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.

The A chain comprised by the proinsulin provided herein may be any A chain as defined herein above for the insulin analogs provided herein. In some embodiments, the A chain of said proin sulin comprises a mutation at position A14 which is substituted with an amino acid selected from glutamic acid (Glu), aspartic acid (Asp) and histidine (His).

In addition to the insulin A chain and/or the insulin B chain, the proinsulins provided herein may comprise further elements such as leader sequences or a C-peptide. In some embodiments, the proinsulin may further comprise a C-peptide which is located between the insulin B chain and the insulin A chain. The C-peptide may have a length of 4-10 amino acids, such as a length of 4 to 9 amino acids. The orientation may be as follows (from N-terminus to C-terminus): B chain, C-peptide, A chain.

Provided herein are polynucleotides encoding the insulin analogs, insulin B chains, and the pro insulins provided herein. Said polynucleotide may be operably linked to a promoter which allows for the expression of said polynucleotide. In some embodiments, the promoter is heterologous with respect to said polynucleotide. In some embodiments, the promoter is a constitutive pro moter. In another embodiment, the promoter is an inducible promoter. Further, provided herein are vectors comprising the polynucleotide encoding the insulin analogs provided herein. In some embodiments, said vector is an expression vector.

Provided herein are host cells comprising nucleic acids encoding the insulin analogs, insulin B chains, and proinsulins, the polynucleotides, and/or the vectors provided herein. In some em bodiments, the host cell is a bacterial cell such as a cell of belonging to the genus Escherichia, e.g. an E. coli cell. In another embodiment, the host cell is a yeast cell, such as a Pichia pastoris cell or Klyveromyces lactis cell.

Provided herein are pharmaceutical compositions comprising a pharmaceutically effective amount of an insulin analog provided herein and a pharmaceutically acceptable excipient.

Provided herein are methods for treating a disease comprising administering a pharmaceutically effective amount of one or more insulin analogs provided herein or the pharmaceutical composi tion thereof to a subject.

In some embodiments, the disease is diabetes mellitus such as diabetes type II mellitus.

Provided herein are insulin analogs or the pharmaceutical composition thereof for use in medi cine.

Provided herein are insulin analogs or the pharmaceutical composition thereof for use in the treatment of diabetes mellitus, such as of diabetes type II mellitus.

Finally, provided herein are uses of the insulin analogs provided herein or the pharmaceutical compositions thereof for the preparation of a medicament or drug for the treatment of diabetes mellitus, such as of diabetes type II mellitus.

The insulin analogs, insulin B chains, proinsulins, and uses are further illustrated by the follow ing embodiments and combinations of embodiments as indicated by the respective dependen cies and back-references. The definitions and explanations given herein above apply mutatis mutandis to the following embodiments.

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 hy drophobic amino acid and/or a mutation at position B25 which is substituted with a hy drophobic amino acid.

2. The insulin analog of embodiment 1 , wherein the parent insulin is human insulin, porcine insulin, or bovine insulin. 3. The insulin analog of embodiments 1 and 2, wherein the hydrophobic amino acid at posi tion B16 and/or position B25 is an aliphatic amino acid.

4. The insulin analog of any one of embodiments 1 to 3, wherein said aliphatic amino acid at position B16 and/or position B25 is a branched-chain amino acid, such as a branched- chain amino acid selected from the group consisting of valine (Val), isoleucine (lie), and leucine (Leu).

5. The insulin analog of any one of embodiments 1 to 3, wherein said insulin analog further comprises 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).

6. The insulin analog of any one of embodiments 1 to 5, wherein said insulin analog further comprises a mutation at position B30, e.g. 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 embodiments 1 to 6, wherein said insulin analog further comprises a mutation at position B3 which is substituted with a glutamic acid (Glu).

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

9. The insulin analog of any one of embodiments 1 to 8, wherein the B chain of the insulin analog comprises or consists of an amino acid sequence selected from the group con- sisting of

FVNQH LCGSH LVEALYLVCGERGFLYTPK (SEQ ID NO: 22)

FVNQH LCGSH LVEALYLVCGERGFI YTPK (SEQ ID NO: 44)

FVNQH LCGSH LVEALYLVCGERGFVYTPK (SEQ ID NO: 48)

FVEQHLCGSHLVEALYLVCGERGFVYTPK (SEQ ID NO: 50)

FVNQH LCGSH LVEALI LVCGERGFI YTPK (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)

FVNQH LCGSH LVEALYLVCGERGFLYTPKT (SEQ ID NO: 85)

FVNQH LCGSHLVEALYLVCGERGFVYTPKT (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), and

FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 97). The insulin analog of any one of embodiments 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). 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. 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 hydrophobic amino acid, and/or a mutation at position B25 which is substituted with a hydrophobic amino acid. The insulin B chain according to embodiment 12, wherein the parent insulin is human insulin, porcine insulin, or bovine insulin. The insulin B chain according to embodiments 12 and 13, wherein the hydrophobic ami no acid at position B16 and/or position B25 is an aliphatic amino acid. The insulin B chain according to any one of embodiments 12 to 14, wherein said aliphat ic amino acid in a branched-chain amino acid, such as a branched-chain amino acid se lected from the group consisting of valine (Val), isoleucine (lie), and leucine (Leu).

The insulin B chain according to any one of embodiments 12 to 15, wherein said insulin B chain further comprises a mutation at position B3 which is substituted with a glutamic acid (Glu). The insulin B chain according to any one of embodiments 12 to 16, wherein said insulin B chain further comprises a mutation at position B30, wherein the mutation at position B30 is the deletion of the amino acid at position B30 of the parent insulin (Des(B30)- mutation). 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 hydrophobic amino acid and/or a mutation at position B25 which is substituted with a hydrophobic amino acid. The proinsulin of embodiment 18, wherein the insulin A chain of said proinsulin compris es a mutation at position A14 which is substituted with an amino acid selected from glu tamic acid (Glu), aspartic acid (Asp) and histidine (His). The proinsulin according to embodiments 18 and 19, wherein the parent insulin is human insulin, porcine insulin, or bovine insulin. The proinsulin according to any one of embodiments 18 to 20, wherein the hydrophobic amino acid at position B16 and/or position B25 is an aliphatic amino acid. The proinsulin according to any one of embodiments 18 to 21 , wherein said aliphatic amino acid in a branched-chain amino acid, such as a branched-chain amino acid se lected from the group consisting of valine (Val), isoleucine (lie), and leucine (Leu). The proinsulin according to any one of embodiments 18 to 22, wherein said proinsulin further comprises a mutation at position B3 which is substituted with a glutamic acid (Glu). The proinsulin according to any one of embodiments 18 to 23, wherein said proinsulin further comprises a mutation at position B30, wherein the mutation at position B30 is the deletion of the amino acid at position B30 of the parent insulin (Des(B30)-mutation). 25. A polynucleotide encoding the insulin analog of any one of embodiments 1 to 11 , the insulin B chain of any one of embodiments 12 to 17, and/or the proinsulin of any one of embodiment 18 to 24.

26. An expression vector comprising the polynucleotide of embodiment 25.

27. A host cell comprising insulin analog of any one of embodiments 1 to 11 , the insulin B chain of any one of embodiments 12 to 17, the proinsulin of any one of embodiments 18 to 24, the polynucleotide of embodiment 25, and/or the expression vector of embodiment 26.

28. A method for treating a patient having diabetes mellitus comprising administering to the patient one or more insulin analog as defined in any one of embodiments 1 to 11.

29. The insulin analog as defined in any one of embodiments 1 to 11 for use in treating dia betes mellitus.

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specifica tion.

Example 1 : Production of human insulin and insulin analogs

Human insulin as well as insulin analogs were produced recombinantly. Polynucleotides encod ing pre-pro-insulin were ordered from Geneart ^® . The designed polynucleotides were optimized for expression in yeast. They were inserted into an expression vector by classical restriction cloning enabling functional expression and secretion in Klyveromyces lactis K. As secretion leader, the gene was C-terminally fused to a DNA sequence encoding the alpha mating factor signal of Saccharomyces cerevisiae. The recombinant gene expression was controlled by a lactose inducible K. lactis promoter.

Human insulin as well as insulin analogs were manufactured as a pre-pro-insulin. A genetically fused N-terminal pre-sequence was used to improve expression and secretion yields and to stabilize the peptide in the culture broth. A broad variety of sequences can be used for this pur pose and were tested for efficiency. The proinsulin itself consists of a B-chain fused to a C- peptide followed by the C-terminal A-chain. As C-peptide a variety of amino acid combinations are described. It was shown that short peptides of 1-10 amino acids work well as C-sequences. For later processing of the insulin the recognition sites for specific proteases, which flank the C- peptide to enable its excision, are important.

K. lactis cells were made competent by chemical means. Subsequently, the cells were trans- formed with the expression plasmid coding for the respective pre-pro-insulin. After insertion of the plasmid, cells were plated on selective agar plates containing geneticin. Grown colonies were isolated and tested for recombinant gene expression. Cells were grown to sufficiently high cell densities in yeast peptone dextrose medium supplemented with geneticin. After an initial growth phase, a salt-buffered yeast extract medium with geneticin supplemented with lactose was added to the cultures to induce expression of the recombinant gene. Cultures were grown several days and supernatants were harvested by centrifugation.

Purification of the functional insulin or insulin analogs was started by a filtration procedure. Initial chromatographic capturing procedure was made with an ion-exchange resin. Cleavage of pre- pro-insulin to insulin was performed with a highly specific protease. Depletion of host cell pro tein, pre-sequence and product related products were made by a cascade of two additional chromatographic steps. Next to a hydrophobic interaction chromatography another ion- exchange procedure was applied to achieve this goal. Final polishing was made by reverse phase chromatography. Filtration, precipitation and freeze drying were used to finish the pro- duction process of the insulin molecule.

After coupling reactions with an activated carboxylic acid derivative, the solution with conjugated insulin molecules was filtered. Final purification was made by reverse phase chromatography. Filtration, precipitation and freeze drying were used to finish the synthesis of the target mole- cule.

Various insulins analogs with mutations e.g. at positions B16, B25 and/or A14 were generated. Table 1 provides an overview of the generated insulins.

Table 1: Generated analogs of human Insulin

Example 2: Insulin receptor binding affinity assays / Insulin receptor autophosphorylation assays

Insulin binding and signal transduction of various generated insulin analogs were determined by a binding assay and a receptor autophosphorylation assay.

A) Insulin receptor binding affinity assay

Insulin receptor binding affinity for the analogs listed in Table 1 was determined as described in Hartmann et al. (Effect of the long-acting insulin analogs glargine and degludec on cardiomyo- cyte cell signaling and function. Cardiovasc Diabetol. 2016; 15:96): Isolation of insulin receptor embedded plasma membranes (M-IR) and competition binding experiments were performed as previously described (Sommerfeld et al., PLoS One. 2010; 5(3): e9540). Briefly, CHO-cells overexpressing the IR were collected and re-suspended in ice-cold 2.25 STM buffer (2.25 M sucrose, 5 mM Tris-HCI pH 7.4, 5 mM MgCh, complete protease inhibitor) and disrupted using a Dounce homogenizer followed by sonication. The homogenate was overlaid with 0.8 STM buffer (0.8 M sucrose, 5 mM Tris-HCI pH 7.4, 5 mM MgCh, complete protease inhibitor) and ultra-centrifuged for 90 min at 100,000g. Plasma membranes at the interface were collected and washed twice with phosphate buffered saline (PBS). The final pellet was re-suspended in dilu tion buffer (50 mM Tris-HCI pH 7.4, 5 mM MgCh, complete protease inhibitor) and again ho mogenized with a Dounce homogenizer. Competition binding experiments were performed in a binding buffer (50 mM Tris-HCI, 150 mM NaCI, 0.1 % BSA, complete protease inhibitor, adjust ed to pH 7.8) in 96-well microplates. In each well 2 pg isolated membrane was incubated with 0.25 mg wheat germ agglutinin polyvinyltoluene polyethylenimine scintillation proximity assay (SPA) beads. Constant concentrations of [125l]-labelled human insulin (100 pM) and various concentrations of respective unlabelled insulin (0.001-1000 nM) were added for 12 h at room temperature (23 °C). The radioactivity was measured at equilibrium in a microplate scintillation counter (Wallac Microbeta, Freiburg, Germany).”

The results of the insulin receptor binding affinity assays for the tested analogs relative to hu man insulin are shown in Table 2.

B) Insulin receptor autophosphorylation assays (as a measure for signal transduction)

In order to determine signal transduction of an insulin analog binding to insulin receptor B, auto phosphorylation was measured in vitro.

CHO cells expressing human insulin receptor isoform B (IR-B) were used for IR autophosphory lation assays using In-Cell Western technology as previously described (Sommerfeld et al., PLoS One. 2010; 5(3): e9540). For the analysis of IGF1 R autophosphorylation, the receptor was overexpressed in a mouse embryo fibroblast 3T3 Tet off cell line (BD Bioscience, Heidel berg, Germany) that was stably transfected with IGF1 R tetracycline-regulatable expression plasmid. In order to determine the receptor tyrosine phosphorylation level, cells were seeded into 96-well plates and grown for 44 h. Cells were serum starved with serum-free medium Ham’s F12 medium (Life Technologies, Darmstadt, Germany) for 2 h. The cells were subse quently treated with increasing concentrations of either human insulin or the insulin analog for 20 min at 37°C. After incubation the medium was discarded and the cells fixed in 3.75% freshly prepared para-formaldehyde for 20 min. Cells were permeabilised with 0.1 % Triton X-100 in PBS for 20 min. Blocking was performed with Odyssey blocking buffer (LICOR, Bad Homburg, Germany) for 1 hour at room temperature. Anti-pTyr 4G10 (Millipore, Schwalbach, Germany) was incubated for 2 h at room temperature. After incubation of the primary antibody, cells were washed with PBS + 0.1 % Tween 20 (Sigma-Aldrich, St Louis, MO, USA). The secondary anti- mouse-lgG-800-CW antibody (LICOR, Bad Homburg, Germany) was incubated for 1 h. Results were normalized by the quantification of DNA with TO-PR03 dye (Invitrogen, Karlsruhe, Ger many). Data were obtained as relative units (RU).

The results of the insulin receptor autophosphorylation assays for the tested analogs relative to human insulin are shown in Table 2.

Table 2: Relative insulin receptor binding affinities and autophosphorylation activities of tested analogs of human insulin (for the sequences, please see Table 1).

* relative to human insulin, nd\ not determined

** a value of 0 means that the binding affinity was below the detection limit

C) Conclusions

As it can be derived from Table 2, various hydrophobic substitutions at positions B16 and/or B25 were tested (tryptophan, alanine, valine, leucine and isoleucine). Albeit to a different extent, all tested insulin analogs with hydrophobic substitutions at these positions showed a decrease of insulin receptor binding activity. As compared to Trp substitutions (see e.g. Analogs 4, 15 and 23), substitutions with aliphatic amino acids such as alanine, valine, leucine and isoleucine had a stronger impact on insulin receptor binding activity. The strongest effects were observed for valine, leucine and isoleucine which are branched-chain amino acids. Substitutions with isoleu cine, valine and leucine resulted in a significant decrease of insulin receptor binding activity. Interestingly, insulin analogs with such substitutions at position B25 (such as valine, leucine or isoleucine substitution at B25, Analogs 11 , 12, 22, 24, 25, 29, 30, 32, 33, 35 38, 39, 40) showed up to 6-fold enhancement in signal transduction than expected based on their IR-B binding affin ities. Specifically, Leu(B25)Des(B30)-lnsulin and Val(B25)Des(B30)-lnsulin (Analogs 11 and 12, respectively) showed only 1 % binding to insulin receptor B and 6% auto phosphorylation rela tive to human insulin. Similarly, a single leucine substitution at position B16 (Analog 3) also showed a similar enhancement in signal transduction albeit to a slightly lower extent. By com parison, with the exception of Analog 26, analogs bearing a histidine B25 substitution (Analogs 10, 13, 14, 21 , 28) also showed reduced receptor binding, however a concomitant reduction in auto phosphorylation. In some cases (Analogs 30, 32, 35, 38, 39), insulin receptor binding was 0% whilst still showing activity in the auto phosphorylation assay. All of these analogs have combinations of valine and/or isoleucine substitutions at positions B16 and B25 in common, suggesting that the combi nation is responsible for the further drop in insulin receptor binding. Insulins with no substitution at position B25 but with exchanges at position B16 exhibited slightly higher binding affinities in comparison to their autophosphorylation values (Analogs 3, 4, 16, 17, 18, 19, 20).

Alanine in position B25 shows similar effects as valine, leucine or isoleucine substitution (Ana logs 11 , 12, 22), although to a lower extent. The receptor binding affinity and autophosphoryla- tion activity of analogs with valine, leucine or isoleucine substitution is lower than analogs with an alanine substitution.

Example 3: Determination of in vitro stability in different recombinant proteases and gastric simulated fluid

The insulin analogs were tested for proteolytic stability (a-chymotrypsin, cathepsin D, insulin degrading enzyme (IDE) and simulated gastric fluid.

A) Assay conditions

B) Preparation of simulated gastric fluid Two grams of sodium chloride and 3.2 g of purified pepsin (from porcine stomach mucosa, with an activity of 800 to 2500 units per mg of protein) were dissolved in 7.0 ml of hydrochloric acid. The volume was adjusted with water up to 1000 ml. The resulting solution was mixed and ad justed with either 0.2 N sodium hydroxide or 0.2 N hydrochloric acid to a pH of 1.2 ± 0.1.

C) General assay procedure

The stability determination was done using appropriate time points (for SIF and SGF 15, 30, 60, 120 and 240 minutes; for proteases 15, 30, 60 and 120 minutes). The incubation was done at 37°C and the % of remaining parent compound was calculated in reference to a TO time point. For the determination of the parent compound an appropriate bioanalytical LC-MS/MS or LC- HRMS method was used, using the supernatant, after protein precipitation with ethanol (1 eq. v/v) and a centrifugation step.

D) Preparation of samples

Compounds were dissolved in diluted hydrochloric acid at a final concentration of 40 mM. The compound concentration in the assay was 2 pM. A 1 :20 dilution of the working solution was done into the protease buffer and samples are then incubated at 37°C, under stirring. At the appropriate time point and aliquot was taken, the reaction was quenched ethanol (1 eq. v/v), than centrifuged. The supernatant was analyzed.

E) Conclusions

In particular, lipophilic amino acid substitutions such as valine and isoleucine at positions 16 and 25 in the B-chain were investigated. Of the tested Analogs (2, 7, 11 , 12, 14, 16, 19, 22, 23, 24 and 38), only minor differences in stability were observed against the proteases trypsin, car- boxypeptidase A and carboxypeptidase B relative to human insulin (data not shown). In general, all analogs bearing A14 and B25 (Analogs 22, 24, 38) substitution showed improved proteolytic stability against a-chymotrypsin, cathepsin D and insulin degrading enzyme (IDE). For example, in the case of a-chymotrypsin, human insulin was completely degraded within 2 hours whereas Analog 22 was almost completely resistant. Similarly, all B25-substituted analogs tested showed improved stability against cathepsin D, albeit with Analog 38 (a B16/B25 variant) showing supe rior stability compared to the other B25 variants.

One notable exception was observed in the case of IDE, in which Analog 19 with A14/B16 sub stitutions showed improved performance compared to the B25 variants. The data suggests however, that a substitution at A14, tested here with glutamic acid, is important for increased stability. Other substitutions were also shown to be beneficial for increased stability: such as substitutions at position B16 and at position B25. For example, Analog 7 with an amino acid exchange in position B25, lead to increased instability.

Table 3: Percent remaining insulin analog after incubation of different insulin analogs for 30 or 120 minutes with four different proteases (for the sequences of the tested analogs, see Table

nd: not determined CLAUSES

The following clauses are part of the description:

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 clause 1 , wherein the parent insulin is human insulin, porcine insu lin, or bovine insulin.

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

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

6. The insulin analog of clause 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 clauses 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 clauses 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 clauses 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 LVEALILVCGERGFVYTPK (SEQ ID NO: 64) FVEQHLCGSHLVEALILVCGERGFVYTPK (SEQ ID NO: 66)

FVNQHLCGSHLVEALVLVCGERGFIYTPK (SEQ ID NO: 70)

FVNQHLCGSHLVEALVLVCGERGFVYTPK (SEQ ID NO: 76)

FVEQHLCGSHLVEALVLVCGERGFVYTPK (SEQ ID NO: 78)

FVEQHLCGSHLVEALVLVCGERGFVYTPK (SEQ ID NO: 80)

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), and

FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 97).

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