, wherem the composition further

comprises one or more buffering agents, one or more surfactants, and one or more tsotonicity agents.

18. The pharmaceutical composition of claim 16, further comprising citrate, citric acid, polysorbate 80, and mannitol.

19. The pharmaceutical composition of any of claims 16-18 wherein the pH ranges from about 5.5 to about 8.0.

20. The pharmaceutical composition of any of claims 16-19 wherem the pH ranges from about 6.0 to about 6.75.

21. The pharmaceutical composition of any of claims 16-20, further comprising an

additional active ingredient.

22. The pharmaceutical composition of claim 21, wherein the additional active ingredient is an incretin-based therapy.

23. The pharmaceutical composition of claim 23, wherein the incretin-based therapy is a GLP-1 R agonist.

24. The pharmaceutical composition of claim 23 wherein the GLP-1R agonist is

dulaglutide.

25. A pharmaceutical composition comprising a homodimer of claim 15 and dulaglutide.

26. A method of treating a patient with diabetes mellitus comprising administering to a patient in need thereof a therapeutically effective amount of the fusion protein of any of claims 1-14.

27. The method of claim 26, wherein the fusion protein of any of claims 1 -14 is

administered in combination with an additional active ingredient.

28. The method of claim 27, wherein the additional active ingredient is an incretin-based therapy.

29. The method of claim 28, wherein the incretin-based therapy is a GLP-1 R agonist.

30. The method of claim 29, wherem the GLP-1R agonist is dulaglutide.

31. A method of treating a patient with diabetes mellitus comprising administering to a patient in need thereof a homodimer of claim 15 in combination with dulaglutide.

32. The fusion protein of any of claims 1 -14 for use in therapy.

33. The fusion protein of any of claims 1-14 for use in the treatment of diabetes mellitus.

34. The fusion protein of any of claims 1-14 in for use in simultaneous, separate or

sequential combination with an additional active ingredient.

35. The fusion protein of any of claims 1-14 in for use in simultaneous, separate or

sequential combination with dulaglutide.

36. The homodimer of claim 15 for use in simultaneous, separate or sequential

combination with dulaglutide.

37. A polynucleotide encoding the fusion protein of any of claims 1-14.