RU2816595C2 - Insulin analogues characterized by reduced affinity of binding to insulin receptors - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to production of insulin analogues and can be used for recombinant production of human proinsulin and insulin with substitution of Glu(A14) in combination with additional modifications, including substitution in position B16 and/or B25 with a branched-chain amino acid.

EFFECT: invention enables to obtain an analogue of human proinsulin and insulin, having a reduced affinity of binding to insulin receptors.

9 cl, 3 tbl, 3 ex

Description

Provided herein are insulin analogs containing at least one mutation from the parent insulin, wherein the insulin analogs contain a mutation at position B16, which is a hydrophobic amino acid substitution, and/or a mutation at position B25, which is a hydrophobic amino acid substitution.

BACKGROUND OF THE INVENTION

More than 400 million people worldwide have type 1 or type 2 diabetes. Type 1 diabetes is treated with insulin replacement. Unlike type 1 diabetes, type 2 diabetes is not essentially insulin deficient, but in many cases, especially in the later stages, patients with type 2 diabetes are treated with insulin.

In a healthy person, the release of insulin by the pancreas is closely related to the concentration of glucose in the blood. Elevated blood glucose levels, such as those occurring after a meal, are quickly compensated for by a corresponding increase in insulin secretion. In the fasting state, plasma insulin levels fall to a basal value that is sufficient to ensure a continuous supply of glucose to insulin-sensitive organs and tissues and to maintain low levels of hepatic glucose production at night. Often, replacing endogenous insulin secretion with exogenous one, mainly through subcutaneous administration of insulin, does not ensure the achievement of the quality of physiological regulation of blood glucose levels described above. Increases and decreases in blood glucose levels may occur, which in their most severe forms can be life-threatening. It follows that improved diabetes therapy should primarily be aimed at maintaining blood glucose levels within the physiological range as accurately as possible.

Human insulin is a 51 amino acid polypeptide that is divided into 2 amino acid chains: an A chain of 21 amino acids and a B chain of 30 amino acids. The chains are connected to each other using 2 disulfide bridges. The third disulfide bridge is located between cysteine residues at positions 6 and 11 of the A chain. Some products currently used to treat diabetes mellitus are insulin analogues, i.e. insulin variants whose sequence differs from the sequence 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 administered frequently, which is associated with inconvenience for the patient. Therefore, insulin analogues with an extended in vivo half-life and thus a prolonged duration of action are needed.

Currently, there are different approaches to increase the half-life of insulin variants.

One approach is based on developing a soluble formulation at low pH, but with reduced solubility compared to native insulin at physiological pH. The isoelectric point of the insulin analog is increased by the addition of two arginine residues to the C-terminus of the B chain. The addition of two arginine residues in combination with a glycine substitution at position A21 (insulin glargine) provides insulin with an increased duration of action. The insulin analogue precipitates in the presence of zinc when injected into subcutaneous sites and slowly solubilizes, resulting in the persistent presence of insulin glargine.

In another approach, a long-chain fatty acid group is conjugated to the LysB29 epsilon amino group of insulin. The presence of this group allows insulin to attach to serum albumin through non-covalent, reversible binding. As a consequence, this insulin analog has a significantly prolonged time action profile compared to human insulin (see, for example, Mayer et al., Inc. Biopolymers (Pept Sci) 88: 687-713, 2007; or WO 2009/115469).

WO 2016/006963 discloses insulin analogues characterized by a reduced rate of insulin receptor-mediated elimination compared to human insulin.

WO 2018/056764 discloses insulin analogues characterized by a reduced rate of insulin receptor-mediated elimination compared to human insulin.

WO 2008/034881 discloses insulin analogues stabilized against proteases.

To increase the duration of action of a drug, the half-life plays an important role. The half-life (t _1/2 ) is proportional to the volume of distribution divided by the clearance. In the case of human insulin, clearance is primarily due to binding to the insulin receptor, internalization, and subsequent degradation.

Accordingly, there is a need for insulin analogs that have reduced insulin receptor binding activity and therefore a reduced rate of receptor-mediated elimination, but which have signal transduction activity to sufficiently lower blood glucose levels in vivo .

SHORT DESCRIPTION

Provided herein are long-acting insulin analogues that have very low binding affinities (thus lower clearance rates) while still maintaining high levels of signal transduction.

Surprisingly, in the context of the studies underlying the present invention, it was shown that 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 in insulin receptor binding activity (by compared with the insulin receptor binding activity of the parent insulin, see "Examples"). The strongest effects on insulin receptor binding activity were observed with branched chain amino acid substitutions (leucine, isoleucine, and valine). Interestingly, insulin analogues with such substitutions at these positions (such as at position B25) showed increased signal transduction by up to 6-fold compared to what was expected based on their isoform B binding affinity (IR-B) values. ) insulin receptor (see "Examples"). In addition, some insulin analogues tested showed improved proteolytic stability towards α-chymotrypsin, cathepsin D and insulin degrading enzyme (see Examples).

Accordingly, provided herein are insulin analogs containing at least one mutation compared to the parent insulin, wherein the insulin analogs contain a mutation at position B16, which is a substitution of a hydrophobic amino acid, and/or a mutation at position B25, which is a substitution of a hydrophobic amino acid. In some embodiments, an insulin analogue is provided containing at least one mutation compared to the parent insulin, wherein the insulin analogue contains a mutation at position B16, which is a substitution with a branched chain amino acid, and/or a mutation at position B25, which is a substitution branched chain amino acid.

The expression “insulin analogue” as used herein refers to a peptide having a molecular structure that can formally be derived from the structure of naturally occurring insulin (herein also referred to as “parent insulin,” e.g., human insulin) by deletion and/ or replacing at least one amino acid residue contained in naturally occurring insulin, and/or adding at least one amino acid residue. The added and/or replacement amino acid residues may be encoded amino acid residues or other naturally occurring residues or amino acid residues obtained solely by synthetic means. The analogue mentioned herein is capable of reducing blood glucose levels in vivo , such as in a human subject.

In some embodiments, an insulin analog provided herein contains two peptide chains, an A chain and a B chain. Typically, the two chains are connected by disulfide bridges between cysteine residues. For example, in some embodiments, the insulin analogs provided herein contain three disulfide bridges: one disulfide bridge between the cysteine residues at positions A6 and A11, 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, the insulin analogues provided herein may contain cysteine residues at positions A6, A7, A11, A20, B7 and B19.

In some embodiments provided herein, the insulin analog is single chain insulin. Single chain insulin is a single polypeptide chain in which the insulin B chain is continuously linked to the insulin A chain through a non-cleavable linking peptide.

Insulin mutations, i.e. mutations of the parent insulin are indicated herein by reference to the chain, i.e. on the A-chain or on the B-chain of the analogue, the position of the mutant amino acid residue in the A- or B-chain (such as A14, B16 and B25) and the three-letter code of the amino acid replacing the native amino acid in the original insulin. The term "desB30" refers to an analogue that does not have the B30 amino acid from the parent insulin (ie, the amino acid at position B30 is missing). For example, human insulin Glu(A14)Ile (B16)desB30 is a human insulin analogue in which the amino acid residue at position 14 of the A chain (A14) of human insulin is replaced by glutamic acid, the amino acid residue at position 16 of the B chain (B16) is replaced isoleucine, and the amino acid at position 30 of the B chain is deleted (i.e., missing).

The insulin analogues provided herein contain at least one mutation (substitution, deletion or addition of an amino acid) compared to the parent insulin. As used herein, the term “at least one” means one or more than one, such as “at least two,” “at least three,” “at least four,” “at least five,” and etc. In some embodiments, the insulin analogs provided herein contain 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 contain at least two mutations in the B chain and at least one mutation in the A chain. For example, an insulin analog may contain a substitution at position B16, a deletion at position B30, and a substitution at position A14. Alternatively, the insulin analog may contain a substitution at position B25, a deletion at position B30, and a substitution at position A14. In addition, the insulin analog may contain a substitution at position B16, a substitution at position B25, a deletion at position B30, and a substitution at position A14.

The insulin analogues provided herein may contain mutations in addition to the mutations listed above. In some embodiments, the number of mutations does not exceed a certain number. In some embodiments, the insulin analogues contain fewer than twelve mutations (i.e., deletions, substitutions, additions) compared to the parent insulin. In another embodiment, the analog contains fewer than ten mutations compared to the parent insulin. In another embodiment, the analog contains fewer than eight mutations compared to the parent insulin. In another embodiment, the analog contains fewer than seven mutations compared to the parent insulin. In another embodiment, the analog contains fewer than six mutations compared to the parent insulin. In another embodiment, the analog contains fewer than five mutations compared to the parent insulin. In another embodiment, the analog contains fewer than four mutations compared to the parent insulin. In another embodiment, the analog contains fewer than three mutations compared to the parent insulin.

The expression "original insulin" as used herein refers to naturally occurring insulin, i.e. to non-mutant wild-type insulin. In some embodiments, the parent insulin is animal-derived 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 is shown in Table 1 in the Examples section. Human insulin contains an A chain having the amino acid sequence shown under SEQ ID NO: 1 (GIVEQCCTSICSLYQLENYCN) and a B chain having the amino acid sequence shown under 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 contains an A chain having the amino acid sequence shown under SEQ ID NO: 81 (GIVEQCCASVCSLYQLENYCN) and a B chain having the amino acid sequence shown under 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 contains an A chain having the amino acid sequence shown under SEQ ID NO: 83 (GIVEQCCTSICSLYQLENYCN) and a B chain having the amino acid sequence shown under SEQ ID NO: 84 (FVNQHLCGSHLVEALYLVC GERGFFYTPKA).

Human, bovine, and porcine insulins contain three disulfide bridges: one disulfide bridge between the cysteine residues at positions A6 and A11, 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. -chain and cysteine at position B19 of the B-chain.

The insulin analogues provided herein have an insulin receptor binding affinity that is reduced compared to the insulin receptor binding affinity of the corresponding parent insulin, eg human insulin.

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

The human insulin analogues provided herein advantageously have significantly reduced binding affinity for the human insulin receptor compared to the binding affinity of human insulin for the human insulin receptor (see Examples). Thus, insulin analogues are characterized by a very low elimination rate, i.e. very low rate of insulin receptor-mediated elimination.

In some embodiments, insulin analogues have, i.e. characterized by less than 20% binding affinity to the corresponding insulin receptor compared to their parent insulin. In another embodiment, the insulin analogs provided herein have less than 10% binding affinity for the corresponding insulin receptor compared to their parent insulin. In another embodiment, the insulin analogs provided herein have less than 5% binding affinity for the corresponding insulin receptor compared to their parent insulin, such as less than 3% binding affinity compared to their parent insulin. For example, insulin analogues provided herein may have 0.1% to 10%, such as 0.3% to 5%, binding affinity for the corresponding insulin receptor compared to their parent insulin. Also, the insulin analogues provided herein may have 0.5% to 3%, such as 0.5% to 2%, binding affinity for the corresponding insulin receptor compared to their parent insulin.

Methods for determining the binding affinity of an insulin analog to the insulin receptor are well known in the art. For example, binding affinity to insulin receptors can be determined using a proximity scintillation assay, which is based on assessing the competitive binding between a parent [125I]-labeled insulin, such as [125I]-labeled human insulin, and an (unlabeled) insulin analogue at the insulin receptor . The insulin receptor may be present in the membrane of a cell, such as a CHO (Chinese hamster ovary) cell, that overexpresses a recombinant insulin receptor. In an embodiment, the binding affinity for insulin receptors is determined as described in the Examples section.

Binding of naturally occurring insulin or an insulin analogue to the insulin receptor activates the insulin signaling pathway. The insulin receptor has tyrosine kinase activity. The binding of insulin to its receptor induces a conformational change that stimulates autophosphorylation of the receptor at tyrosine residues. Autophosphorylation of the insulin receptor stimulates the tyrosine kinase activity of the receptor towards intracellular substrates involved in signal transduction. Autophosphorylation of the insulin receptor by an insulin analogue is thus considered to be a measure of the signal transduction induced by the analogue.

The insulin analogs from Table 1 in the Examples section were subjected to autophosphorylation assays. Interestingly, insulin analogues with aliphatic amino acid substitutions at positions B16 and B25 caused higher levels of insulin receptor autophosphorylation than expected based on their insulin receptor binding affinity values. Thus, the insulin analogues provided herein have low binding activity and therefore a lower rate of receptor-mediated clearance, but are nevertheless capable of producing relatively high levels of signal transduction. Therefore, the insulin analogues provided herein can be used as long-acting insulin options. In some embodiments, an insulin analog provided herein is capable of inducing autophosphorylation of insulin receptors at a level of 1 to 10%, such as 2 to 8%, compared to the parent insulin (such as human insulin). Additionally, in some embodiments, the insulin analogs provided herein are capable of inducing autophosphorylation of insulin receptors at a level of 3 to 7%, such as 5 to 7%, compared to parent insulin (such as human insulin). Autophosphorylation of insulin receptors relative to parent insulin can be determined as described in the Examples section.

The insulin analogues provided herein were subjected to protease stability assays. As shown in Table 3, the insulin analogues provided herein exhibited greater stability against at least some of the proteases tested compared to human insulin. Improved proteolytic stability was observed against α-chymotrypsin, cathepsin D and insulin degrading enzyme (IDE). Accordingly, the insulin analogs provided herein are generally proteolytically stable insulin analogs. Thus, they are destroyed more slowly by proteases compared to the original insulin. In some embodiments, the insulin analog provided herein is stabilized against degradation by α-chymotrypsin, cathepsin D, and insulin degrading enzyme (IDE) compared to parent insulin.

As stated above, the insulin analog contains at least one mutation compared to the parent insulin.

In some embodiments, the insulin analogs provided herein contain a mutation at position B16, which is a hydrophobic amino acid substitution. Thus, the amino acid at position B16 (tyrosine in human, bovine and porcine insulin) is replaced by a hydrophobic amino acid.

In another embodiment, the insulin analogs provided herein contain a mutation at position B25, which is a hydrophobic amino acid substitution. Thus, the amino acid at position B25 (phenylalanine in human, bovine and porcine insulin) is replaced by a hydrophobic amino acid.

In another embodiment, the insulin analogs provided herein contain a mutation at position B16, which is a hydrophobic amino acid substitution, and a mutation at position B25, which is a hydrophobic amino acid substitution.

The hydrophobic amino acid can be any hydrophobic amino acid. For example, the hydrophobic 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 substitution at position B16 and/or B25 is isoleucine, valine, leucine, alanine, tryptophan, methionine, proline, glycine, phenylalanine, or tyrosine (or a derivative the above amino acids).

Some versions of parent insulin, such as human, bovine, and porcine insulin, contain tyrosine at position B16 and phenylalanine at position B25. Thus, the amino acid at position B16 of the original insulin can be replaced by isoleucine, valine, leucine, alanine, tryptophan, methionine, proline, glycine or phenylalanine (or a derivative of the above amino acids). In addition, the amino acid at position B25 of the original insulin can be replaced by isoleucine, valine, leucine, alanine, tryptophan, methionine, proline, glycine or tyrosine (or a derivative of the above amino acids).

Derivatives of the above amino acids are known from the prior art.

Leucine derivatives include, but are not limited to, homoleucine and tert -leucine. Thus, the amino acid at position B16 and/or B25 can be replaced by homoleucine or tert -leucine.

The valine derivative is, for example, 3-ethylnorvaline. Thus, the amino acid at position B16 and/or B25 can be replaced by 3-ethylnorvaline.

Glycine derivatives include, but are not limited to, cyclohexylglycine, cyclopropylglycine, and trifluoroethylglycine.

Alanine derivatives include, but are not limited to, beta-tert-butylalanine, cyclobutylalanine, cyclopropylalanine, and homocyclohexylalanine.

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

In some embodiments, the hydrophobic amino acid used for 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 derivatives thereof) used, for example, for substitution at positions 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 contain a mutation at position B16, which is an aliphatic amino acid substitution, and a mutation at position B25, which is an aliphatic amino acid substitution (and optionally additional mutations, including but not limited to Des(B30) and Glu(A14)).

Aliphatic amino acids are non-polar and hydrophobic amino acids containing an aliphatic side chain functional group. Hydrophobicity increases with the number of carbon atoms in the hydrocarbon chain. A measure of the hydrophobicity of an aliphatic structure is the hydropathicity index according to the Kyte-Doolittle scale, which, for example, can be determined as described in 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 hydropathicity index (according to the Kyte-Doolittle scale) greater than 2.0, such as greater than 3.0 or greater than 3.5.

Aliphatic amino acids include, but are not limited to, isoleucine, valine, leucine, alanine and glycine. For example, the aliphatic amino acid may be an amino acid selected from isoleucine, valine, 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 contain a mutation at position B16, which is a branched chain amino acid substitution, and a mutation at position B25, which is a branched chain amino acid substitution (and optionally additional mutations, including without limitation Des(B30) and Glu(A14)).

BCAA are amino acids such as isoleucine, valine and leucine, which are amino acids with aliphatic side chains that are non-linear, i.e. Branched chain amino acids are amino acids having a branched aliphatic side chain (a central carbon atom linked to three or more carbon atoms).

The branched chain amino acid may be a proteinogenic BCAA, i.e. an amino acid that is biosynthesized into proteins during translation, or non-proteinogenic BCAA, i.e. an amino acid that is not naturally encoded or found in the genetic code of any organism. For example, proteinogenic BCAAs are leucine, isoleucine and valine. Thus, the hydrophobic/aliphatic branched chain amino acid may be leucine, isoleucine, or valine (or a derivative of leucine, isoleucine, or valine, such as a leucine or valine derivative 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 described above, the insulin analogues provided herein may contain additional mutations compared to the parent insulin.

For example, the insulin analog may further contain a mutation at position A14. Such mutations are known to increase stability to proteases (see, for example, WO 2008/034881). In some embodiments, the amino acid at position A14 is replaced with glutamic acid (Glu). In some embodiments, the amino acid at position A14 is replaced with aspartic acid (Asp). In some embodiments, the amino acid at position A14 is replaced with histidine (His).

In addition, the insulin analogues provided herein may contain a mutation at position B30. In some embodiments, the mutation at position B30 is a deletion of threonine at position B30 of the parent insulin (also referred to as a Des(B30) mutation).

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

In an embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 22 (FVNQHLCGSHLVEALYLVCGERGFLYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 24 (FVNQHLCGSHLVEALYLVCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 44 (FVNQHLCGSHLVEALYLVCGERGFIYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 48 (FVNQHLCGSHLVEALYLVCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 50 (FVEQHLCGSHLVEALYLVCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 58 (FVNQHLCGSHLVEALILVCGERGFIYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 60 (FVEQHLCGSHLVEALILVCGERGFIYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 64 (FVNQHLCGSHLVEALILVCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 66 (FVEQHLCGSHLVEALILVCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 70 (FVNQHLCGSHLVEALVLVCGERGFIYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 78 (FVEQHLCGSHLVEALVLVCGERGFVYTPK).

In another embodiment, the B chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 80 (FVEQHLCGSHLVEALVLVCGERGFVYTPK).

The B chains summarized above contain the Des(B30) mutation. Accordingly, the amino acid that is present at position B30 of the original insulin (threonine in human insulin and alanine in porcine and bovine insulin) is removed, i.e. is absent. However, it is also contemplated that the B chains of the analogues of the present invention do not contain this mutation, i.e. contain threonine at position 30. Accordingly, the B chain of the insulin analog of the present invention may contain 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),

FVNQHLCGSHLVEALILVCGERGFIYTPKT (SEQ ID NO: 90),

FVEQHLCGSHLVEALILVCGERGFIYTPKT (SEQ ID NO: 91),

FVNQHLCGSHLVEALILVCGERGFVYTPKT (SEQ ID NO: 92),

FVEQHLCGSHLVEALILVCGERGFVYTPKT (SEQ ID NO: 93),

FVNQHLCGSHLVEALVLVCGERGFIYTPKT (SEQ ID NO: 94),

FVNQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 95),

FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 96),

FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 97), or consist of it.

In an embodiment, the A chain of the insulin analog of the present invention contains or consists of the amino acid sequence shown under SEQ ID NO: 1 (GIVEQCCTSICSLYQLENYCN).

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

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

Typically, the insulin analog of the present invention contains an A chain and a B chain as described above.

For example, the insulin analogue of the present invention is selected from the group consisting of:

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

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

Ile(B16)-insulin (for example, human insulin),

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

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

Ile(B16)Des(B30)-insulin (for example, human insulin),

Leu(B25)-insulin (for example, human insulin),

Val(B25)-insulin (for example, human insulin),

Ile(B25)-insulin (for example, human insulin),

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

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

Ile(B25)Des(B30)-insulin (for example, human insulin),

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

Glu(A14)Ile(B16)Des(B30)-insulin (for example, human insulin),

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

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

Glu(A14)Ile(B16)-insulin (for example, human insulin),

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

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

Glu(A14)Ile(B25)Des(B30)-insulin (for example, human insulin),

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

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

Glu(A14)Ile(B25)-insulin (for example, human insulin),

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

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

Glu(A14)Ile(B16)Ile(B25)Des(B30)-insulin (for example, human insulin),

Glu(A14)Glu(B3)Ile(B16)Ile(B25)Des(B30)-insulin (for example, human insulin),

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

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

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

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

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

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

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

Glu(A14)Ile(B16)Ile(B25)-insulin (for example, human insulin),

Glu(A14)Glu(B3)Ile(B16)Ile(B25)-insulin (for example, human insulin),

Glu(A14)Ile(B16)Val(B25)-insulin (for example, human insulin),

Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)-insulin (for example, human insulin),

Glu(A14)Val(B16)Ile(B25)-insulin (for example, human insulin),

Glu(A14)Val(B16)Val(B25)-insulin (for example, 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 (for example, human insulin).

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

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

Asp(A14)Ile(B16)Des(B30)-insulin (for example, human insulin),

Asp(A14)Val(B16)Des(B30)-insulin (for example, human insulin),

Asp(A14)Leu(B16)-insulin (for example, human insulin),

Asp(A14)Ile(B16)-insulin (for example, human insulin),

Asp(A14)Val(B16)-insulin (for example, human insulin),

Asp(A14)Leu(B25)Des(B30)-insulin (for example, human insulin),

Asp(A14)Ile(B25)Des(B30)-insulin (for example, human insulin),

Asp(A14)Val(B25)Des(B30)-insulin (for example, human insulin),

Asp(A14)Leu(B25)-insulin (for example, human insulin),

Asp(A14)Ile(B25)-insulin (for example, human insulin),

Asp(A14)Val(B25)-insulin (for example, human insulin),

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

Asp(A14)Ile(B16)Ile(B25)Des(B30)-insulin (for example, human insulin),

Asp(A14)Glu(B3)Ile(B16)Ile(B25)Des(B30)-insulin (for example, human insulin),

Asp(A14)Ile(B16)Val(B25)Des(B30)-insulin (for example, human insulin),

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

Asp(A14)Val(B16)Ile(B25)Des(B30)-insulin (for example, human insulin),

Asp(A14)Val(B16)Val(B25)Des(B30)-insulin (for example, human insulin),

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

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

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

Asp(A14)Ile(B16)Ile(B25)-insulin (for example, human insulin),

Asp(A14)Glu(B3)Ile(B16)Ile(B25)-insulin (for example, human insulin),

Asp(A14)Ile(B16)Val(B25)-insulin (for example, human insulin),

Asp(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)-insulin (for example, human insulin),

Asp(A14)Val(B16)Ile(B25)-insulin (for example, human insulin),

Asp(A14)Val(B16)Val(B25)-insulin (for example, 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 (eg human insulin).

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

His(A14)Leu(B16)Des(B30)-insulin (for example, human insulin),

His(A14)Ile(B16)Des(B30)-insulin (for example, human insulin),

His(A14)Val(B16)Des(B30)-insulin (for example, human insulin),

His(A14)Leu(B16)-insulin (for example, human insulin),

His(A14)Ile(B16)-insulin (for example, human insulin),

His(A14)Val(B16)-insulin (for example, human insulin),

His(A14)Leu(B25)Des(B30)-insulin (for example, human insulin),

His(A14)Ile(B25)Des(B30)-insulin (for example, human insulin),

His(A14)Val(B25)Des(B30)-insulin (for example, human insulin),

His(A14)Leu(B25)-insulin (for example, human insulin),

His(A14)Ile(B25)-insulin (for example, human insulin),

His(A14)Val(B25)-insulin (for example, human insulin),

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

His(A14)Ile(B16)Ile(B25)Des(B30)-insulin (for example, human insulin),

His(A14)Glu(B3)Ile(B16)Ile(B25)Des(B30)-insulin (for example, human insulin),

His(A14)Ile(B16)Val(B25)Des(B30)-insulin (for example, human insulin),

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

His(A14)Val(B16)Ile(B25)Des(B30)-insulin (for example, human insulin),

His(A14)Val(B16)Val(B25)Des(B30)-insulin (for example, human insulin),

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

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

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

His(A14)Ile(B16)Ile(B25)-insulin (for example, human insulin),

His(A14)Glu(B3)Ile(B16)Ile(B25)-insulin (for example, human insulin),

His(A14)Ile(B16)Val(B25)-insulin (for example, human insulin),

His(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)-insulin (for example, human insulin),

His(A14)Val(B16)Ile(B25)-insulin (for example, human insulin),

His(A14)Val(B16)Val(B25)-insulin (for example, 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 (eg human insulin).

In another embodiment, the insulin analog is Leu(B25)Des(B30) insulin (such as human Leu(B25)Des(B30) insulin). The sequence of this analogue, for example, is shown in Table 1 of the “Examples” section (see analogue 11). For example, Leu(B25)Des(B30)-insulin contains an A chain having the amino acid sequence shown at SEQ ID NO: 21 (GIVEQCCTSICSLYQLENYCN) and a B chain having the amino acid sequence shown at SEQ ID NO: 22 (FVNQHLCGSHLVEALYLVCGERGFLYTPK ).

In another embodiment, the insulin analogue is Val(B25)Des(B30)-insulin (such as human Val(B25)Des(B30)-insulin). The sequence of this analogue, for example, is shown in Table 1 of the "Examples" section (see analogue 12). For example, Val(B25)Des(B30)-insulin contains an A chain having the amino acid sequence shown at SEQ ID NO: 23 (GIVEQCCTSICSLYQLENYCN) and a B chain having the amino acid sequence shown at SEQ ID NO: 24 (FVNQHLCGSHLVEALYLVCGERGFVYTPK ).

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

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

In another embodiment, the insulin analogue is Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-insulin (such as human Glu(A14)Gly(A21)Glu(B3)Val(B25) Des(B30)-insulin). The sequence of this analogue, for example, is shown in Table 1 of the Examples section (see analogue 25). For example, Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30) insulin contains an A chain having the amino acid sequence shown in SEQ ID NO: 49 (GIVEQCCTSICSLEQLENYCG) and a B chain having the amino acid sequence shown under SEQ ID NO: 50 (FVEQHLCGSHLVEALYLVCGERGFVYTPK).

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

In another embodiment, the insulin analogue is Glu(A14)Glu(B3)Ile(B16)Ile(B25)Des(B30)-insulin (such as human Glu(A14)Glu(B3)Ile(B16)Ile(B25) Des(B30)-insulin). The sequence of this analogue, for example, is shown in Table 1 of the Examples section (see analogue 30). For example, Glu(A14)Glu(B3)Ile(B16)Ile(B25)Des(B30) insulin contains an A chain having the amino acid sequence shown in SEQ ID NO: 56 (GIVEQCCTSICSLEQLENYCN) and a B chain having the amino acid sequence shown under SEQ ID NO: 60 (FVEQHLCGSHLVEALILVCGERGFIYTPK).

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

In another embodiment, the insulin analog is Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)Des(B30)-insulin (such as human Glu(A14)Gly(A21)Glu(B3) Ile(B16)Val(B25)Des(B30)-insulin). The sequence of this analogue, for example, is shown in Table 1 of the Examples section (see analogue 33). For example, Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)Des(B30)-insulin contains an A chain having the amino acid sequence shown under SEQ ID NO: 65 (GIVEQCCTSICSLEQLENYCG), and B -chain having the amino acid sequence shown under SEQ ID NO: 66 (FVEQHLCGSHLVEALILVCGERGFVYTPK).

In another embodiment, the insulin analog is Glu(A14)Val(B16)Ile(B25)Des(B30)-insulin (such as human Glu(A14)Val(B16)Ile(B25)Des(B30)-insulin). The sequence of this analogue, for example, is shown in Table 1 of the Examples section (see analogue 35). For example, Glu(A14)Val(B16)Ile(B25)Des(B30)-insulin contains an A chain having the amino acid sequence shown under SEQ ID NO: 69 (GIVEQCCTSICSLEQLENYCN) and a B chain having the amino acid sequence shown under SEQ ID NO: 70 (FVNQHLCGSHLVEALVLVCGERGFIYTPK).

In another embodiment, the insulin analog is Glu(A14)Val(B16)Val(B25)Des(B30)-insulin (such as human Glu(A14)Val(B16)Val(B25)Des(B30)-insulin). The sequence of this analogue, for example, is shown in Table 1 of the Examples section (see analogue 38). For example, Glu(A14)Val(B16)Val(B25)Des(B30)-insulin contains an A chain having the amino acid sequence shown under SEQ ID NO: 75 (GIVEQCCTSICSLEQLENYCN) and a B chain having the amino acid sequence shown under SEQ ID NO: 76 (FVNQHLCGSHLVEALVLVCGERGFVYTPK).

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

In another embodiment, the insulin analogue is Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin (such as human Glu(A14)Gly(A21)Glu(B3) Val(B16)Val(B25)Des(B30)-insulin). The sequence of this analogue, for example, is shown in Table 1 of the Examples section (see analogue 40). For example, Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin contains an A chain having the amino acid sequence shown under SEQ ID NO: 79 (GIVEQCCTSICSLEQLENYCG), and B -chain having the amino acid sequence shown under SEQ ID NO: 80 (FVEQHLCGSHLVEALVLVCGERGFVYTPK).

The insulin analogue can be prepared by any method deemed suitable. For example, an insulin analog can be produced by recombinant methods or by solid phase synthesis.

The definitions and explanations set forth in this document above apply mutatis mutandis to the following.

This document provides for the insulin B chains, i.e. insulin B chain peptides as defined herein above in relation to the B chain insulin analog provided herein. Accordingly, provided herein are insulin B chains that contain at least one mutation relative to the insulin B chain in the parent insulin, wherein the B chains contain a mutation at position B16, which is a hydrophobic amino acid substitution, and/or a mutation at position B25, which is a hydrophobic amino acid substitution. The insulin B chain may contain additional mutations described herein above, such as the des(B30) deletion.

Also provided herein are proinsulin variants comprising an insulin A chain and/or an insulin B chain from the insulin analogues provided herein. The B chain may be any B chain as defined herein above for the insulin analogues provided herein. For example, provided herein are variants of proinsulin comprising an insulin A chain and an insulin B chain, wherein said B chain contains at least one mutation from the parent insulin B chain, wherein the mutation is at position B16, wherein it is a substitution with a hydrophobic amino acid, and/or the mutation is at position B25, wherein it is a substitution with a hydrophobic amino acid. The insulin B chain may contain additional mutations described herein above for the B chain.

The A chain contained in the proinsulin provided herein may be any A chain defined herein above for the insulin analogues provided herein. In some embodiments, the A chain of said proinsulin contains a mutation at position A14, which is a substitution 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 proinsulin variants provided herein may contain additional elements such as leader sequences or a C peptide. In some embodiments, proinsulin may further comprise a C-peptide that is located between the insulin B chain and the insulin A chain. The C-peptide may be 4-10 amino acids long, such as 4-9 amino acids long. The orientation may be (N-terminus to C-terminus): B chain, C peptide, A chain.

Provided herein are polynucleotides encoding insulin analogs, insulin B chains, and proinsulin variants provided herein. Said polynucleotide may be operably linked to a promoter that allows expression of said polynucleotide. In some embodiments, the promoter is heterologous to the specified polynucleotide. In some embodiments, the promoter is a constitutive promoter. In another embodiment, the promoter is an inducible promoter.

In addition, provided herein are vectors containing a polynucleotide encoding the insulin analogues provided herein. In some embodiments, said vector is an expression vector.

Provided herein are host cells containing nucleic acids encoding insulin analogues, insulin B chains and proinsulin variants, polynucleotides and/or vectors provided herein. In some embodiments, the host cell is a bacterial cell, such as a cell belonging to the genus Escherichia , such as an E. coli cell. In another embodiment, the host cell is a yeast cell, such as a Pichia pastoris cell or a Kluyveromyces 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 of treating a disease comprising administering to a subject a pharmaceutically effective amount of one or more insulin analogues provided herein, or a pharmaceutical composition based thereon.

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

This document provides insulin analogues or a pharmaceutical composition based on them for use in medicine.

Provided herein are insulin analogues or a pharmaceutical composition based thereon for use in the treatment of diabetes mellitus, such as type II diabetes mellitus.

Finally, provided herein are ways of using the insulin analogues provided herein, or pharmaceutical compositions based thereon, to prepare a medicament or medicament for the treatment of diabetes mellitus, such as type II diabetes mellitus.

Insulin analogs, insulin B chains, proinsulin variants, and routes of administration thereof are further illustrated by the following embodiments and combinations of embodiments indicated by appropriate relationships and references. The definitions and explanations set forth in this document above apply mutatis mutandis to the following embodiments.

1. An insulin analog containing at least one mutation compared to the parent insulin, where the insulin analog contains a mutation at position B16, which is a substitution with a hydrophobic amino acid, and/or a mutation at position B25, which is a substitution with a hydrophobic amino acid.

2. An insulin analogue according to embodiment 1, wherein the parent insulin is human insulin, porcine insulin or bovine insulin.

3. An insulin analog according to embodiments 1 and 2, wherein the hydrophobic amino acid at position B16 and/or position B25 is an aliphatic amino acid.

4. The insulin analog according to any one of embodiments 1-3, 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 (Ile) and leucine (Leu).

5. The insulin analogue according to any one of embodiments 1-3, wherein said insulin analogue further comprises a mutation at position A14 which is a substitution with an amino acid selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp) and histidine ( His).

6. The insulin analogue of any one of embodiments 1-5, wherein said insulin analogue further comprises a mutation at position B30, for example, wherein the mutation at position B30 is a deletion of the amino acid at position B30 of the parent insulin (Des(B30) mutation).

7. The insulin analog according to any one of embodiments 1-6, wherein said insulin analog further contains a mutation at position B3 that is a glutamic acid (Glu) substitution.

8. The insulin analogue of any one of embodiments 1-7, wherein said insulin further contains a mutation at position A21 that is a glycine (Gly) substitution.

9. An insulin analog according to any one of embodiments 1-8, wherein the insulin analog B chain comprises an amino acid sequence selected from the group consisting of:

FVNQHLCGSHLVEALYLVCGERGFLYTPK (SEQ ID NO: 22),

FVNQHLCGSHLVEALYLVCGERGFIYTPK (SEQ ID NO: 44),

FVNQHLCGSHLVEALYLVCGERGFVYTPK (SEQ ID NO: 48),

FVEQHLCGSHLVEALYLVCGERGFVYTPK (SEQ ID NO: 50),

FVNQHLCGSHLVEALILVCGERGFIYTPK (SEQ ID NO: 58),

FVEQHLCGSHLVEALILVCGERGFIYTPK (SEQ ID NO: 60),

FVNQHLCGSHLVEALILVCGERGFVYTPK (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),

FVNQHLCGSHLVEALILVCGERGFIYTPKT (SEQ ID NO: 90),

FVEQHLCGSHLVEALILVCGERGFIYTPKT (SEQ ID NO: 91),

FVNQHLCGSHLVEALILVCGERGFVYTPKT (SEQ ID NO: 92),

FVEQHLCGSHLVEALILVCGERGFVYTPKT (SEQ ID NO: 93),

FVNQHLCGSHLVEALVLVCGERGFIYTPKT (SEQ ID NO: 94),

FVNQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 95),

FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 96) and

FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 97), or consists of it.

10. An insulin analogue according to any of embodiments 1-9, containing:

An A chain having the amino acid sequence shown under SEQ ID NO: 43 (GIVEQCCTSICSLEQLENYCN), and a B chain having the amino acid sequence shown under SEQ ID NO: 44 (FVNQHLCGSHLVEALYLVCGERGFIYTPK),

An A chain having the amino acid sequence shown under SEQ ID NO: 47 (GIVEQCCTSICSLEQLENYCN), and a B chain having the amino acid sequence shown under SEQ ID NO: 48 (FVNQHLCGSHLVEALYLVCGERGFVYTPK), or

An A chain having the amino acid sequence shown under SEQ ID NO: 77 (GIVEQCCTSICSLEQLENYCN), and a B chain having the amino acid sequence shown under SEQ ID NO: 78 (FVEQHLCGSHLVEALVLVCGERGFVYTPK).

11. An insulin analog selected from the group consisting of:

human Leu(B16)-insulin,

human Val(B16)-insulin,

human Ile(B16)-insulin,

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

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

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

human Leu(B25)-insulin,

human Val(B25)-insulin,

human Ile(B25)-insulin,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

12. An insulin B chain containing at least one mutation compared to the parent insulin B chain, wherein the B chain contains a mutation at position B16, which is a substitution of a hydrophobic amino acid, and/or a mutation at position B25, which is replacement with a hydrophobic amino acid.

13. The insulin B chain of Embodiment 12, wherein the parent insulin is human insulin, porcine insulin, or bovine insulin.

14. The insulin B chain of embodiments 12 and 13, wherein the hydrophobic amino acid at position B16 and/or position B25 is an aliphatic amino acid.

15. The insulin B chain according to any one of embodiments 12-14, wherein said aliphatic amino acid is a branched chain amino acid, such as a branched chain amino acid selected from the group consisting of valine (Val), isoleucine (Ile) and leucine (Leu).

16. The insulin B chain of any one of embodiments 12-15, wherein said insulin B chain further comprises a mutation at position B3 that is a glutamic acid (Glu) substitution.

17. The insulin B chain of any one of embodiments 12-16, wherein said insulin B chain further comprises a mutation at position B30, wherein the mutation at position B30 is a deletion of the amino acid at position B30 of the parent insulin (Des(B30) mutation).

18. Proinsulin comprising an insulin A chain and an insulin B chain, wherein the insulin B chain contains at least one mutation compared to the parent insulin B chain, wherein the B chain contains a mutation at position B16 that is a substitution of a hydrophobic amino acid, and/or a mutation at position B25, which is a substitution with a hydrophobic amino acid.

19. The proinsulin of embodiment 18, wherein the insulin A chain of said proinsulin contains a mutation at position A14 which is a substitution with an amino acid selected from glutamic acid (Glu), aspartic acid (Asp) and histidine (His).

20. Proinsulin according to embodiments 18 and 19, wherein the parent insulin is human insulin, porcine insulin or bovine insulin.

21. Proinsulin according to any one of embodiments 18-20, wherein the hydrophobic amino acid at position B16 and/or position B25 is an aliphatic amino acid.

22. Proinsulin according to any one of embodiments 18-21, wherein said aliphatic amino acid is a branched chain amino acid, such as a branched chain amino acid selected from the group consisting of valine (Val), isoleucine (Ile) and leucine (Leu) .

23. The proinsulin of any one of embodiments 18-22, wherein said proinsulin further comprises a mutation at position B3 that is a glutamic acid (Glu) substitution.

24. The proinsulin of any one of embodiments 18-23, wherein the proinsulin further comprises a mutation at position B30, wherein the mutation at position B30 is a deletion of the amino acid at position B30 of the parent insulin (Des(B30) mutation).

25. A polynucleotide encoding an insulin analogue according to any one of embodiments 1-11, an insulin B chain according to any one of embodiments 12-17, and/or a proinsulin according to any one of embodiments 18-24.

26. An expression vector containing the polynucleotide according to embodiment 25.

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

28. A method of treating a patient who has diabetes mellitus, comprising administering to the patient one or more insulin analogues specified in any of embodiments 1-11.

29. An insulin analogue as specified in any of embodiments 1-11, for use in the treatment of diabetes mellitus.

All references cited herein are incorporated herein by reference with respect to the entire content of their disclosure and the content of the disclosure specifically referred to herein.

Example 1: Preparation of Human Insulin and Insulin Analogues

Human insulin, as well as insulin analogues, were obtained recombinantly. Polynucleotides encoding preproinsulin were ordered from GeneArt ^® . The designed polynucleotides were optimized for expression in yeast. They were inserted into an expression vector by classical restriction cloning, allowing functional expression and secretion in Kluyveromyces lactis K. As a secretion leader sequence, the gene was fused at the C-terminus to a DNA sequence encoding the mating signal alpha factor of Saccharomyces cerevisiae . Expression of the recombinant gene was controlled using the lactose-inducible K. lactis promoter.

Human insulin, as well as insulin analogues, were produced in the form of preproinsulin. A genetically fused N-terminal presequence was used to improve expression and secretion yields and to stabilize the peptide in the culture broth. A wide variety of sequences that have been tested for effectiveness can be used for this purpose. Proinsulin itself consists of a B chain fused to a C peptide, followed by a C-terminal A chain. Various combinations of amino acids have been described as the C-peptide. Short peptides of 1-10 amino acids in length have been shown to work well as C-sequences. For further processing of insulin, recognition sites for specific proteases are important, which flank the C-peptide to allow its excision.

K. lactis cells were made competent using chemical means. The cells were then transformed with an expression plasmid encoding the appropriate preproinsulin. 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 fairly high cell densities in dextrose-peptone yeast growth medium supplemented with geneticin. After the initial growth phase, salt-buffered yeast extract medium with geneticin supplemented with lactose was added to the cultures to induce recombinant gene expression. Cultures were grown for several days, and supernatant samples were collected by centrifugation.

Purification of functional insulin or insulin analogues was initiated using a filtration procedure. The initial chromatographic capture procedure was performed using an ion exchange resin. The cleavage of preproinsulin to insulin was performed using a highly specific protease. Depletion of host cell protein, presequence, and co-products was accomplished through a cascade of two additional chromatographic steps. Along with hydrophobic interaction chromatography, another ion exchange procedure was used to achieve this goal. Final deep purification was carried out by reverse phase chromatography. Filtration, precipitation and freeze-drying were used to complete the process of obtaining the insulin molecule.

After coupling reactions with the activated carboxylic acid derivative, the solution with conjugated insulin molecules was filtered. Final purification was carried out by reverse phase chromatography. Filtration, precipitation, and freeze-drying were used to complete the synthesis of the target molecule.

Various analogs of insulin variants with mutations, for example at positions B16, B25 and/or A14, have been obtained. Table 1 provides an overview of the resulting insulin variants.

Table 1. Obtained analogues of human insulin

Analogue skeleton A14 B16 B25 Amino acid sequence of the A chain SEQ ID NO (A-chain) B chain amino acid sequence SEQ ID NO (B-circuit) W.T. Human insulin (wild type) Tyr Tyr Phe GIVEQCCTSICS
LYQLENYCN 1 FVNQHLCGSHLVEAL
YLVCGERGFFYTPKT 2 2 Glu(A14)Des(B30)-insulin Glu Tyr Phe GIVEQCCTSICS
LEQLENYCN 3 FVNQHLCGSHLVEAL
YLVCGERGFYTPK 4 3 Leu(B16)Des(B30)-insulin Tyr Leu Phe GIVEQCCTSICS
LYQLENYCN 5 FVNQHLCGSHLVEAL
LLVCGERGFFYTPK 6 4 Gly(A21)Trp(B16)Des(B30)-insulin Tyr Trp Phe GIVEQCCTSICS
LYQLENYCG 7 FVNQHLCGSHLVEAL
WLVCGERGFYTPK 8 5 His(B16)Des(B30)-insulin Tyr His Phe GIVEQCCTSICS
LYQLENYCN 9 FVNQHLCGSHLVEAL
HLVCGERGFFYTPK 10 6 Val(B16)Des(B30)-insulin Tyr Val Phe GIVEQCCTSICS
LYQLENYCN eleven FVNQHLCGSHLVEAL
VLVCGERGFYTPK 12 7 Ala(B25)-insulin Tyr Tyr Ala GIVEQCCTSICS
LYQLENYCN 13 FVNQHLCGSHLVEAL
YLVCGERGFAYTPKT 14 8 Ala(B25)Des(B30)-insulin Tyr Tyr Ala GIVEQCCTSICS
LYQLENYCN 15 FVNQHLCGSHLVEAL
YLVCGERGFAYTPK 16 9 Glu(B25)Des(B30)-insulin Tyr Tyr Glu GIVEQCCTSICS
LYQLENYCN 17 FVNQHLCGSHLVEAL
YLVCGERGFEYTPK 18 10 His(B25)Des(B30)-insulin Tyr Tyr His GIVEQCCTSICS
LYQLENYCN 19 FVNQHLCGSHLVEAL
YLVCGERGFHYTPK 20 eleven Leu(B25)Des(B30)-insulin Tyr Tyr Leu GIVEQCCTSICS
LYQLENYCN 21 FVNQHLCGSHLVEAL
YLVCGERGFLYTPK 22 12 Val(B25)Des(B30)-insulin Tyr Tyr Val GIVEQCCTSICS
LYQLENYCN 23 FVNQHLCGSHLVEAL
YLVCGERGFVYTPK 24 13 His(B16)His(B25)Des(B30)-insulin Tyr His His GIVEQCCTSICS
LYQLENYCN 25 FVNQHLCGSHLVEAL
HLVCGERGFHYTPK 26 14 Gly(A21)Trp(B16)His(B25)Des(B30)-insulin Tyr Trp His GIVEQCCTSICS
LYQLENYCG 27 FVNQHLCGSHLVEAL
WLVCGERGFHYTPK 28 15 Gly(A21)Trp(B16)Trp(B25)Des(B30)-insulin Tyr Trp Trp GIVEQCCTSICS
LYQLENYCG 29 FVNQHLCGSHLVEAL
WLVCGERGFWYTPK thirty 16 Glu(A14)His(B16)Des(B30)-insulin Glu His Phe GIVEQCCTSICS
LEQLENYCN 31 FVNQHLCGSHLVEAL
HLVCGERGFFYTPK 32 17 Glu(A14)Gly(A21)Trp(B16)Des(B30)-insulin Glu Trp Phe GIVEQCCTSICS
LEQLENYCG 33 FVNQHLCGSHLVEAL
WLVCGERGFYTPK 34 18 Glu(A14)Ile(B16)Des(B30)-insulin Glu Ile Phe GIVEQCCTSICS
LEQLENYCN 35 FVNQHLCGSHLVEAL
ILVCGERGFFYTPK 36 19 Glu(A14)Val(B16)Des(B30)-insulin Glu Val Phe GIVEQCCTSICS
LEQLENYCN 37 FVNQHLCGSHLVEA
LVLVCGERGFYTPK 38 20 Glu(A14)Val(B3)Val(B16)Des(B30)-insulin Glu Val Phe GIVEQCCTSICS
LEQLENYCN 39 FVEQHLCGSHLVEA
LVLVCGERGFYTPK 40 21 Glu(A14)His(B25)Des(B30)-insulin Glu Tyr His GIVEQCCTSICS
LEQLENYCN 41 FVNQHLCGSHLVEA
LYLVCGERGFHYTPK 42 22 Glu(A14)Ile(B25)Des(B30)-insulin Glu Tyr Ile GIVEQCCTSICS
LEQLENYCN 43 FVNQHLCGSHLVEA
LYLVCGERGFIYTPK 44 23 Glu(A14)Gly(A21)Trp(B25)Des(B30)-insulin Glu Tyr Trp GIVEQCCTSICS
LEQLENYCG 45 FVNQHLCGSHLVEA
LYLVCGERGFWYTPK 46 24 Glu(A14)Val(B25)Des(B30)-insulin Glu Tyr Val GIVEQCCTSICS
LEQLENYCN 47 FVNQHLCGSHLVEA
LYLVCGERGFVYTPK 48 25 Glu(A14)Gly(A21)Glu(B3)-Val(B25)Des(B30)-insulin Glu Tyr Val GIVEQCCTSIC
SLEQLENYCG 49 FVEQHLCGSHLVEA
LYLVCGERGFVYTPK 50 26 Glu(A14)Glu(B16)His(B25)Des(B30)-insulin Glu Glu His GIVEQCCTSIC
SLEQLENYCN 51 FVNQHLCGSHLVEA
LELVCGERGFHYTPK 52 27 Glu(A14)His(B16)Ala(B25)Des(B30)-insulin Glu His Ala GIVEQCCTSIC
SLEQLENYCN 53 FVNQHLCGSHLVEA
LHLVCGERGFAYTPK 54 28 Glu(A14)His(B16)His(B25)Des(B30)-insulin Glu His His GIVEQCCTSIC
SLEQLENYCN 55 FVNQHLCGSHLVEA
LHLVCGERGFHYTPK 56 29 Glu(A14)Ile(B16)Ile(B25)-Des(B30)-insulin Glu Ile Ile GIVEQCCTSIC
SLEQLENYCN 57 FVNQHLCGSHLVEA
LILVCGERGFIYTPK 58 thirty Glu(A14)Glu(B3)Ile(B16)-Ile(B25)Des(B30)-insulin Glu Ile Ile GIVEQCCTSIC
SLEQLENYCN 59 FVEQHLCGSHLVEA
LILVCGERGFIYTPK 60 31 Glu(A14)Gly(A21)Glu(B3)-Ile(B16)Trp(B25)Des(B30)-insulin Glu Ile Trp GIVEQCCTSIC
SLEQLENYCG 61 FVEQHLCGSHLVEA
LILVCGERGFWYTPK 62 32 Glu(A14)Ile(B16)Val(B25)-Des(B30)-insulin Glu Ile Val GIVEQCCTSIC
SLEQLENYCN 63 FVNQHLCGSHLVEA
LILVCGERGFVYTPK 64 33 Glu(A14)Gly(A21)Glu(B3)-Ile(B16)Val(B25)Des(B30)-insulin Glu Ile Val GIVEQCCTSIC
SLEQLENYCG 65 FVEQHLCGSHLVEA
LILVCGERGFVYTPK 66 34 Glu(A14)Leu(B16)Ala(B25)Des(B30)-insulin Glu Leu Ala GIVEQCCTSIC
SLEQLENYCN 67 FVNQHLCGSHLVEA
LLLVCGERGFAYTPK 68 35 Glu(A14)Val(B16)Ile(B25)-Des(B30)-insulin Glu Val Ile GIVEQCCTSIC
SLEQLENYCN 69 FVNQHLCGSHLVEA
LVLVCGERGFIYTPK 70 36 Glu(A14)Gly(A21)Val(B16)Trp(B25)Des(B30)-insulin Glu Val Trp GIVEQCCTSIC
SLEQLENYCG 71 FVNQHLCGSHLVEA
LVLVCGERGFWYTPK 72 37 Glu(A14)Gly(A21)Glu(B3)-Val(B16)Trp(B25)Des(B30)-insulin Glu Val Trp GIVEQCCTSIC
SLEQLENYCG 73 FVEQHLCGSHLVEAL
VLVCGERGFWYTPK 74 38 Glu(A14)Val(B16)Val(B25)Des(B30)-insulin Glu Val Val GIVEQCCTSIC
SLEQLENYCN 75 FVNQHLCGSHLVEAL
VLVCGERGFVYTPK 76 39 Glu(A14)Glu(B3)Val(B16)-Val(B25)Des(B30)-insulin Glu Val Val GIVEQCCTSIC
SLEQLENYCN 77 FVEQHLCGSHLVEAL
VLVCGERGFVYTPK 78 40 Glu(A14)Gly(A21)Glu(B3)-Val(B16)Val(B25)Des(B30)-insulin Glu Val Val GIVEQCCTSIC
SLEQLENYCG 79 FVEQHLCGSHLVEAL
VLVCGERGFVYTPK 80

Example 2: Insulin Receptor Binding Affinity Assays/Insulin Receptor Autophosphorylation Assays

Insulin binding and signaling by various prepared insulin analogues was determined using binding assays and receptor autophosphorylation assays.

A) Insulin receptor binding affinity assay

The insulin receptor binding affinities for the analogues listed in Table 1 were determined as described in Hartmann et al. (Effect of the long-acting insulin analogs glargine and degludec on cardiomyocyte cell signaling and function. Cardiovasc Diabetol. 2016;15:96). Isolation of plasma membrane-integrated insulin receptors (M-IR) and competitive binding experiments were performed as previously described (Sommerfeldet al., PLoS One. 2010; 5(3): e9540). Briefly, CHO cells overexpressing IR were harvested and resuspended in ice-cold 2.25 STM buffer (2.25 M sucrose, 5 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, cOmplete protease inhibitor) and destroyed using a Dounce homogenizer followed by sonication. The homogenate was covered with 0.8 STM buffer (0.8 M sucrose, 5 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, cOmplete protease inhibitor) and ultracentrifuged for 90 min. at 100000 g. Interfacial plasma membranes were collected and washed twice with phosphate-buffered saline (PBS). The final pellet was resuspended in dilution buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, cOmplete protease inhibitor) and homogenized again using a Dounce homogenizer. Competition binding experiments were performed in binding buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% BSA, cOmplete protease inhibitor adjusted to pH 7.8) in 96-well microplates. In each well, 2 μg of isolated membranes were incubated with 0.25 mg of polyvinyltoluene and polyethylenimine scintillation proximity assay (SPA) beads coated with wheat germ agglutinin. [125I]-labeled human insulin at constant concentrations (100 pM) and corresponding unlabeled insulin at varying concentrations (0.001-1000 nM) were added for 12 hours at room temperature (23°C). Radioactivity was measured at steady state in a microplate scintillation counter (Wallac MicroBeta, Freiburg, Germany).

The results of insulin receptor binding affinity assays for the tested analogs compared to human insulin are shown in Table 2.

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

To determine signal transduction upon binding of an insulin analog to the insulin B receptor, the level of autophosphorylation was measured in vitro .

CHO cells expressing human insulin receptor isoform B (IR-B) were used for IR autophosphorylation assays using In-Cell Western technology described previously (Sommerfeld et al. , PLoS One. 2010;5(3):e9540). To analyze IGF1R autophosphorylation, the receptor was overexpressed in the 3T3 Tet-off mouse embryonic fibroblast cell line (BD Bioscience, Heidelberg, Germany), which was stably transfected with a tetracycline-regulated IGF1R expression plasmid. To determine the level of receptor tyrosine phosphorylation, cells were seeded in 96-well plates and grown for 44 hours. Cells were serum starved in serum-free Ham's F12 medium (Life Technologies, Darmstadt, Germany) for 2 hours. Cells were then treated with human insulin or an insulin analogue in increasing concentrations for 20 minutes. at 37°C. After incubation, the medium was discarded and the cells were fixed in freshly prepared 3.75% paraformaldehyde for 20 min. Cells were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Blocking was performed using Odyssey blocking buffer (LICOR, Bad Homburg, Germany) for 1 hour at room temperature. Antibody to pTyr 4G10 (Millipore, Schwalbach, Germany) was incubated for 2 hours at room temperature. After primary antibody incubation, cells were washed with PBS+0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA). Secondary anti-mouse IgG antibody 800-CW (LICOR, Bad Homburg, Germany) was incubated for 1 h. Results were normalized by DNA quantification using TO-PRO3 dye (Invitrogen, Karlsruhe, Germany). Data were obtained as relative units (RU).

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

Table 2. Relative values of insulin receptor binding affinity and autophosphorylation activity for the tested human insulin analogues (see Table 1 for sequences).

Analogue skeleton A14 B16 B25 Binding affinity for insulin receptors* Autophosphorylation activity W.T. Human insulin (wild type) Tyr Tyr Phe 1 1 2 Glu(A14)Des(B30)-insulin Glu Tyr Phe 1.05 0.87 3 Leu(B16)Des(B30)-insulin Tyr Leu Phe 0.24 0.34 4 Gly(A21)Trp(B16)Des(B30)-insulin Tyr Trp Phe 0.57 0.4 5 His(B16)Des(B30)-insulin Tyr His Phe nd nd 6 Val(B16)Des(B30)-insulin Tyr Val Phe nd 0.32 7 Ala(B25)-insulin Tyr Tyr Ala 0.05 0.2 8 Ala(B25)Des(B30)-insulin Tyr Tyr Ala nd 0.17 9 Glu(B25)Des(B30)-insulin Tyr Tyr Glu nd nd 10 His(B25)Des(B30)-insulin Tyr Tyr His 0.37 0.31 eleven Leu(B25)Des(B30)-insulin Tyr Tyr Leu 0.01 0.06 12 Val(B25)Des(B30)-insulin Tyr Tyr Val 0.01 0.06 13 His(B16)His(B25)Des(B30)-insulin Tyr His His 0.11 0.1 14 Gly(A21)Trp(B16)His(B25)Des(B30)-insulin Tyr Trp His 0.4 0.35 15 Gly(A21)Trp(B16)Trp(B25)Des(B30)-insulin Tyr Trp Trp 0.43 0.38 16 Glu(A14)His(B16)Des(B30)-insulin Glu His Phe 0.36 0.29 17 Glu(A14)Gly(A21)Trp(B16)Des(B30)-insulin Glu Trp Phe 0.63 0.38 18 Glu(A14)Ile(B16)Des(B30)-insulin Glu Ile Phe 0.23 0.18 19 Glu(A14)His(B16)Des(B30)-insulin Glu Val Phe nd 0.32 20 Glu(A14)Val(B3)Val(B16)Des(B30)-insulin Glu Val Phe 0.4 0.28 21 Glu(A14)His(B25)Des(B30)-insulin Glu Tyr His nd nd 22 Glu(A14)Ile(B25)Des(B30)-insulin Glu Tyr Ile 0.01 0.04 23 Glu(A14)Gly(A21)Trp(B25)Des(B30)-insulin Glu Tyr Trp 0.56 0.37 24 Glu(A14)His(B25)Des(B30)-insulin Glu Tyr Val 0.01 0.04 25 Glu(A14)Gly(A21)Glu(B3)Val(B25)-Des(B30)-insulin Glu Tyr Val 0.02 0.03 26 Glu(A14)Glu(B16)His(B25)Des(B30)-insulin Glu Glu His 0.01 0.07 27 Glu(A14)His(B16)Ala(B25)Des(B30)-insulin Glu His Ala 0.11 28 Glu(A14)His(B16)His(B25)Des(B30)-insulin Glu His His 0.12 0.11 29 Glu(A14)Ile(B16)Ile(B25)Des(B30)-insulin Glu Ile Ile 0.01 thirty Glu(A14)Glu(B3)Ile(B16)Ile(B25)-Des(B30)-insulin Glu Ile Ile 0** 0.01 31 Glu(A14)Gly(A21)Glu(B3)Ile(B16)-Trp(B25)Des(B30)-insulin Glu Ile Trp 0.12 0.13 32 Glu(A14)Ile(B16)Val(B25)Des(B30)-insulin Glu Ile Val 0 0.04 33 Glu(A14)Gly(A21)Glu(B3)Ile(B16)-Val(B25)Des(B30)-insulin Glu Ile Val 0.01 0.02 34 Glu(A14)Leu(B16)Ala(B25)Des(B30)-insulin Glu Leu Ala 0.01 0.04 35 Glu(A14)Val(B16)Ile(B25)Des(B30)-insulin Glu Val Ile 0 0.01 36 Glu(A14)Gly(A21)Val(B16)Trp(B25)-Des(B30)-insulin Glu Val Trp 0.17 0.23 37 Glu(A14)Gly(A21)Glu(B3)Val(B16)-Trp(B25)Des(B30)-insulin Glu Val Trp 0.21 0.19 38 Glu(A14)Val(B16)Val(B25)Des(B30)-insulin Glu Val Val 0 0.03 39 Glu(A14)Glu(B3)Val(B16)Val(B25)-Des(B30)-insulin Glu Val Val 0 0.02 40 Glu(A14)Gly(A21)Glu(B3)Val(B16)-Val(B25)Des(B30)-insulin Glu Val Val 0.01 0.01

* compared to human insulin, n.d .: not determined

**value 0 means the binding affinity was below the detection limit

C) Conclusions

As can be seen from Table 2, various substitutions at positions B16 and/or B25 with hydrophobic amino acids (tryptophan, alanine, valine, leucine and isoleucine) were tested. Although to varying degrees, all tested insulin analogues with hydrophobic amino acid substitutions at these positions showed a decrease in binding activity to insulin receptors. Compared to substitutions with Trp (see, for example, analogues 4, 15 and 23), substitutions with aliphatic amino acids such as alanine, valine, leucine and isoleucine had a stronger effect on insulin receptor binding activity. The strongest effects were observed for valine, leucine and isoleucine, which are branched-chain amino acids. Substitutions with isoleucine, valine, and leucine resulted in a significant decrease in insulin receptor binding activity. Interestingly, insulin analogs with such substitutions at position B25 (such as the valine, leucine or isoleucine substitution at B25, analogs 11, 12, 22, 24, 25, 29, 30, 32, 33, 35, 38, 39, 40) exhibited up to 6-fold enhancement of signal transduction compared to what was expected based on their IR-B binding affinity values. In particular, Leu(B25)Des(B30)-insulin and Val(B25)Des(B30)-insulin (analogs 11 and 12, respectively) showed only 1% binding to the insulin B receptor and 6% autophosphorylation compared to human insulin. Likewise, a single leucine substitution at position B16 (analogue 3) also showed a similar increase in signal transduction, although to a slightly lesser extent. In comparison, analogues containing a histidine substitution at B25 (analogs 10, 13, 14, 21, 28), with the exception of analogue 26, also showed reduced receptor binding but also a concomitant reduction in autophosphorylation.

In some cases (analogues 30, 32, 35, 38, 39) binding to insulin receptors was 0% and still showed activity in the autophosphorylation assay. All of these analogues have combinations of valine and/or isoleucine substitutions at positions B16 and B25 together, suggesting that the combination is responsible for additional reduction in insulin receptor binding. Insulin variants without a substitution at position B25, but with substitutions at position B16, were characterized by slightly higher binding affinity values compared to their autophosphorylation values (analogues 3, 4, 16, 17, 18, 19, 20).

Alanine at position B25 shows effects similar to substitution with valine, leucine, or isoleucine (analogs 11, 12, 22), although to a lesser extent. The receptor binding affinity and autophosphorylation activity of analogues with valine, leucine or isoleucine substitution is lower than that of analogues with alanine substitution.

Example 3 Determination of in vitro stability in various recombinant proteases and artificial gastric juice

Insulin analogues were tested for proteolytic stability (to α-chymotrypsin, cathepsin D, insulin degrading enzyme (IDE), and artificial gastric acid).

A) Analysis conditions

Protease Final protease concentration in the assay [µg/ml] Buffer system used Trypsin 2 0.1 M ammonium bicarbonate, pH 8.3 α-Chymotrypsin 0.5 0.1 M ammonium bicarbonate, pH 8.3 Carboxypeptidase A 0.1 Pre-activation (60 min.) with trypsin in TCNB buffer, analytical Tris buffer 7.5 Carboxypeptidase B 0.1 Pre-activation (30 min.) with trypsin in TCNB buffer, analytical Tris buffer 7.5 Cathepsin D 2 Acetate buffer, pH 4.5 IDE 2 Tris buffer, pH 7.5

B) Obtaining artificial gastric juice

Two grams of sodium chloride and 3.2 g of purified pepsin (from porcine gastric 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 brought to 1000 ml with water. The resulting solution was stirred and diluted with 0.2 N. sodium hydroxide or 0.2 N. hydrochloric acid to pH 1.2±0.1.

C) General analysis procedure

Stability determinations were performed using appropriate time points (for SIF and SGF, 15, 30, 60, 120, and 240 minutes; for proteases, 15, 30, 60, and 120 minutes). Incubation was carried out at 37°C, and the % of starting compound remaining was calculated relative to time T0.

An appropriate LC-MS/MS or LC-HRMS bioanalytical method was used to determine the parent compound using the supernatant after ethanol precipitation (1 eq v/v) and centrifugation step.

D) Obtaining samples

The compounds were dissolved in dilute hydrochloric acid to a final concentration of 40 μM. The concentration of the compound in the assay was 2 μM. A 1:20 dilution of the working solution was carried out in protease buffer, and then the samples were incubated at 37°C with mixing. At the appropriate time point after aliquoting, the reaction mixture was quenched with ethanol (1 eq. v/v), then centrifuged. The supernatant was analyzed.

E) Conclusions

In particular, substitutions with lipophilic amino acids such as valine and isoleucine at positions 16 and 25 in the B chain were investigated. The tested analogues (2, 7, 11, 12, 14, 16, 19, 22, 23, 24 and 38) showed only minor differences in stability towards the proteases trypsin, carboxypeptidase A and carboxypeptidase B compared with human insulin (data not shown). In general, all analogues containing a substitution at positions A14 and B25 (analogues 22, 24, 38) showed improved proteolytic stability towards α-chymotrypsin, cathepsin D and insulin degrading enzyme (IDE). For example, in the case of α-chymotrypsin, human insulin was completely destroyed within 2 hours, while analogue 22 was almost completely stable. Similarly, all analogues tested with a substitution in B25 showed improved stability towards cathepsin D, although analogue 38 (variant B16/B25) showed improved stability compared to other B25 variants.

One notable exception was observed in the case of IDE, where analog 19 with substitutions in A14/B16 showed improved performance compared to the B25 variants. However, data suggest that the glutamic acid substitution in A14 tested in this case is important for increasing stability. Other substitutions have also been shown to be useful in increasing stability, such as the substitutions at position B16 and at position B25. For example, in analogue 7, an amino acid change at position B25 leads to increased instability.

Table 3. Percentage of insulin analog remaining after incubation of different insulin analogs for 30 or 120 minutes with four different proteases (see Table 1 for sequences of analogues tested).

Analogue skeleton A14 B16 B25 Gastric juice (30 minutes) [%] α-Chymotrypsin (120 minutes) [%] Cathepsin D
(30 minutes) [%] Insulin Degrading Enzyme - IDE
(30 minutes) [%] W.T. Insulin Tyr Tyr Phe 0 5 0 38 2 Glu(A14)Des-(B30)-insulin Glu Tyr Phe 0 35 0 81 7 Ala(B25)-insulin Tyr Tyr Ala thirty 58 52 2 eleven Leu(B25)Des-(B30)-insulin Tyr Tyr Leu nd nd nd 12 12 Val(B25)Des-(B30)-insulin Tyr Tyr Val 44 31 43 eleven 14 Gly(A21)Trp(B16)His(B25)Des-(B30)-insulin Tyr Trp His nd nd nd 41 16 Glu(A14)His(B16)Des(B30)-insulin Glu His Phe nd nd nd 88 19 Glu(A14)His(B16)Des(B30)-insulin Glu Val Phe 0 thirty 0 98 22 Glu(A14)Ile(B25)Des(B30)-insulin Glu Tyr Ile 52 99 20 77 23 Glu(A14)Gly(A21)Trp(B25)Des-(B30)-insulin Glu Tyr Trp nd nd nd 80 24 Glu(A14)His(B25)Des(B30)-insulin Glu Tyr Val 52 40 59 86 38 Glu(A14)Val(B16)Val(B25)Des-(B30)-insulin Glu Val Val 36 34 62 86

nd: not defined

ITEMS DESCRIPTION

The following points are part of the description.

1. An insulin analog containing at least one mutation compared to the parent insulin, wherein the insulin analog contains a mutation at position B16, which is a substitution with a branched chain amino acid, and/or a mutation at position B25, which is a substitution with a branched chain amino acid chain.

2. An insulin analogue according to paragraph 1, where the original insulin is human insulin, porcine insulin or bovine insulin.

3. An insulin analog according to paragraphs 1 and 2, wherein the branched chain amino acid is selected from the group consisting of valine (Val), isoleucine (Ile) and leucine (Leu).

4. An insulin analogue according to any one of claims 1-3, wherein said insulin analogue further comprises a mutation at position A14 which is a substitution with an amino acid selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp) and histidine (His) ).

5. An insulin analogue according to any one of claims 1-4, wherein said insulin analogue further contains a mutation at position B30.

6. An insulin analog according to paragraph 5, where the mutation at position B30 is a deletion of the amino acid at position B30 of the parent insulin (Des(B30) mutation).

7. An insulin analog according to any one of claims 1 to 6, wherein said insulin analog further contains a mutation at position B3 that is a glutamic acid (Glu) substitution.

8. An insulin analogue according to any one of claims 1 to 7, wherein said insulin further contains a mutation at position A21 which is a glycine (Gly) substitution.

9. An insulin analogue according to any one of claims 1-8, wherein the B-chain of the insulin analogue contains an amino acid sequence selected from the group consisting of:

FVNQHLCGSHLVEALYLVCGERGFLYTPK (SEQ ID NO: 22),

FVNQHLCGSHLVEALYLVCGERGFIYTPK (SEQ ID NO: 44),

FVNQHLCGSHLVEALYLVCGERGFVYTPK (SEQ ID NO: 48),

FVEQHLCGSHLVEALYLVCGERGFVYTPK (SEQ ID NO: 50),

FVNQHLCGSHLVEALILVCGERGFIYTPK (SEQ ID NO: 58),

FVEQHLCGSHLVEALILVCGERGFIYTPK (SEQ ID NO: 60),

FVNQHLCGSHLVEALILVCGERGFVYTPK (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),

FVNQHLCGSHLVEALILVCGERGFIYTPKT (SEQ ID NO: 90),

FVEQHLCGSHLVEALILVCGERGFIYTPKT (SEQ ID NO: 91),

FVNQHLCGSHLVEALILVCGERGFVYTPKT (SEQ ID NO: 92),

FVEQHLCGSHLVEALILVCGERGFVYTPKT (SEQ ID NO: 93),

FVNQHLCGSHLVEALVLVCGERGFIYTPKT (SEQ ID NO: 94),

FVNQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 95),

FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 96) and

FVEQHLCGSHLVEALVLVCGERGFVYTPKT (SEQ ID NO: 97), or consists of it.

10. An insulin analogue according to any of paragraphs 1-9, containing:

An A chain having the amino acid sequence shown under SEQ ID NO: 43 (GIVEQCCTSICSLEQLENYCN), and a B chain having the amino acid sequence shown under SEQ ID NO: 44 (FVNQHLCGSHLVEALYLVCGERGFIYTPK),

An A chain having the amino acid sequence shown under SEQ ID NO: 47 (GIVEQCCTSICSLEQLENYCN), and a B chain having the amino acid sequence shown under SEQ ID NO: 48 (FVNQHLCGSHLVEALYLVCGERGFVYTPK), or

An A chain having the amino acid sequence shown under SEQ ID NO: 77 (GIVEQCCTSICSLEQLENYCN), and a B chain having the amino acid sequence shown under SEQ ID NO: 78 (FVEQHLCGSHLVEALVLVCGERGFVYTPK).

11. An insulin analog selected from the group consisting of:

human Leu(B16)-insulin,

human Val(B16)-insulin,

human Ile(B16)-insulin,

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

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

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

human Leu(B25)-insulin,

human Val(B25)-insulin,

human Ile(B25)-insulin,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

12. An insulin B chain containing at least one mutation compared to the parent insulin B chain, wherein the B chain contains a mutation at position B16, which is a substitution with a branched chain amino acid, and/or a mutation at position B25, which is a substitution with a branched chain amino acid, and wherein said insulin B chain optionally further comprises a mutation at position B30, wherein the mutation at position B30 is a deletion of threonine at position B30 of the parent insulin (Des(B30) mutation).

13. Proinsulin comprising an insulin A chain and an insulin B chain, wherein the insulin B chain contains at least one mutation compared to the parent insulin B chain, wherein the B chain contains a mutation at position B16 that is an amino acid substitution branched chain, and/or a mutation at position B25, which is a substitution with a branched chain amino acid, and wherein the insulin A chain in said proinsulin optionally contains a mutation at position A14, which is a substitution with an amino acid selected from glutamic acid (Glu) , aspartic acid (Asp) and histidine (His).

14. A polynucleotide encoding an insulin analogue according to any of paragraphs 1-11, an insulin B chain according to paragraph 12 and/or proinsulin according to paragraph 13.

15. A host cell containing an insulin analog according to any of paragraphs 1-11, an insulin B chain according to paragraph 12, a proinsulin according to paragraph 13 and/or a polynucleotide according to paragraph 14.

--->

LIST OF SEQUENCES

<110> Sanofi

<120> Insulin analogs with reduced affinity

binding to insulin receptors

<130>DE2018/059

<150> EP 18 306 657.0

<151> 2018-12-11

<150> EP 18 306 658.8

<151> 2018-12-11

<150> EP 18 306 659.6

<151> 2018-12-11

<160> 97

<170> BiSSAP 1.3.6

<210> 1

<211> 21

<212> PROTEIN

<213> Homo sapiens

<220>

<223> Circuit A

<400> 1

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 2

<211> 30

<212> PROTEIN

<213> Homo sapiens

<220>

<223> Circuit B

<400> 2

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

20 25 30

<210> 3

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 3

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 4

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 4

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25

<210> 5

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 5

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 6

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 6

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Leu

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25

<210> 7

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 7

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 8

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 8

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Trp

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25

<210> 9

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 9

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 10

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 10

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25

<210> 11

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 11

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 12

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 12

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25

<210> 13

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 13

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 14

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 14

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ala Tyr Thr Pro Lys Thr

20 25 30

<210> 15

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 15

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 16

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 16

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ala Tyr Thr Pro Lys

20 25

<210> 17

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 17

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 18

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 18

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Glu Tyr Thr Pro Lys

20 25

<210> 19

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 19

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 20

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 20

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys

20 25

<210> 21

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 21

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 22

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 22

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Leu Tyr Thr Pro Lys

20 25

<210> 23

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 23

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 24

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 24

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 25

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 25

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 26

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 26

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys

20 25

<210> 27

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 27

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 28

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 28

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Trp

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys

20 25

<210> 29

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 29

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 30

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 30

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Trp

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Trp Tyr Thr Pro Lys

20 25

<210> 31

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 31

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 32

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 32

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25

<210> 33

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 33

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 34

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 34

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Trp

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25

<210> 35

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 35

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 36

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 36

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ile

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25

<210> 37

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 37

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 38

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 38

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25

<210> 39

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 39

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 40

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 40

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25

<210> 41

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 41

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 42

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 42

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys

20 25

<210> 43

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 43

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 44

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 44

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ile Tyr Thr Pro Lys

20 25

<210> 45

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 45

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 46

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 46

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Trp Tyr Thr Pro Lys

20 25

<210> 47

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 47

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 48

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 48

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 49

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 49

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 50

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 50

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 51

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 51

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 52

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 52

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Glu

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys

20 25

<210> 53

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 53

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 54

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 54

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ala Tyr Thr Pro Lys

20 25

<210> 55

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 55

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 56

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 56

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys

20 25

<210> 57

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 57

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 58

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 58

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ile

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ile Tyr Thr Pro Lys

20 25

<210> 59

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 59

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 60

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 60

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ile

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ile Tyr Thr Pro Lys

20 25

<210> 61

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 61

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 62

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 62

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ile

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Trp Tyr Thr Pro Lys

20 25

<210> 63

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 63

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 64

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 64

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ile

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 65

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 65

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 66

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 66

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ile

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 67

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 67

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 68

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 68

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Leu

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ala Tyr Thr Pro Lys

20 25

<210> 69

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 69

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 70

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 70

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ile Tyr Thr Pro Lys

20 25

<210> 71

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 71

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 72

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 72

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Trp Tyr Thr Pro Lys

20 25

<210> 73

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 73

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 74

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 74

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Trp Tyr Thr Pro Lys

20 25

<210> 75

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 75

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 76

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 76

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 77

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 77

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 78

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 78

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 79

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit A

<400> 79

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu

1 5 10 15

Glu Asn Tyr Cys Gly

20

<210> 80

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 80

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 81

<211> 21

<212> PROTEIN

<213>Bos

<220>

<223> Circuit A

<400> 81

Gly Ile Val Glu Gln Cys Cys Ala Ser Val Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 82

<211> 30

<212> PROTEIN

<213>Bos

<220>

<223> Circuit B

<400> 82

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ala

20 25 30

<210> 83

<211> 21

<212> PROTEIN

<213> Sus

<220>

<223> Circuit A

<400> 83

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 84

<211> 30

<212> PROTEIN

<213> Sus

<220>

<223> Circuit B

<400> 84

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ala

20 25 30

<210> 85

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 85

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Leu Tyr Thr Pro Lys

20 25

<210> 86

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 86

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 87

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 87

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ile Tyr Thr Pro Lys

20 25

<210> 88

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 88

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 89

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 89

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 90

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 90

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ile

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ile Tyr Thr Pro Lys

20 25

<210> 91

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 91

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ile

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ile Tyr Thr Pro Lys

20 25

<210> 92

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 92

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ile

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 93

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 93

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ile

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 94

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 94

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ile Tyr Thr Pro Lys

20 25

<210> 95

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 95

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 96

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 96

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<210> 97

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Circuit B

<400> 97

Phe Val Glu Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Val

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Val Tyr Thr Pro Lys

20 25

<---

Claims (42)

1. An insulin analog having a reduced binding affinity to insulin receptors, containing (a) an A chain having the amino acid sequence shown under SEQ ID NO: 43 (GIVEQCCTSICSLEQLENYCN), and a B chain having the amino acid sequence shown under SEQ ID NO: 44 (FVNQHLCGSHLVEALYLVCGERGFIYTPK), (b) an A chain having the amino acid sequence shown under SEQ ID NO: 47 (GIVEQCCTSICSLEQLENYCN), and a B chain having the amino acid sequence shown under SEQ ID NO: 48 (FVNQHLCGSHLVEALYLVCGERGFVYTPK), or (c) an A chain having the amino acid sequence shown under SEQ ID NO: 77 (GIVEQCCTSICSLEQLENYCN), and a B chain having the amino acid sequence shown under SEQ ID NO: 78 (FVEQHLCGSHLVEALVLVCGERGFVYTPK). 2. An insulin analogue having a reduced binding affinity to insulin receptors, where said analogue is selected from the group consisting of: human Glu(A14)Leu(B16)Des(B30)-insulin, human Glu(A14)Ile(B16)Des(B30)-insulin, human Glu(A14)Val(B16)Des(B30)-insulin, human Glu(A14)Leu(B16)-insulin, human Glu(A14)Ile(B16)-insulin, human Glu(A14)Val(B16)-insulin, human Glu(A14)Leu(B25)Des(B30)-insulin, human Glu(A14)Ile(B25)Des(B30)-insulin, human Glu(A14)Val(B25)Des(B30)-insulin, human Glu(A14)Leu(B25)-insulin, human Glu(A14)Ile(B25)-insulin, human Glu(A14)Val(B25)-insulin, human Glu(A14)Gly(A21)Glu(B3)Val(B25)Des(B30)-insulin, human Glu(A14)Ile(B16)Ile(B25)Des(B30)-insulin, human Glu(A14)Glu(B3)Ile(B16)Ile(B25)Des(B30)-insulin, human Glu(A14)Ile(B16)Val(B25)Des(B30)-insulin, human Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)Des(B30)-insulin, human Glu(A14)Val(B16)Ile(B25)Des(B30)-insulin, human Glu(A14)Val(B16)Val(B25)Des(B30)-insulin, human Glu(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin, human Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)Des(B30)-insulin, human Glu(A14)Gly(A21)Glu(B3)Val(B25)-insulin, human Glu(A14)Ile(B16)Ile(B25)-insulin, human Glu(A14)Glu(B3)Ile(B16)Ile(B25)-insulin, human Glu(A14)Ile(B16)Val(B25)-insulin, human Glu(A14)Gly(A21)Glu(B3)Ile(B16)Val(B25)-insulin, human Glu(A14)Val(B16)Ile(B25)-insulin, human Glu(A14)Val(B16)Val(B25)-insulin, human Glu(A14)Glu(B3)Val(B16)Val(B25)-insulin and human Glu(A14)Gly(A21)Glu(B3)Val(B16)Val(B25)-insulin. 3. Proinsulin for producing an insulin analogue according to claim 1 or 2, where said proinsulin contains an A-chain and a B-chain of an insulin analogue according to claim 1 or 2. 4. Polynucleotide for producing an insulin analog according to any one of claims. 1, 2, where said polynucleotide contains a nucleotide sequence encoding an insulin analogue according to any one of claims. 12. 5. A polynucleotide for producing proinsulin according to claim 3, where said polynucleotide contains a nucleotide sequence encoding proinsulin according to claim 3. 6. Polynucleotide for producing an insulin analog according to any one of claims. 1, 2 and proinsulin according to claim 3, where said polynucleotide contains a nucleotide sequence encoding an insulin analogue according to any one of claims. 1, 2 and proinsulin according to paragraph 3. 7. Host cell for producing an insulin analog according to any one of paragraphs. 1, 2, where said cell contains an expression vector containing a polynucleotide according to claim 4. 8. A host cell for producing proinsulin according to claim 3, where said cell contains an expression vector containing a polynucleotide according to claim 5. 9. Host cell for producing an insulin analog according to any one of paragraphs. 1, 2 and proinsulin according to claim 3, where said cell contains an expression vector containing a polynucleotide according to claim 6.