Description

Provided herein are insulin analogs comprising at least one mutation relative to the parent insu lin, wherein the insulin analogs comprise a mutation at position B16 which is substituted with a hydrophobic amino acid and/or a mutation at position B25 which is substituted with a hydropho bic amino acid.

BACKGROUND

Worldwide, more than 400 million people suffer from type 1 or type 2 diabetes mellitus. Type 1 diabetes is treated with insulin substitution. In contrast to type 1 diabetes, there is basically no deficiency of insulin in type 2 diabetes, but in a large number of cases, especially in the ad vanced stage, type 2 diabetes patients are treated with insulin.

In a healthy person, the release of insulin by the pancreas is strictly coupled to the concentra tion of the blood glucose. Elevated blood glucose levels, such as occur after meals, and are rapidly compensated by a corresponding increase in insulin secretion. In the fasting state, the plasma insulin level falls to a basal value which is adequate to guarantee a continuous supply of insulin-sensitive organs and tissue with glucose and to keep hepatic glucose production low in the night. Often, the replacement of the endogenous insulin secretion by exogenous, mostly subcutaneous administration of insulin does not achieve the quality of the physiological regula tion of the blood glucose described above. Deviations of the blood glucose upward or downward can occur, which in their severest forms can be life-threatening. It is to be derived from this that an improved therapy of diabetes is primarily to be aimed at keeping the blood glucose as close ly as possible in the physiological range.

Human insulin is a polypeptide of 51 amino acids, which are divided into 2 amino acid chains: the A chain having 21 amino acids and the B chain having 30 amino acids. The chains are con nected to one another by means of 2 disulfide bridges. A third disulfide bridge exists between the cysteines at position 6 and 11 of the A chain. Some products in current use for the treatment of diabetes mellitus are insulin analogs, i.e. insulin variants whose sequence differs from that of human insulin by one or more amino acid substitutions in the A chain and/or in the B chain.

Like many other peptide hormones, human insulin has a short half-life in vivo. Thus, it is admin istered frequently which is associated with discomfort for the patient. Therefore, insulin analogs are desired which have an increased half-life in vivo and, thus, a prolonged duration of action.

There are currently different approaches for extending the half-life of insulins.

One approach is based on the development of a soluble formulation at low pH, but of reduced solubility relative to native insulin at physiologic pH. The isoelectric point of the insulin analog is increased through the addition of two arginines to the C-terminus of the B-chain. The addition of two arginines in combination with a glycine substitution at A21 (insulin glargine) provides an insulin with extended duration of action. The insulin analog precipitates in the presence of zinc upon injection in subcutaneous sites and slowly solubilizes, resulting a sustained presence of insulin glargine.

In another approach, a long chain fatty acid group is conjugated to the epsilon amino group of LysB29 of insulin. The presence of this group allows the attachment of the insulin to serum al bumin by noncovalent, reversible binding. As a consequence, this insulin analog has a signifi cantly prolonged time-action profile relative to human insulin (see e.g. Mayer et al., Inc. Biopol ymers (Pept Sci) 88: 687-713, 2007; or WO 2009/115469).

WO 2016/006963 discloses insulin analogs having a reduced insulin receptor-mediated clear ance rate, compared to human insulin.

WO 2018/056764 discloses insulin analogs having a reduced insulin receptor-mediated clear ance rate, compared to human insulin.

WO 2008/034881 discloses protease stabilized insulin analogs.

In order to increase the duration of action of a drug, half-life plays a major role. Half-life (L/2) is proportional to the volume of distribution divided by clearance. In the case of human insulin, clearance is mainly driven by binding to the insulin receptor, internalization and subsequent degradation.

Accordingly, there is a need for insulin analogs which have a reduced insulin receptor-binding activity, and thus a reduced receptor-mediated clearance rate, but which have a signal trans duction activity which allow for sufficiently lowering the blood glucose level in vivo.

SUMMARY

Provided herein are long-acting insulin analogs having a very low binding affinity (hence a lower clearance rate) whilst still maintaining high signal transduction.

Surprisingly, it was shown in the context of the studies underlying the present invention that a substitution at position B16 and/or B25 of human insulin with a hydrophobic amino acid (such as leucine, isoleucine, valine, alanine and tryptophan) resulted in a decrease of insulin receptor binding activity (as compared to the insulin receptor binding activity of the parent insulin, see Examples). The strongest effects on insulin receptor binding activity were observed for substitu tions with branched-chain amino acids (leucine, isoleucine and valine). Interestingly, insulin ana logs with such substitutions at these positions (such as at position B25) showed up to 6-fold enhancement in signal transduction than expected based on their insulin receptor isoform B (IR-B) binding affinities (see Examples). Further, some tested insulin analogs showed improved proteolytic stability against a-chymotrypsin, cathepsin D and insulin degrading enzyme (see Examples).

Accordingly, provided herein are insulin analogs comprising at least one mutation relative to the parent insulin, wherein the insulin analogs comprise a mutation at position B16 which is substi tuted with a hydrophobic amino acid, and/or a mutation at position B25 which is substituted with a hydrophobic amino acid. In some embodiments, an insulin analog is provided comprising at least one mutation relative to the parent insulin, wherein the insulin analog comprises a muta tion at position B16 which is substituted with a branched-chain amino acid and/or a mutation at position B25 which is substituted with a branched-chain amino acid.

The expression“insulin analog” as used herein refers to a peptide which has a molecular struc ture which formally can be derived from the structure of a naturally occurring insulin (herein also referred to as“parent insulin”, e.g. human insulin) by deleting and/or substituting at least one amino acid residue occurring in the naturally occurring insulin and/or adding at least one amino acid residue. The added and/or exchanged amino acid residue can either be codable amino acid residues or other naturally occurring residues or purely synthetic amino acid residues. The analog as referred to herein is capable of lowering blood glucose levels in vivo, such as in a human subject.

In some embodiments, the insulin analog provided herein comprises two peptide chains, an A-chain and a B-chain. Typically, the two chains are connected by disulfide bridges between cys teine residues. For example, in some embodiments, insulin analogs provided herein comprise three disulfide bridges: one disulfide bridge between the cysteines at position A6 and A1 1 , one disulfide bridge between the cysteine at position A7 of the A-chain and the cysteine at position B7 of the B-chain, and one between the cysteine at position A20 of the A-chain and the cysteine at position B19 of the B-chain. Accordingly, insulin analogs provided herein may comprise cys teine residues at positions A6, A7, A11 , A20, B7 and B19.

In some embodiments provided herein, the insulin analog is a single-chain insulin. A single chain insulin is a single polypeptide chains in which the insulin B-chain is linked contiguously with the insulin A-chain via an uncleaved connecting peptide.

Mutations of insulin, i.e. mutations of a parent insulin, are indicated herein by referring to the chain, i.e. either the A-chain or the B-chain of the analog, the position of the mutated amino acid residue in the A- or B-chain (such as A14, B16 and B25), and the three letter code for the amino acid substituting the native amino acid in the parent insulin. The term“desB30” refers to an ana log lacking the B30 amino acid from the parent insulin (i.e. the amino acid at position B30 is absent). For example, Glu(A14)lle(B16)desB30 human insulin, is an analog of human insulin in which the amino acid residue at position 14 of the A-chain (A14) of human insulin is substituted with glutamic acid, the amino acid residue at position 16 of the B-chain (B16) is substituted with isoleucine, and the amino acid at position 30 of the B chain is deleted (i.e. is absent).

Insulin analogs provided herein comprise at least one mutation (substitution, deletion, or addi tion of an amino acid) relative to parent insulin. The term“at least one”, as used herein means one, or more than one, such as“at least two”,“at least three”,“at least four”,”at least five”, etc. In some embodiments, the insulin analogs provided herein comprise at least one mutation in the B-chain and at least one mutation in the A-chain. In a further embodiment, the insulin analogs provided herein comprise at least two mutations in the B-chain and at least one mutation in the A-chain. For example, the insulin analog may comprise a substitution at position B16, a deletion at position B30 and a substitution at position A14. Alternatively, the insulin analog may comprise a substitution at position B25, a deletion at position B30 and a substitution at position A14. Fur ther, the insulin analog may comprise a substitution at position B16, a substitution at position B25, a deletion at position B30 and a substitution at position A14.

The insulin analogs provided herein may comprise mutations in addition to the mutations above. In some embodiments, the number of mutations does not exceed a certain number. In some embodiments, the insulin analogs comprise less than twelve mutations (i.e. deletions, substitu tion, additions) relative to the parent insulin. In another embodiment, the analog comprises less than ten mutations relative to the parent insulin. In another embodiment, the analog comprises less than eight mutations relative to the parent insulin. In another embodiment, the analog com prises less than seven mutations relative to the parent insulin. In another embodiment, the ana log comprises less than six mutations relative to the parent insulin. In another embodiment, the analog comprises less than five mutations relative to the parent insulin. In another embodiment, the analog comprises less than four mutations relative to the parent insulin. In another embodi ment, the analog comprises less than three mutations relative to the parent insulin.

The expression“parent insulin” as used herein refers to naturally occurring insulin, i.e. to an unmutated wild-type insulin. In some embodiments, the parent insulin is animal insulin, such as mammalian insulin. For example, the parent insulin may be human insulin, porcine insulin, or bovine insulin.

In some embodiments, the parent insulin is human insulin. The sequence of human insulin is well known in the art and shown in Table 1 in the Example section. Human insulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 1 (GIVEQCCTSICSLYQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 2 (FVNQHLCGSHLVEALYLVCGERGFFYTPKT).

In another embodiment, the parent insulin is bovine insulin. The sequence of bovine insulin is well known in the art. Bovine insulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 81 (GIVEQCCASVCSLYQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 82 (FVNQHLCGSHLVEALYLVC-GERGFFYTPKA).

In another embodiment, the parent insulin is porcine insulin. The sequence of porcine insulin is well known in the art. Porcine insulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 83 (GIVEQCCTSICSLYQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 84 (FVNQHLCGSHLVEALYLVC GERGFFYTPKA).

Human, bovine, and porcine insulin comprises three disulfide bridges: one disulfide bridge be tween the cysteines at position A6 and A11 , one disulfide bridge between the cysteine at posi tion A7 of the A-chain and the cysteine at position B7 of the B-chain, and one between the cys teine at position A20 of the A-chain and the cysteine at position B19 of the B-chain.

The insulin analogs provided herein have an insulin receptor binding affinity which is reduced as compared to the insulin receptor binding affinity of the corresponding parent insulin, e.g. of hu man insulin.

The insulin receptor can be any mammalian insulin receptor, such as a bovine, porcine or hu man insulin receptor. In some embodiments, the insulin receptor is a human insulin receptor, e.g. human insulin receptor isoform A or human insulin receptor isoform B (which was used in the Examples section).

Advantageously, the human insulin analogs provided herein have a significantly reduced bind ing affinity to the human insulin receptor as compared to the binding affinity of human insulin to the human insulin receptor (see Examples). Thus, the insulin analogs have a very low clearance rate, i.e. a very low insulin-receptor-mediated clearance rate.

In some embodiments, the insulin analogs have, i.e. exhibit, less than 20 % of the binding affini ty to the corresponding insulin receptor compared to its parent insulin. In another embodiment, the insulin analogs provided herein have less than 10 % of the binding affinity to the corre sponding insulin receptor compared to its parent insulin. In another embodiment, the insulin analogs provided herein have less than 5 % of the binding affinity to the corresponding insulin receptor compared to its parent insulin, such as less than 3 % of the binding affinity compared to its parent insulin. For example, the insulin analogs provided herein may have between 0.1 % to 10 %, such as between 0.3 % to 5 % of the of the binding affinity to the corresponding insulin receptor compared to its parent insulin. Also, the insulin analogs provided herein may have be tween 0.5% to 3 %, such as between 0.5 % to 2 % of the of the binding affinity to the corre sponding insulin receptor compared to its parent insulin.

Methods for determining the binding affinity of an insulin analog to an insulin receptor are well known in the art. For example, the insulin receptor binding affinity can be determined by a scin tillation proximity assay which is based on the assessment of competitive binding between [125l]-labelled parent insulin, such as [125l]-labelled human insulin, and the (unlabeled) insulin analog to the insulin receptor. The insulin receptor can be present in a membrane of a cell, e.g. of CHO (Chinese Hamster Ovary) cell, which overexpresses a recombinant insulin receptor. In an embodiment, the insulin receptor binding affinity is determined as described in the Examples section.

Binding of a naturally occurring insulin or an insulin analog to the insulin receptor activates the insulin signaling pathway. The insulin receptor has tyrosine kinase activity. Binding of insulin to its receptor induces a conformational change that stimulates the autophosphorylation of the receptor on tyrosine residues. The autophosphorylation of the insulin receptor stimulates the receptor’s tyrosine kinase activity toward intracellular substrates involved in the transduction of the signal. The autophosphorylation of the insulin receptor by an insulin analog is therefore con sidered as a measure for signal transduction caused by said analog.

The insulin analogs in Table 1 of the Examples section were subjected to autophosphorylation assays. Interestingly, insulin analogs with aliphatic substitutions at positions B16 and B25 caused higher than expected insulin receptor autophosphorylation based on their insulin recep tor binding affinities. Thus, the insulin analogs provided herein have a low binding activity, and consequently a lower receptor-mediated clearance rate, but are nevertheless capable of caus ing a relatively high signal transduction. Therefore, the insulin analogs provided herein could be used as long-acting insulins. In some embodiments, the insulin analog provided herein are ca pable of inducing 1 to 10 %, such as 2 to 8 %, insulin receptor autophosphorylation relative to the parent insulin (such as human insulin). Further, in some embodiments, the insulin analogs provided herein are capable of inducing 3 to 7 %, such as 5 to 7% insulin receptor autophos phorylation relative to the parent insulin (such as human insulin). The insulin receptor auto phosphorylation relative to a parent insulin can be determined as described in the Examples section.

Insulin analogs provided herein were subjected to protease stability assays. As shown in Table 3, insulin analogs provided herein had higher stability towards at least some of the tested prote ases as compared to human insulin. Improved proteolytic stability was observed against a-chymotrypsin, cathepsin D and insulin degrading enzyme (IDE). Accordingly, insulin analogs provided herein are, typically, proteolytically stable insulin analogs. Thus, they are slower de graded by proteases relative to the parent insulin. In some embodiments, the insulin analog provided herein are stabilized against degradation by a-chymotrypsin, cathepsin D and insulin degrading enzyme (IDE) compared to parent insulin.

As set forth above, the insulin analog comprises at least one mutation as compared to the par ent insulin.

In some embodiments insulin analogs provided herein comprise a mutation at position B16 which is substituted with a hydrophobic amino acid. Thus, the amino acid at position B16 (tyro sine in human, bovine and porcine insulin) is replaced with a hydrophobic amino acid.

In another embodiment, insulin analogs provided herein comprise a mutation at position B25 which is substituted with a hydrophobic amino acid. Thus, the amino acid at position B25 (phe nylalanine in human, bovine and porcine insulin) is replaced with a hydrophobic amino acid.

In another embodiment, insulin analogs provided herein comprise a mutation at position B16 which is substituted with a hydrophobic amino acid and a mutation at position B25 which is sub stituted with a hydrophobic amino acid.

The hydrophobic amino acid may be any hydrophobic amino acid. For example, the hydropho bic amino acid may be an aliphatic amino acid such as a branched-chain amino acid.

In some embodiments of the insulin analogs provided herein, the hydrophobic amino acid used for the substitution at position B16 and/or B25 is isoleucine, valine, leucine, alanine, tryptophan, methionine, proline, glycine, phenylalanine or tyrosine (or with a derivative of the aforemen tioned amino acids).

Several parent insulins such as human, bovine and porcine insulin comprise tyrosine at position B16 and phenylalanine at position B25. Thus, the amino acid at position B16 of the parent insu lin may be substituted with isoleucine, valine, leucine, alanine, tryptophan, methionine, proline, glycine or phenylalanine (or with a derivative of the aforementioned amino acids). Further, the amino acid at position B25 of the parent insulin may be substituted with isoleucine, valine, leu cine, alanine, tryptophan, methionine, proline, glycine, or tyrosine (or with a derivative of the aforementioned amino acids).

Derivatives of the aforementioned amino acids are known in the art.

Derivatives of leucine include, but are not limited to, homo-leucine and tert-leucine. Thus, the amino acid at position B16 and/or B25 may be substituted with homo-leucine or tert-leucine.

A derivative of valine is, e.g., 3-ethyl norvaline. Thus, the amino acid at position B16 and/or B25 may be substituted with 3-ethyl norvaline.

Derivatives of glycine include, but are not limited to, cyclohexyl-glycine cyclopropylglycine, and trifluorethylglycine.

Derivatives of alanine include, but are not limited to, beta-t-butylalanine, cyclobutyl-alanine, cy-clopropyl-alanine and homo-cyclohexylalanine.

In some embodiments, the hydrophobic amino acid used for the substitution at position B16 and/or B25 is isoleucine, valine, leucine, alanine, or tryptophan.

In some embodiments, the aliphatic amino acid is not alanine. Accordingly, the hydrophobic amino acid used for the substitution at position B16 and/or B25 may be isoleucine, valine, leu cine, or tryptophan.

In some embodiments, the hydrophobic amino acid used for the substitution at position B16 and/or B25 is isoleucine, valine, or leucine.

In some embodiments, the amino acids referred to herein are L-amino acids (such as L-isoleucine, L-valine, or L-leucine). Accordingly, the amino acids (or the derivatives thereof) used for e.g. the substitution at position B16, B25 and/or A14 are typically L-amino acids.

In some embodiments, the hydrophobic amino acid is an aliphatic amino acid. Accordingly, the insulin analogs provided herein comprise a mutation at position B16 which is substituted with an aliphatic amino acid and a mutation at position B25 which is substituted with an aliphatic amino acid (and optionally further mutations including but not limited to Des(B30) and Glu(A14)).

Aliphatic amino acids are non-polar and hydrophobic amino acids comprising an aliphatic side chain functional group. Hydrophobicity increases with the number of carbon atoms on the hy drocarbon chain increases. A measure for the hydrophobicity of an aliphatic is the hydropathy index according to the Kyte and Doolittle scale which e.g. can be determined as disclosed by Kyte J. et al. Journal of Molecular Biology. 1982 157 (1): 105-32. In some embodiments, the aliphatic amino acid is an aliphatic amino acid having a hydropathy index (according to the Kyte and Doolittle scale) of larger than 2.0, such as larger than 3.0 or larger than 3.5.

Aliphatic amino acids include, but are not limited to, isoleucine, valine, leucine, alanine and gly cine. For example, the aliphatic amino acid may be an amino acid selected from isoleucine, va line, leucine, and glycine, such as an amino acid selected from isoleucine, valine and leucine.

Isoleucine, valine and leucine are branched-chain amino acids (abbreviated BCAA). Thus, the aliphatic amino acid may be a branched-chain amino acid. In some embodiments the insulin analogs provided herein comprise a mutation at position B16 which is substituted with a branched-chain amino acid and a mutation at position B25 which is substituted with a branched-chain amino acid (and optionally further mutations including but not limited to Des(B30) and Glu(A14)).

BCAAs are amino acids such as isoleucine, valine, and leucine are amino acids having aliphatic side chains that are non-linear, i.e. branched-chain amino acids are amino acid having an ali phatic side-chain with a branch (a central carbon atom bound to three or more carbon atoms).

The branched-chain amino acid may be a proteinogenic BCAA, i.e. an amino acid that is incor porated biosynthetically into proteins during translation, or a non-proteinogenic BCAA, i.e. an amino acid that is not naturally encoded or found in the genetic code of any organism. For ex ample, proteinogenic BCAAs are leucine, isoleucine, and valine. Thus, the hydrophobic/aliphatic amino acid branched-chain amino acid may be leucine, isoleucine or valine (or a derivative of the leucine, isoleucine or valine, such as a derivative of leucine or valine as set forth above).

In some embodiments, the branched-chain amino acid is isoleucine.

In some embodiments, the branched-chain amino acid is valine.

In some embodiments, the branched-chain amino acid is leucine.

In addition to the mutation at position B16 and/or the mutation at position B25 as described above, insulin analogs provided herein may comprise further mutations relative to the parent insulin.

For example, the insulin analog may further comprise a mutation at position A14. Such muta tions are known to increase protease stability (see e.g. WO 2008/034881). In some embodi ments, the amino acid at position A14 is substituted with glutamic acid (Glu). In some embodi ments, the amino acid at position A14 is substituted with aspartic acid (Asp). In some embodi ments, the amino acid at position A14 is substituted with histidine (His).

Further, the insulin analogs provided herein may comprise a mutation at position B30. In some embodiment, the mutation at position B30 is the deletion of threonine at position B30 of the par ent insulin (also referred to as Des(B30)-mutation).

Further, the insulin analog of the present invention may further comprise a mutation at position B3 which is substituted with a glutamic acid (Glu), and/or a mutation at position A21 which is substituted with glycine (Gly).

In an embodiment, the B chain of the insulin analog of the present invention comprises or con sists of the amino acid sequence shown in SEQ ID NO: 22 (FVNQHLCGSHLVEALYL-VCGERGFLYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 24 (FVNQHLCGSHLVEALYL-VCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 44 (FVNQHLCGSHLVEALYL-VCGERGFIYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 48 (FVNQHLCGSHLVEALYL-VCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 50 (FVEQHLCGSHLVEALYL-VCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 58 (FVNQHLCGSHLVEAL-ILVCGERGFIYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 60 (FVEQHLCGSHLVEAL-ILVCGERGFIYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 64 (FVNQHLCGSHLVEAL-ILVCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 66 (FVEQHLCGSHLVEAL-ILVCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 70

(FVNQHLCGSHLVEALVLVCGERGFIYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 78

(FVEQHLCGSHLVEALVLVCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 80

(FVEQHLCGSHLVEALVLVCGERGFVYTPK).

The B chains summarized above comprise the Des(B30) mutation. Accordingly, the amino acid which is present at position B30 of the parent insulin (threonine in human insulin, and alanine in porcine and bovine insulin) is deleted, i.e. not present. However, it is also envisaged that the B chains of the analogs of the present invention do not comprise this mutation, i.e. comprise a threonine at position 30. Accordingly, the B chain of the insulin analog of the present invention may comprise or consist of an amino acid sequence selected from the group consisting of:

• FVNQHLCGSHLVEALYLVCGERGFLYTPKT (SEQ ID NO: 85)

• FVNQHLCGSHLVEALYLVCGERGFVYTPKT (SEQ ID NO: 86)

• FVNQHLCGSHLVEALYLVCGERGFIYTPKT (SEQ ID NO: 87)

• FVNQHLCGSHLVEALYLVCGERGFVYTPKT (SEQ ID NO: 88)

• FVEQHLCGSHLVEALYLVCGERGFVYTPKT (SEQ ID NO: 89)

• FVNQH LCGSH LVEALI LVCGERGFI YTPKT (SEQ ID NO: 90)

• FVEQHLCGSHLVEALILVCGERGFIYTPKT (SEQ ID NO: 91)

• FVNQH LCGSH LVEALI LVCGERGFVYTPKT (SEQ ID NO: 92)

• FVEQHLCGSHLVEALILVCGERGFVYTPKT (SEQ ID NO: 93)

• FVNQH LCGSHLVEALVLVCGERGFIYTPKT (SEQ ID NO: 94)

• FVNQH LCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 95)

• FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 96)

• FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 97)

In an embodiment, the A chain of the insulin analog of the present invention comprises or con sists of the amino acid sequence shown in SEQ ID NO: 1 (GIVEQCCTSICSLYQLENYCN).

In another embodiment, the A chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 43 (GIVEQCCTSICSLEQLENYCN).

In another embodiment, the A chain of the insulin analog of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 45 (GIVEQCCTSICSLEQLENYCG).

Typically, the insulin analog of the present invention comprises an A-chain and a B-chain as set forth above.

For example, the insulin analog of the present invention is selected from the group consisting of: Leu(B16)-insulin (e.g. human insulin, i.e. Leu(B16)-human insulin),

Val(B16)-insulin (e.g. human insulin, i.e. Val(B16)-human insulin),

lle(B16)-insulin (e.g. human insulin),

Leu(B16)Des(B30)-insulin (e.g. human insulin),

Val(B16)Des(B30)-insulin (e.g. human insulin),

lle(B16)Des(B30)-insulin (e.g. human insulin),

Leu(B25)-insulin (e.g. human insulin),

Val(B25)-insulin (e.g. human insulin),

lle(B25)-insulin (e.g. human insulin),

Leu(B25)Des(B30)-insulin (e.g. human insulin),

Val(B25)Des(B30)-insulin (e.g. human insulin),

lle(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Leu(B16)Des(B30)-insulin (e.g. human insulin),

Glu(A14)lle(B16)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Val(B16)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Leu(B16)-insulin (e.g. human insulin),

Glu(A14)lle(B16)-insulin (e.g. human insulin),

Glu(A14)Val(B16)-insulin (e.g. human insulin),

Glu(A14)Leu(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)lle(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Val(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Leu(B25)-insulin (e.g. human insulin),

Glu(A14)lle(B25)-insulin (e.g. human insulin),

Glu(A14)Val(B25)-insulin (e.g. human insulin),

Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)lle(B16)lle(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Glu(B3)lle(B16)lle(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)lle(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Val(B16)lle(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Val(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

Glu(A14)Gly(A21)Glu(B3)Val(B25)-insulin (e.g. human insulin),

Glu(A14)lle(B16)lle(B25)-insulin (e.g. human insulin),

Glu(A14)Glu(B3)lle(B16)lle(B25)-insulin (e.g. human insulin),

Glu(A14)lle(B16)Val(B25)-insulin (e.g. human insulin),

Glu(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)-insulin (e.g. human insulin),

Glu(A14)Val(B16)lle(B25)-insulin (e.g. human insulin),

Glu(A14)Val(B16)Val(B25)-insulin (e.g. human insulin),

Glu(A14)Glu(B3)Val(B16)Val(B25)-insulin (e.g. human insulin), and

Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)-insulin (e.g. human insulin).

In another embodiment, the insulin analogs provided herein are selected from the group con sisting of:

Asp(A14)Leu(B16)Des(B30)-insulin (e.g. human insulin, i.e. Asp(A14)Leu(B16)Des(B30)-human insulin),

Asp(A14)lle(B16)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Val(B16)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Leu(B16)-insulin (e.g. human insulin),

Asp(A14)lle(B16)-insulin (e.g. human insulin),

Asp(A14)Val(B16)-insulin (e.g. human insulin),

Asp(A14)Leu(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)lle(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Val(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Leu(B25)-insulin (e.g. human insulin),

Asp(A14)lle(B25)-insulin (e.g. human insulin),

Asp(A14)Val(B25)-insulin (e.g. human insulin),

Asp(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)lle(B16)lle(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Glu(B3)lle(B16)lle(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)lle(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Val(B16)lle(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Val(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

Asp(A14)Gly(A21)Glu(B3)Val(B25)-insulin (e.g. human insulin),

Asp(A14)lle(B16)lle(B25)-insulin (e.g. human insulin),

Asp(A14)Glu(B3)lle(B16)lle(B25)-insulin (e.g. human insulin),

Asp(A14)lle(B16)Val(B25)-insulin (e.g. human insulin),

Asp(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)-insulin (e.g. human insulin),

Asp(A14)Val(B16)lle(B25)-insulin (e.g. human insulin),

Asp(A14)Val(B16)Val(B25)-insulin (e.g. human insulin),

Asp(A14)Glu(B3)Val(B16)Val(B25)-insulin (e.g. human insulin), and

Asp(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)-insulin (e.g. human insulin).

In another embodiment, the insulin analogs provided herein are selected from the group con sisting of:

His(A14)Leu(B16)Des(B30)-insulin (e.g. human insulin),

His(A14)lle(B16)Des(B30)-insulin (e.g. human insulin),

His(A14)Val(B16)Des(B30)-insulin (e.g. human insulin),

His(A14)Leu(B16)-insulin (e.g. human insulin),

His(A14)lle(B16)-insulin (e.g. human insulin),

His(A14)Val(B16)-insulin (e.g. human insulin),

His(A14)Leu(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)lle(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)Val(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)Leu(B25)-insulin (e.g. human insulin),

His(A14)lle(B25)-insulin (e.g. human insulin),

His(A14)Val(B25)-insulin (e.g. human insulin),

His(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)lle(B16)lle(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)Glu(B3)lle(B16)lle(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)lle(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)Val(B16)lle(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)Val(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin (e.g. human insulin),

His(A14)Gly(A21)Glu(B3)Val(B25)-insulin (e.g. human insulin),

His(A14)lle(B16)lle(B25)-insulin (e.g. human insulin),

His(A14)Glu(B3)lle(B16)lle(B25)-insulin (e.g. human insulin),

His(A14)lle(B16)Val(B25)-insulin (e.g. human insulin),

His(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)-insulin (e.g. human insulin),

His(A14)Val(B16)lle(B25)-insulin (e.g. human insulin),

His(A14)Val(B16)Val(B25)-insulin (e.g. human insulin),

His(A14)Glu(B3)Val(B16)Val(B25)-insulin (e.g. human insulin), and

His(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)-insulin (e.g. human insulin).

In another embodiment, the insulin analog is Leu(B25)Des(B30)-lnsulin (such as

Leu(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 11). For example, Leu(B25)Des(B30)-lnsulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 21

(GIVEQCCTSICSLYQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 22 (FVNQHLCGSHLVEALYLVCGERGFLYTPK).

In another embodiment, the insulin analog is Val(B25)Des(B30)-lnsulin (such as

Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 12). For example, Val(B25)Des(B30)-lnsulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 23

(GIVEQCCTSICSLYQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 24 (FVNQHLCGSHLVEALYLVCGERGFVYTPK).

In another embodiment, the insulin analog is Glu(A14)lle(B25)Des(B30)-lnsulin (such as Glu(A14)lle(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 22). For example, Glu(A14)lle(B25)Des(B30)-lnsulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 43 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 44 (FVNQHLCGSHLVEALYLVCGERGFIYTPK).

In another embodiment, the insulin analog is Glu(A14)Val(B25)Des(B30)-lnsulin (such as Glu(A14)Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Ta- ble 1 of the Examples section (see Analog 24). For example, Glu(A14)Val(B25)Des(B30)-lnsulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 47 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 48 (FVNQHLCGSHLVEALYLVCGERGFVYTPK).

In another embodiment, the insulin analog is Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-Insulin (such as Glu(A14)Gly(A21)Glu(B3) Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 25). For example, Glu(A14)Gly(A21)Glu(B3) Val(B25)Des(B30)-lnsulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 49 (GIVEQCCTSICSLEQLENYCG) and a B chain hav ing an amino acid sequence as shown in SEQ ID NO: 50 (FVEQHLCGSHLVEALYL-VCGERGFVYTPK).

In another embodiment, the insulin analog is Glu(A14)lle(B16)lle(B25)Des(B30)-lnsulin(such as Glu(A14)lle(B16)lle(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 29). For example, Glu(A14)lle(B16)lle(B25)Des(B30)-lnsulin comprises an A chain having an amino acid se quence as shown in SEQ ID NO: 57 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 58 (FVNQHLCGSHLVEALILVCGERGFIYTPK).

In another embodiment, the insulin analog is Glu(A14)Glu(B3)lle(B16)lle(B25)Des(B30)-lnsulin(such as Glu(A14)Glu(B3)lle(B16) lle(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 30). For example, Glu(A14)Glu(B3) lle(B16)lle(B25)Des(B30)-lnsulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 56 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 60 (FVEQHLCGSHLVEALILVCGERG-FIYTPK).

In another embodiment, the insulin analog is Glu(A14)lle(B16)Val(B25)Des(B30)-lnsulin (such as Glu(A14)lle(B16)Val(B25)Des(B30)-human insulin) The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 32). For example, Glu(A14)lle(B16)Val(B25)Des(B30)-lnsulin comprises an A chain having an amino acid se quence as shown in SEQ ID NO: 63 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 64 (FVNQHLCGSHLVEALILVCGERGFVYTPK).

In another embodiment, the insulin analog is

Glu(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)Des(B30)-lnsulin (such as Glu(A14)Gly(A21) Glu(B3)lle(B16)Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 33). For example,

Glu(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)Des(B30)-lnsulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 65 (GIVEQCCTSICSLEQLENYCG) and a B chain having an amino acid sequence as shown in SEQ ID NO: 66 (FVEQHLCGSHLVEAL-ILVCGERGFVYTPK).

In another embodiment, the insulin analog is Glu(A14)Val(B16)lle(B25)Des(B30)-lnsulin (such as Glu(A14)Val(B16)lle(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 35). For example,

Glu(A14)Val(B16)lle(B25)Des(B30)-lnsulin comprises an A chain having an amino acid se quence as shown in SEQ ID NO: 69 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 70 (FVNQHLCGSHLVEALVLVCGERGFIYTPK).

In another embodiment, the insulin analog is Glu(A14)Val(B16)Val(B25)Des(B30)-lnsulin (such as Glu(A14)Val(B16)Val(B25) Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 38). For example,

Glu(A14)Val(B16)Val(B25)Des(B30)-lnsulin comprises an A chain having an amino acid se quence as shown in SEQ ID NO: 75 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 76

(FVNQHLCGSHLVEALVLVCGERGFVYTPK).

In another embodiment, the insulin analog is Glu(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-Insulin (such as Glu(A14)Glu(B3)Val(B16) Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 39). For example, Glu(A14)Glu(B3)Val(B16) Val(B25)Des(B30)-lnsulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 77 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 78

(FVEQHLCGSHLVEALVLVCGERGFVYTPK).

In another embodiment, the insulin analog is

Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-lnsulin (such as Glu(A14)Gly(A21) Glu(B3)Val(B16)Val(B25)Des(B30)-human insulin). The sequence of this analog is, e.g., shown in Table 1 of the Examples section (see Analog 40). For example,

Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-lnsulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 79 (GIVEQCCTSICSLEQLENYCG) and a B chain having an amino acid sequence as shown in SEQ ID NO: 80

(FVEQHLCGSHLVEALVLVCGERGFVYTPK).

The insulin analog can be prepared by any method deemed appropriate. For example, the insu lin analog can be prepared by recombinant methods or by solid-phase synthesis.

The definitions and explanations given herein above apply mutatis mutandis to the following.

Provided herein are insulin B chains, i.e. insulin B chain peptides, as defined herein above in connection with the B chain of the insulin analog provided herein. Accordingly, provided herein are insulin B chains which comprise at least one mutation relative to the insulin B chain of the parent insulin, wherein the B chains comprise a mutation at position B16 which is substituted with a hydrophobic amino acid, and/or a mutation at position B25 which is substituted with a hydrophobic amino acid. The insulin B chain may comprise further mutations as described here in above such as the des(B30) deletion.

Also provided herein are proinsulins comprising an insulin A chain and/or an insulin B chain of the insulin analogs provided herein. The B chain may be any B chain as defined herein above for the insulin analogs provided herein. For example, provided herein are proinsulins comprising an insulin A chain and an insulin B chain, wherein said B chain comprises at least one mutation relative to B chain of a parent insulin, wherein the mutation is in position B16 which is substitut ed with a hydrophobic amino acid and/or wherein the mutation is in position B25 which is substi tuted with a hydrophobic amino acid. The insulin B chain may comprise further mutations as described herein above for the B chain.

Claims

1. An insulin analog comprising at least one mutation relative to the parent insulin, wherein the insulin analog comprises a mutation at position B16 which is substituted with a branched-chain amino acid and/or a mutation at position B25 which is substituted with a branched-chain amino acid.

2. The insulin analog of claim 1 , wherein the parent insulin is human insulin, porcine insulin, or bovine insulin.

3. The insulin analog of claims 1 and 2, wherein the branched-chain amino acid is selected from the group consisting of valine (Val), isoleucine (lie), and leucine (Leu).

4. The insulin analog of any one of claims 1 to 3, wherein said insulin analog further com prises a mutation at position A14 which is substituted with an amino acid selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp) and histidine (His).

5. The insulin analog of any one of claims 1 to 4, wherein said insulin analog further com prises a mutation at position B30.

6. The insulin analog of claim 5, wherein the mutation at position B30 is the deletion of the amino acid at position B30 of the parent insulin (Des(B30)-mutation).

7. The insulin analog of any one of claims 1 to 6, wherein said insulin analog further com prises a mutation at position B3 which is substituted with a glutamic acid (Glu).

8. The insulin analog of any one of claims 1 to 7, wherein said insulin further comprises a mutation at position A21 which is substituted with glycine (Gly).

9. The insulin analog of any one of claims 1 to 8, wherein the B chain of the insulin analog comprises or consists of an amino acid sequence selected from the group consisting of FVNQH LCGSH LVEALYLVCGERGFLYTPK (SEQ ID NO: 22)

FVNQHLCGSHLVEALYLVCGERGFIYTPK (SEQ ID NO: 44)

FVNQH LCGSH LVEALYLVCGERGFVYTPK (SEQ ID NO: 48)

FVEQHLCGSHLVEALYLVCGERGFVYTPK (SEQ ID NO: 50)

FVNQH LCGSH LVEALILVCGERGFIYTPK (SEQ ID NO: 58)

FVEQHLCGSHLVEALILVCGERGFIYTPK (SEQ ID NO: 60)

FVNQH LCGSH LVEALI LVCGERGFVYTPK (SEQ ID NO: 64)

FVEQHLCGSHLVEALILVCGERGFVYTPK (SEQ ID NO: 66)

FVNQH LCGSH LVEALVLVCGERGFIYTPK (SEQ ID NO: 70)

FVNQH LCGSH LVEALVLVCGERGFVYTPK (SEQ ID NO: 76)

FVEQHLCGSHLVEALVLVCGERGFVYTPK (SEQ ID NO: 78)

FVEQHLCGSHLVEALVLVCGERGFVYTPK (SEQ ID NO: 80)

FVNQHLCGSHLVEALYLVCGERGFLYTPKT (SEQ ID NO: 85)

FVNQH LCGSHLVEALYLVCGERGFVYTPKT (SEQ ID NO: 86)

FVNQHLCGSHLVEALYLVCGERGFIYTPKT (SEQ ID NO: 87)

FVNQH LCGSHLVEALYLVCGERGFVYTPKT (SEQ ID NO: 88)

FVEQHLCGSHLVEALYLVCGERGFVYTPKT (SEQ ID NO: 89)

FVNQH LCGSH LVEALI LVCGERGFI YTPKT (SEQ ID NO: 90)

FVEQHLCGSHLVEALILVCGERGFIYTPKT(SEQ ID NO: 91)

FVNQH LCGSH LVEALI LVCGERGFVYTPKT (SEQ ID NO: 92)

FVEQHLCGSHLVEALILVCGERGFVYTPKT (SEQ ID NO: 93)

FVNQH LCGSHLVEALVLVCGERGFIYTPKT (SEQ ID NO: 94)

FVNQH LCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 95)

FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 96), and

FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 97).

10. The insulin analog of any one of claims 1 to 9, comprising

(a) an A chain having an amino acid sequence as shown in SEQ ID NO: 43 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 44 (FVNQHLCGSHLVEALYLVCGERGFIYTPK),

(b) an A chain having an amino acid sequence as shown in SEQ ID NO: 47 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 48 (FVNQHLCGSHLVEALYLVCGERGFVYTPK), or

(c) an A chain having an amino acid sequence as shown in SEQ ID NO: 77 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 78 (FVEQHLCGSHLVEALVLVCGERGFVYTPK).

11. An insulin analog selected from the group consisting of

Leu(B16)-human insulin,

Val(B16)-human insulin,

lle(B16)-human insulin,

Leu(B16)Des(B30)-human insulin,

Val(B16)Des(B30)-human insulin,

lle(B16)Des(B30)-human insulin,

Leu(B25)-human insulin,

Val(B25)-human insulin,

lle(B25)-human insulin,

Leu(B25)Des(B30)-human insulin,

Val(B25)Des(B30)-human insulin,

lle(B25)Des(B30)-human insulin,

Glu(A14)Leu(B16)Des(B30)-human insulin,

Glu(A14)lle(B16)Des(B30)-human insulin,

Glu(A14)Val(B16)Des(B30)-human insulin,

Glu(A14)Leu(B16)-human insulin,

Glu(A14)lle(B16)-human insulin,

Glu(A14)Val(B16)-human insulin,

Glu(A14)Leu(B25)Des(B30)-human insulin,

Glu(A14)lle(B25)Des(B30)-human insulin,

Glu(A14)Val(B25)Des(B30)-human insulin,

Glu(A14)Leu(B25)-human insulin,

Glu(A14)lle(B25)-human insulin,

Glu(A14)Val(B25)-human insulin,

Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-human insulin,

Glu(A14)lle(B16)lle(B25)Des(B30)-human insulin,

Glu(A14)Glu(B3)lle(B16)lle(B25)Des(B30)-human insulin,

Glu(A14)lle(B16)Val(B25)Des(B30)-human insulin,

Glu(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)Des(B30)-human insulin,

Glu(A14)Val(B16)lle(B25)Des(B30)-human insulin,

Glu(A14)Val(B16)Val(B25)Des(B30)-human insulin,

Glu(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-human insulin,

Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-human insulin,

Glu(A14)Gly(A21)Glu(B3)Val(B25)-human insulin,

Glu(A14)lle(B16)lle(B25)-human insulin,

Glu(A14)Glu(B3)lle(B16)lle(B25)-human insulin,

Glu(A14)lle(B16)Val(B25)-human insulin,

Glu(A14)Gly(A21)Glu(B3)lle(B16)Val(B25)-human insulin,

Glu(A14)Val(B16)lle(B25)-human insulin,

Glu(A14)Val(B16)Val(B25)-human insulin,

Glu(A14)Glu(B3)Val(B16)Val(B25)-human insulin, and

Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)-human insulin.

12. An insulin B chain comprising at least one mutation relative to the B chain of the parent insulin, wherein the B chain comprises a mutation at position B16 which is substituted with a branched-chain amino acid, and/or a mutation at position B25 which is substituted with a branched-chain amino acid, and optionally wherein said insulin B chain further comprises a mutation at position B30, wherein the mutation at position B30 is the dele tion of threonine at position B30 of the parent insulin (Des(B30)-mutation).

13. A proinsulin comprising an insulin A chain and an insulin B chain, wherein the insulin B chain comprises at least one mutation relative to B chain of a parent insulin, wherein the B chain comprises a mutation at position B16 which is substituted with a branched-chain amino acid and/or a mutation at position B25 which is substituted with a branched-chain amino acid, and optionally wherein the insulin A chain of said proinsulin comprises a mu tation at position A14 which is substituted with an amino acid selected from glutamic acid (Glu), aspartic acid (Asp) and histidine (His).

14. A polynucleotide encoding the insulin analog of any one of claims 1 to 11 , the insulin B chain of claim 12, and/or the proinsulin of claim 13.

15. A host cell comprising insulin analog of any one of claims 1 to 11 , the insulin B chain of claims 12, the proinsulin of claim 13, and/or the polynucleotide of claim 14.