Insulin analogs and methods of use thereof

Abstract

Insulin (INS) analogs are disclosed, which include modifications compared to native human INS that increase half-life, maintain selectivity for the insulin receptor (IR), and provide in vitro and in vivo stability to improve drugability and reduce immunogenicity. Pharmaceutical compositions are also disclosed that include one or more of the INS analogs described herein in a pharmaceutically acceptable carrier. Methods of preparing and using the INS analogs are also disclosed, particularly for treating metabolic conditions, diseases, or disorders, especially diseases such as diabetes and obesity.

Description

[0001] The present disclosure generally relates to biology and medicine, and more specifically it relates to insulin (INS) analogs, particularly long-acting single-chain INS (SCI) analogs, which can bind to the INS receptor (IR) and thus act as IR agonists. The present disclosure further relates to compositions containing them and their use in the treatment of metabolic disorders, diseases or conditions such as diabetes and obesity.

[0002] INS is a peptide hormone secreted by pancreatic β (beta) cells, and its physiological role is to maintain normal blood glucose levels by promoting cellular glucose uptake and by concomitantly inhibiting hepatic glycogenolysis, thereby regulating carbohydrate, lipid and protein metabolism. In addition, INS promotes cell division and growth in the fed state.

[0003] Structurally, INS is a heterodimer of two peptide chains of 21 and 30 amino acids (A chain and B chain respectively) linked by two inter-chain disulfide bonds, where the A chain also has an intra-chain disulfide bond. INS is produced from its prohormone proinsulin by cleavage of the C peptide therefrom. See, for example, De Meyts (2004) Bioessays 26:1351-1362; and Wilcox (2005) Clin. Biochem. Rev. 26:19-39.

[0004] Therapeutically, there are six main types of INS, including: (1) rapid-acting INS, (2) short-acting (meal-time) INS, (3) intermediate-acting INS, (4) long-acting (basal) INS, (5) combination / premixed INS and (6) inhaled INS. Effective INS therapy for individuals with diabetes is typically a combination of two types of exogenous INS preparations - short-acting (meal-time) INS administered at mealtimes and long-acting (basal) INS administered once or twice daily to control blood glucose levels between meals.

[0005] There are many INS analogs that have an extended t½ compared to native INS and thus can be administered as long-acting (basal) INS. For example, International Patent Application Publication Nos. WO 1995 / 007931 and WO 2018 / 217573 describe INS analogs that include INS and its variants linked (i.e., acylated) to a fatty acid moiety to improve t½. U.S. Patent No. 5,656,722 describes INS analogs that include an Asp21Gly mutation in the A chain and a carboxyl-terminal extension of the B chain with two Arg residues. International Patent Application Publication No. WO 2005 / 012347 describes INS analogs that are hexamers produced by adding hexadecanedioic acid to Lys at position 29 of the B chain. Duttaroy et al. describe analogs that include human INS linked to human serum albumin to improve t½ (See Duttaroy et al. (2005) Diabetes 54:251-258). Chinese Patent No. 103509118 and International Patent Application Publication No. WO 2016 / 178905 describe INS analogs that are fusions of the A-chain and / or B-chain and further include an Fc portion to improve t½.

[0006] Despite a large number of INS analogs, there is still a need for other INS analogs with improved t½, especially as analogs for long-acting (basal) INS.

[0007] To meet this need, the present disclosure first describes SCI analogs that are active against IR and can thus act as IR agonists. Such SCI analogs include the following basic structures from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus):

[0008] VHH-L1-A-L2-B,

[0009] VHH-L1-B-L2-A,

[0010] A-L2-B-L1-VHH, or

[0011] B-L2-A-L1-VHH,

[0012] wherein VHH is a portion that acts as a pharmacokinetic enhancer, A is the INS A-chain, B is the INS B-chain, L1 is the first linker, and L2 is the second linker.

[0013] In some cases, the VHH portion can have the amino acid sequence of SEQ ID NO:7, 8, or 9. In other cases, the VHH portion can be a variant having one or more additions, deletions, insertions, or substitutions such that the VHH portion has an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:7, 8, or 9.

[0014] In some cases, the A-chain can have the amino acid sequence of SEQ ID NO:3. In other cases, the A-chain can be a variant having one or more additions, deletions, insertions, or substitutions such that the A-chain has an amino acid sequence having at least about 90% to about 99% sequence similarity to any of SEQ ID NO:3. For example, the A-chain can include the E4Q mutation, T8H mutation, Y14E mutation, or N21G mutation of SEQ ID NO:3.

[0015] In some cases, the B chain can have the amino acid sequence of SEQ ID NO:4. In other cases, the B chain can be a variant with one or more additions, deletions, insertions, or substitutions such that the B chain has an amino acid sequence with at least about 90% to about 99% sequence similarity to any of those in SEQ ID NO:4. For example, the B chain can include the N3D mutation, N3K mutation, N3S mutation, S9A mutation, Y16E mutation, Y16F mutation, Y16H mutation, Y16R mutation, Y16W mutation, E21Q mutation, or F25H mutation of SEQ ID NO:4.

[0016] In some cases, L1 can have (GGGGQ) n (SEQ ID NO:10), (GGGQ) n (SEQ ID NO:11), (GGGGS) n (SEQ ID NO:12), (PGPQ) n (SEQ ID NO:13), (PGPA) n (SEQ ID NO:14), (GGE) n GG(SEQ ID NO:15), (GGGGE) n GGGG (SEQ ID NO:16), (GGGGK) n GGGG (SEQ ID NO:17), GGGG(AP) n GGGG (SEQ ID NO:18), GGGG(EP) n GGGG (SEQ ID NO:19), GGGG(KP) n GGGG (SEQ IDNO:20), (PGPE) n PGPQ (SEQ ID NO:21), (PGPK) n PGPQ (SEQ ID NO:22) amino acid sequence, where n can be 1 - 10. In other cases, L1 can have the amino acid sequence of any of SEQ ID NO:23 to 33. In other cases, L1 can be a variant with one or more additions, deletions, insertions, or substitutions such that L1 has an amino acid sequence with at least about 90% to about 99% sequence similarity to any of those in SEQ IDNO:23 to 33.

[0017] In some cases, L2 can have the amino acid sequence of any one of SEQ ID NOs: 34 to 36. In other cases, L2 can be a variant having one or more additions, deletions, insertions or substitutions such that L2 has an amino acid sequence having at least about 90% to about 99% sequence similarity to any one of SEQ ID NOs: 34 to 36.

[0018] In some cases, the INS analogue can have an amino acid sequence comprising: the VHH of SEQ ID NO: 7 or 8; the A chain of SEQ ID NO: 3 or a variant thereof; the B chain of SEQ ID NO: 4 or a variant thereof; L1 of any one of SEQ ID NOs: 23 to 33; and L2 of any one of SEQ ID NOs: 34 to 36. Alternatively, the INS analogue can have an amino acid sequence having at least about 90% to about 99% sequence similarity to an amino acid sequence comprising: the VHH of SEQ ID NO: 7 or 8; the A chain of SEQ ID NO: 3 or a variant thereof; the B chain of SEQ ID NO: 4 or a variant thereof; L1 of any one of SEQ ID NOs: 23 to 33; and L2 of any one of SEQ ID NOs: 34 to 36.

[0019] In certain cases, the INS analogue can have the amino acid sequence of any one of SEQ ID NOs: 37 to 81. Alternatively, the INS analogue can have an amino acid sequence having at least about 90% to about 99% sequence similarity to the amino acid sequence of any one of SEQ ID NOs: 37 to 81.

[0020] In certain cases, the VHH portion can bind to serum albumin, particularly human serum albumin, and can include complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3), wherein CDR1 can have the amino acid sequence of SEQ ID NO:84, 85, or 86, wherein CDR2 can have the amino acid sequence of SEQ ID NO:87, 88, or 89, and CDR3 can have the amino acid sequence of SEQ ID NO:90, 91, or 92. In certain cases, the VHH portion can include CDR1 of SEQ ID NO:84, CDR2 of SEQ ID NO:87, and CDR3 of SEQ ID NO:90; CDR1 of SEQ ID NO:84, CDR2 of SEQ ID NO:88, and CDR3 of SEQ ID NO:90; CDR1 of SEQ ID NO:85, CDR2 of SEQ ID NO:89, and CDR3 of SEQ ID NO:91; or CDR1 of SEQ ID NO:86, CDR2 of SEQ ID NO:89, and CDR3 of SEQ ID NO:92.

[0021] In certain cases, the INS analog has a binding affinity for IR that is comparable to the binding affinity of native human INS (SEQ ID NO:3 and 4). In other cases, the INS analog has a binding affinity for IR that is greater than the binding affinity of native human INS (SEQ ID NO:3 and 4). In other cases, the INS analog has a binding affinity for IR that is weaker than the binding affinity of native human INS (SEQ ID NO:3 and 4).

[0022] In certain cases, the INS analog has a t½ that is longer than the t½ of native human INS (SEQ ID NO:3 and 4), including up to about 20 to about 30 days when administered to humans.

[0023] The above compositions can alternatively be nucleic acid sequences encoding the amino acid sequences herein, as well as vectors for expressing the VHH portion or INS analog herein and host cells containing the same.

[0024] Second, a pharmaceutical composition is described that comprises an INS analogue herein or a pharmaceutically acceptable salt thereof (e.g., trifluoroacetate, acetate, or hydrochloride) and a pharmaceutically acceptable carrier. In certain cases, the pharmaceutically acceptable carrier is a buffer, e.g., normal saline, phosphate-buffered saline, citrate-buffered saline, or histidine-buffered saline. In certain cases, the buffer is histidine, a histidine buffer, or histidine-buffered saline. In other cases, the pharmaceutical composition may further comprise a carrier, diluent, and / or excipient.

[0025] In addition, the pharmaceutical composition may comprise at least one additional therapeutic agent, e.g., an agent used as a standard of care in a metabolic condition, disease, or disorder. In certain cases, the at least one additional therapeutic agent may be a dipeptidyl peptidase 4 (DPP-IV) inhibitor, native amylin or an analogue thereof, a short-acting (meal-time) INS analogue, a native incretin or an analogue thereof, a native insulin-like growth factor (IGF) or an analogue thereof, metformin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, inhibin, a sulfonylurea (SU), a thiazolidinedione (TZD), and / or other antihyperglycemic agents or other anti-obesity agents.

[0026] Third, a method of using an INS analogue herein is described, particularly a method of using an INS analogue to treat a metabolic condition, disease, or disorder. The method comprises at least the step of administering to an individual in need thereof an effective amount of the INS analogue or a pharmaceutically acceptable salt thereof.

[0027] In certain cases, the INS analogue may be administered by any standard route of administration (e.g., intramuscular, intravenous, parenteral, subcutaneous, or transdermal). In certain cases, the INS analogue is administered subcutaneously (SQ), intramuscularly (IM), or intravenously (IV). In a particular case, the INS analogue may be administered subcutaneously or intravenously to an individual.

[0028] Likewise, and in certain cases, the INS analogue may be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), every two weeks (i.e., bi-weekly), once a month (i.e., monthly), once every two months (i.e., every other month), or even once every three months. In certain cases, the INS analogue may be administered subcutaneously every other day, subcutaneously three times a week, subcutaneously twice a week, subcutaneously once a week, subcutaneously every two weeks, or subcutaneously once a month. In a particular case, the INS analogue is administered subcutaneously once a week (QW).

[0029] Alternatively, the INS analog can be administered intravenously to an individual. As above, the INS analog can be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), every two weeks (i.e., bi-weekly), or monthly. In some cases, the INS analog can be administered intravenously every other day, three times a week intravenously, twice a week intravenously, once a week intravenously, every two weeks intravenously, or once a month intravenously. In certain cases, the INS analog is administered intravenously once a week.

[0030] The method can also include the step of administering the INS analog in combination with an effective amount of at least one additional therapeutic agent. Briefly, the standard of care for many of the conditions / diseases / disorders herein includes DPP-IV inhibitors, native amylin or an analog thereof, short-acting (meal) INS analogs, native incretins or an analog thereof, native IGF or an analog thereof, metformin, SGLT2 inhibitors, inhibin, SU, TZD, and / or other anti-hyperglycemic agents or other anti-obesity agents, as well as other therapeutic agents for controlling comorbidities (including, but not limited to, high cholesterol and hypertension). In some cases, the additional therapeutic agent can be administered simultaneously with, separately from, or sequentially to the INS analog.

[0031] For example, the additional therapeutic agent can be administered at the same frequency as the INS analog (i.e., every other day, twice a week, weekly, or even monthly). In other cases, the additional therapeutic agent can be administered at a different frequency than the INS analog. In other cases, the additional therapeutic agent can be administered subcutaneously or intravenously. In other cases, the INS analog is administered subcutaneously, and the additional therapeutic agent can be administered orally or intravenously. Alternatively, the INS analog is administered intravenously, and the additional therapeutic agent is administered subcutaneously.

[0032] In some cases, the individual has diabetes and / or obesity.

[0033] The method can also include steps such as measuring or obtaining blood glucose, HbA1c, cholesterol, triglycerides, and / or body weight and comparing the measured / obtained values with one or more baseline values or previously measured / obtained values to evaluate the effectiveness of the treatment / therapy.

[0034] The method can also be combined with diet and exercise, and / or can be combined with additional therapeutic agents other than those discussed above.

[0035] Fourth, the use of the INS analogs herein is described. For example, an INS analog can be provided for treatment, particularly for treating metabolic conditions, diseases, or disorders, particularly diabetes and / or obesity. The INS analog can optionally be administered simultaneously with, separately from, or sequentially to at least one additional therapeutic agent (i.e., ，Combined administration. Similarly, INS analogs can be provided for the preparation of a medicament for treating a metabolic disorder, disease or condition, wherein the medicament may optionally further comprise one or more additional therapeutic agents as indicated above.

[0036] Fifth, there are provided compounds comprising the following amino acid sequences:

[0037]

[0038] (SEQ ID NO:37). In certain cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:37.

[0039] Alternatively, there are provided compounds comprising the following amino acid sequences:

[0040]

[0041] (SEQ ID NO:38). In certain cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:38.

[0042] Alternatively, there are provided compounds comprising the following amino acid sequences:

[0043]

[0044] (SEQ ID NO:39). In certain cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:39.

[0045] Alternatively, there are provided compounds comprising the following amino acid sequences:

[0046]

[0047] (SEQ ID NO:40). In certain cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:40.

[0048] Alternatively, there are provided compounds comprising the following amino acid sequences:

[0049]

[0050] (SEQ ID NO:41). In certain cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:41.

[0051] Alternatively, a compound is provided that includes the following amino acid sequence:

[0052]

[0053] (SEQ ID NO:42). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:42.

[0054] Alternatively, a compound is provided that includes the following amino acid sequence:

[0055]

[0056] (SEQ ID NO:43). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:43.

[0057] Alternatively, a compound is provided that includes the following amino acid sequence:

[0058]

[0059] (SEQ ID NO:44). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:44.

[0060] Alternatively, a compound is provided that includes the following amino acid sequence:

[0061]

[0062] (SEQ ID NO:45). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:45.

[0063] Alternatively, a compound is provided that includes the following amino acid sequence:

[0064]

[0065] (SEQ ID NO:46). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:46.

[0066] Alternatively, a compound is provided that includes the following amino acid sequence:

[0067]

[0068] (SEQ ID NO:47). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:47.

[0069] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0070]

[0071] (SEQ ID NO:48). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:48.

[0072] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0073]

[0074] (SEQ ID NO:49). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:49.

[0075] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0076]

[0077] (SEQ ID NO:50). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:50.

[0078] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0079]

[0080] (SEQ ID NO:51). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:51.

[0081] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0082]

[0083] (SEQ ID NO:52). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:52.

[0084] Alternatively, a compound is provided that includes the following amino acid sequence:

[0085]

[0086] (SEQ ID NO:53). In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:53.

[0087] Alternatively, a compound is provided that includes the following amino acid sequence:

[0088]

[0089] (SEQ ID NO:54). In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:54.

[0090] Alternatively, a compound is provided that includes the following amino acid sequence:

[0091]

[0092] (SEQ ID NO:55). In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:55.

[0093] Alternatively, a compound is provided that includes the following amino acid sequence:

[0094]

[0095] (SEQ ID NO:56). In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:56.

[0096] Alternatively, a compound is provided that includes the following amino acid sequence:

[0097]

[0098] (SEQ ID NO:57). In some cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:57.

[0099] Alternatively, a compound is provided that includes the following amino acid sequence:

[0100]

[0101] (SEQ ID NO:58). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:58.

[0102] Alternatively, a compound is provided that includes the following amino acid sequence:

[0103]

[0104] (SEQ ID NO:59). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:59.

[0105] Alternatively, a compound is provided that includes the following amino acid sequence:

[0106]

[0107] (SEQ ID NO:60). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:60.

[0108] Alternatively, a compound is provided that includes the following amino acid sequence:

[0109]

[0110] (SEQ ID NO:61). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:61.

[0111] Alternatively, a compound is provided that includes the following amino acid sequence:

[0112]

[0113] (SEQ ID NO:62). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:62.

[0114] Alternatively, a compound is provided that includes the following amino acid sequence:

[0115]

[0116] (SEQ ID NO:63). In certain cases, the compound can have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:63.

[0117] Alternatively, a compound is provided that includes the following amino acid sequence:

[0118]

[0119] (SEQ ID NO:64). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:64.

[0120] Alternatively, a compound is provided that includes the following amino acid sequence:

[0121]

[0122] (SEQ ID NO:65). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:65.

[0123] Alternatively, a compound is provided that includes the following amino acid sequence:

[0124]

[0125] (SEQ ID NO:66). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:66.

[0126] Alternatively, a compound is provided that includes the following amino acid sequence:

[0127]

[0128] (SEQ ID NO:67). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:67.

[0129] Alternatively, a compound is provided that includes the following amino acid sequence:

[0130]

[0131] (SEQ ID NO:68). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:68.

[0132] Alternatively, a compound is provided that includes the following amino acid sequence:

[0133]

[0134] (SEQ ID NO:69). In certain cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:69.

[0135] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0136]

[0137] (SEQ ID NO:70). In certain cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:70.

[0138] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0139]

[0140] (SEQ ID NO:71). In certain cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:71.

[0141] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0142]

[0143] (SEQ ID NO:72). In certain cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:72.

[0144] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0145]

[0146] (SEQ ID NO:73). In certain cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:73.

[0147] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0148]

[0149] (SEQ ID NO:74). In certain cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:74.

[0150] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0151]

[0152] (SEQ ID NO:75). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:75.

[0153] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0154]

[0155] (SEQ ID NO:76). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:76.

[0156] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0157]

[0158] (SEQ ID NO:77). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:77.

[0159] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0160]

[0161] (SEQ ID NO:78). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:78.

[0162] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0163]

[0164] (SEQ ID NO:79). In some cases, the compound can have an amino acid sequence that has at least about 90% to about 99% sequence similarity to SEQ ID NO:79.

[0165] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0166]

[0167] (SEQ ID NO:80). In certain cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:80.

[0168] Alternatively, a compound is provided that comprises the following amino acid sequence:

[0169]

[0170] (SEQ ID NO:81). In certain cases, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO:81.

[0171] One advantage of the INS analogs herein is that they can be chemically or recombinantly produced as single-chain polypeptides (i.e., monomers) and thus do not require endoproteolytic processing to obtain biological activity. However, consider that in certain cases, the VHH moiety can be conjugated not only to single-chain INS but also to double-chain INS (e.g., native). On the VHH moiety, conjugation can occur not only to the N- and C-termini but also to any surface-exposed amino acid of the VHH, provided that such conjugation does not completely abolish albumin binding of the VHH moiety or IR signaling of the INS moiety.

[0172] One advantage of the INS analogs herein is that the VHH moiety can be used not only with native A- and B-chain sequences but also with their modified sequences. In addition, the VHH moiety can be further modified to have enhanced or additional functions, which are achieved by attachment of other peptide / protein fusions or small molecules to the VHH moiety.

[0173] One advantage of the INS analogs herein is that the VHH moiety provides an extended duration of action in mammals such as humans and can have a t½ of about 20 days to about 30 days, thus allowing administration at least weekly or every 2 weeks compared to native human INS, especially native human INS (SEQ ID NO:3 and 4), which can improve compliance.

[0174] One advantage of the INS analogs herein is that they have similar or better selectivity, affinity, and / or efficacy for IR compared to native human INS (SEQ ID NO:3 and 4).

[0175] One advantage of the INS analogs herein is that they have tunable pharmacokinetics, which is achieved by altering the albumin affinity of the VHH moiety.

[0176] One advantage of the INS analogs herein is that, compared to native human INS (SEQ ID NO: 3 and 4) or INS analogs not fused to one of the VHH moieties herein, they have improved stability in formulations, particularly in storage formulations.

[0177] One advantage of the VHH moieties herein is that they have similar binding not only to human serum albumin but also to dog, monkey, mouse, pig, and rat serum albumin, which makes it easier to translate pharmacodynamic, pharmacokinetic, and toxicological studies from these species to humans or other species listed above.

[0178] One advantage of the VHH moieties herein is that they can be used not only to improve the t½ of the INS analogs herein compared to native human INS (SEQ ID NO: 3 and 4), but also to improve the t½ of other bioactive peptides and proteins (e.g., growth differentiation factor 15 (GDF-15), glucose-dependent insulinotropic polypeptide 1 (GLP-1), or relaxin (RLN)).

[0179] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of INS analogs, pharmaceutical compositions, and methods, the preferred methods and materials are described herein.

[0180] Furthermore, the mention of an element by the indefinite article "a" or "an" does not exclude the presence of more than one element, unless the context clearly requires the presence of one and only one element. Thus, the indefinite article "a" or "an" generally refers to "at least one".

[0181] Definition

[0182] As used herein, "about" refers to a statistically significant range within one or more values such as the stated concentration, length, molecular weight, pH, sequence similarity, time range, temperature, volume, etc. Such values or ranges can be within an order of magnitude, typically within 20% of a given value or range, more typically within 10% of a given value or range, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend on the particular system being studied and will be readily understood by those of ordinary skill in the art.

[0183] As used herein and with respect to one or more receptors, "active", "activate", "activation", etc. refer to the ability of a compound such as an INS analog herein to bind to a receptor and induce a response at the receptor, as measured using assays known in the art (such as the in vitro assays described below).

[0184] As used herein, "amino acid" refers to a molecule that, from a chemical perspective, is characterized by the presence of one or more amine groups and one or more carboxylic acid groups and may contain other functional groups. As is known in the art, there is a set of twenty amino acids that are designated as standard amino acids and that can be used as the building blocks of any peptide / protein produced by a living organism. The amino acid sequences in the present disclosure contain the standard single-letter or three-letter codes of the twenty naturally occurring amino acids.

[0185] As used herein, "analogue" refers to a compound, such as a synthetic peptide, polypeptide, or protein, that activates a target receptor and elicits at least one in vivo or in vitro effect elicited by the receptor's natural agonist.

[0186] As used herein, "agonist" refers to a receptor ligand that binds to and activates a receptor.

[0187] As used herein, "conservative substitution" refers to a variant of a reference peptide, polypeptide, or protein that is identical to the reference molecule except for having one or more conservative amino acid substitutions in its amino acid sequence. Generally, conservative modified variants include amino acid sequences having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the reference amino acid sequence. More specifically, conservative substitution means substituting one amino acid with an amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.) and having a minimal impact on the biological activity of the resulting substituted peptide, polypeptide, or protein. Conservative substitutions of functionally similar amino acids are well known in the art and thus need not be described in detail herein.

[0188] As used herein, "effective amount" refers to the amount or dose of one or more INS analogues herein or a pharmaceutically acceptable salt thereof that, upon administration of a single dose or multiple doses to an individual in need thereof, provides the desired effect in such an individual being diagnosed or treated (i.e., may produce a clinically measurable difference in the individual's condition, e.g., reduced blood glucose, reduced HbA1c, reduced cholesterol, reduced triglycerides, or weight loss). By using known techniques and by observing the results obtained in similar situations, one of ordinary skill in the art can readily determine the effective amount. When determining the effective amount for an individual, many factors are considered, including but not limited to the species of mammal, its size, age, and general health, the particular disease or disorder involved, the degree or involvement or severity of the disease or disorder, the individual's response, the particular INS analogue administered, the mode of administration, the bioavailability characteristics of the formulation administered, the dosing regimen selected, the use of concomitant medications, and other relevant circumstances.

[0189] As used herein, "extended duration of action" means that the binding and activity of the INS analogs herein persist for a longer period of time than native INS, particularly native human INS (SEQ ID NO:3 and 4), thereby allowing less frequent administration, such as at least once a day or even three times a week, twice a week or once a week. Using known pharmacokinetic assay methods, such as those used in the following examples, the time-action characteristics of the INS analogs can be measured.

[0190] As used herein, "half-life" or "t½" refers to the time it takes for half of the amount of a compound (such as native INS or an INS analog herein) to be removed from the fluid or other physiological space (such as serum or plasma) of an individual by biological processes. Alternatively, t½ can also refer to the time it takes for a given amount of such a compound to lose half of its pharmacological, physiological or radiological activity.

[0191] As used herein, "half maximal effective concentration" or "EC 50 " refers to the concentration of a compound that results in 50% activation / stimulation of an assay endpoint, such as a dose-response curve (e.g., the IRS-PI3K-Akt and IRS-Raf / Ras / MEK / MAPK signaling pathways).

[0192] As used herein, "in combination with" means administering at least one INS analog herein simultaneously, sequentially or in a single combination formulation with one or more additional therapeutic agents.

[0193] As used herein, "insulin" or "INS" refers to insulin obtained or derived from any species (such as mammalian species, particularly human), wherein the native form is a heterodimeric peptide having two peptide chains (e.g., an A chain and a B chain) linked by two disulfide bonds, and wherein the A chain further has a single intramolecular disulfide bond. In humans, INS processing begins with preproinsulin (SEQ ID NO:1; also see, UniProt / SwissProt database accession number P01308), which is processed into proinsulin (including an A chain, a B chain and a C peptide; native INS has a B-C-A structure), wherein the sequence of native human proinsulin is listed in SEQ ID NO:2. Proinsulin undergoes further processing, wherein the C peptide is cleaved to yield INS. The sequence of the A chain of native human INS is listed in SEQ ID NO:3. Similarly, the sequence of the B chain of native human INS is listed in SEQ ID NO:4.

[0194] INS signaling occurs through the IR, which is a homodimer of two α chains and two β (αβ)2 chains with tyrosine kinase activity. For example, IR has been found in adipose tissue, brain, erythrocytes, fibroblasts, granulocytes, heart, kidney, monocytes, alveoli, pancreatic acini, placenta, vascular endothelium, and skeletal muscle. In humans, there are two IR subtypes - IR-A (SEQ ID NO:5; also see, UniProt / SwissProt database accession number P06213) and IR-B (SEQ ID NO:6; also see, UniProt / SwissProt database accession number P06213). In addition, there are known differences between IR-A and IR-B in tissue expression, ligand binding affinity, receptor internalization, recycling time, and intracellular signaling. See, for example, Belfiore et al. (2009) Endocr. Rev. 30:586-6923; Benyoucef (2007) Biochem. J. 403:603-613; Frasca (1999) Mol. Cell. Biol. 19:3278-3288; Seino et al. (1989) Proc. Natl. Acad. Sci. USA 86:114-118; and Yamaguchi et al. (1993) Endocrinology 132:1132-1138. Stimulation of the IR activates signal transduction networks involving tyrosine kinase, PI3K, or Ras.

[0195] As used herein, "insulin analog" or "INS analog," etc., refers to a compound such as a peptide or polypeptide: which elicits one or more actions of native INS at the IR, but which varies in amino acid sequence in some way compared to native INS due to one or more additions, deletions, insertions, and / or substitutions. INS analogs can also include variants of these compounds that are functionally equivalent to native INS, but have a sequence that is either a fragment or the complete sequence, but which themselves have further additions, deletions, insertions, and / or substitutions. Unless otherwise indicated, all references to amino acid positions in unmodified or modified INS described herein are based on the corresponding positions in the A chain of native human INS of SEQ ID NO:3 or the B chain of SEQ ID NO:4. In some cases, compared to native INS, especially native human INS (SEQ ID NO:3 and 4), INS analogs herein can bind to the IR with higher or lower affinity, but exhibit a longer t½ in vivo or in vitro. In this way, INS analogs herein are synthetic compounds that act as IR agonists.

[0196] As used herein, "insulin resistance" refers to a physiological condition in which normal or elevated levels of INS produce a diminished biological response in an individual.

[0197] As used herein, an "individual in need thereof" refers to a mammal, such as a human, having a disorder, disease, condition, or symptom for which treatment or therapy is needed, including, for example, those listed herein. In particular, a preferred individual to be treated is a human.

[0198] As used herein, "long-acting" means that the binding affinity and activity of the INS analogs herein persist for a longer period of time than native human INS (SEQ ID NO: 3 and 4), thereby allowing for less frequent administration, such as at least once a day or even three times a week, twice a week, once a week, or once a month. Using known pharmacokinetic assay methods, such as those described in the following examples, the time-action characteristics of the INS analogs herein can be measured.

[0199] As used herein, "non-standard amino acid" refers to an amino acid that may occur naturally in cells but does not participate in peptide synthesis. Non-standard amino acids can be components of peptides and are often generated by modifying standard amino acids in a peptide, polypeptide, or protein (i.e., by post-translational modification). Non-standard amino acids can include D-amino acids, which have an absolute chirality opposite to that of the above-mentioned standard amino acids.

[0200] As used herein, "pharmaceutically acceptable buffer" refers to any of the standard pharmaceutical buffers known to those of skill in the art.

[0201] As used herein, "sequence similarity" refers to a quantitative property of two or more nucleic acid sequences or amino acid sequences of a biological compound, e.g., the correspondence of two or more sequences over their entire length or a comparison window. Sequence similarity can be measured by (1) the percentage of identity or (2) the percentage of similarity. The percentage of identity measures the percentage of identical residues between two biological compounds divided by the length of the shortest sequence; while the percentage of similarity measures identity and additionally includes sequence gaps and residue similarity in the evaluation. Methods and algorithms for determining sequence similarity are well known in the art and thus need not be described in detail herein. The specified percentage of identical nucleotide or amino acid positions is at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.

[0202] As used herein, "single-chain insulin", "scINS", "SCI", etc. refer to INS polypeptides in which the A-chain and the B-chain are connected to each other by a non-natural linker (i.e., L2), such as in A-L2-B or B-L2-A. In addition, SCI may include at least one of the native inter-chain and / or intra-chain disulfide bonds to maintain the correct structural folding.

[0203] As used herein, "double-chain insulin", "tcINS", "TCI", etc. refer to INS polypeptides in which the A-chain and the B-chain are connected to each other by one or more inter-chain and / or intra-chain disulfide bonds, but not by any linker, to maintain the correct structural folding, such as native INS.

[0204] As used herein, "treatment" refers to the management and care of an individual having a condition, disease, disorder, or symptom for which the administration of an INS analogue is indicated, with the aim of attenuating, inhibiting, reversing, slowing, or halting the progression or severity of the condition, disease, disorder, or symptom. Treatment includes administering to the individual an INS analogue or a composition containing an INS analogue herein to prevent the onset of symptoms or complications, alleviate symptoms or complications, or eliminate the condition, disease, disorder, or symptom. Treatment includes administering an INS analogue or a composition containing an INS analogue herein to the individual to result in, for example, reduced blood glucose, reduced HbA1c, reduced cholesterol, reduced triglycerides, or reduced body weight. The individual to be treated is a mammal, especially a human.

[0205] As used herein, "patient", "subject", and "individual" are used interchangeably herein and refer to a mammal, especially a human. In certain cases, the individual is further characterized as having a condition, disease, disorder, or symptom that would benefit from the administration of an INS analogue herein.

[0206] As used herein, "VHH" or "VHH moiety" refers to a form of a single-domain antibody, especially an antibody fragment of the single monomer variable region of a heavy-chain-only antibody (HcAb), which has a very small size of approximately 15 kDa. It has been found herein that the VHH moiety can be used as a pharmacokinetic enhancer to extend the duration of action of an INS analogue herein and / or improve its t½. The VHH moiety herein binds to serum albumin, especially human serum albumin; however, the VHH moiety can alternatively be used to bind IgG (including the Fc domain), the neonatal Fc receptor (FcRn), or other long-lived serum proteins. Although the VHH moieties herein are used to improve the t½ of INS, they can equally be used to improve the t½ of other bioactive peptides / proteins such as GDF-15, GLP-1, or RLN.

[0207] Because VHH portions are single-domain heavy-chain antibodies, they have three CDRs that include residues that form specific interactions with an antigen (e.g., human serum albumin). Assignment of residues to the various CDRs can be done by algorithms such as Chothia, IMBT, Kabat, or North. The North CDR definition is based on affinity propagation clustering of a large number of crystal structures (North et al. (2011) J. Mol. Bio. 406:228-256). In this text, the CDRs are preferably defined by the sequences listed in the sequence listing, which are based on a combination of multiple definitions including North and Kabat.

[0208] In some cases, the VHH portion can include at least complementarity determining region (CDR) 1 (CDR1), CDR2, and CDR3, where CDR1 can be one of SEQ ID NO:84, 85, and 86, where CDR2 can be one of SEQ ID NO:87, 88, and 89, and CDR3 can be one of SEQ ID NO:90, 91, and 92.

[0209] Certain abbreviations are defined as follows: "ACR" means albuminuria / creatinine ratio; "amu" means atomic mass unit; "AUC" means area under the curve; "BHI" means biosynthetic human insulin; "Boc" means tert-butoxycarbonyl; "cAMP" means cyclic adenosine monophosphate; "CMV" means cytomegalovirus; "DNA" means deoxyribonucleic acid; "DMF" means dimethylformamide; "DMSO" means dimethyl sulfoxide; "EDC" means 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; "EDTA" means ethylenediaminetetraacetic acid; "EIA / RIA" means enzyme immunoassay / radioimmunoassay; "ETA" means ethanolamine; "GS" means glutamine synthetase; "HIC" means hydrophobic interaction chromatography; "hr" means hour; "HTRF" means homogeneous time-resolved fluorescence; "IV" means intravenous; "kDa" means kilodalton; "LC-MS" means liquid chromatography-mass spectrometry; "min" means minute; "MS" means mass spectrometry; "MSX" means methionine sulfoximine; "NHS" means N-hydroxysuccinimide; "OtBu" means O-tert-butyl; "Pbf" means NG-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; "PEI" means polyethyleneimine; "RP-HPLC" means reverse-phase high performance liquid chromatography; "sec" means second; "NaOAc" means sodium acetate; "RU" refers to resonance unit; "SPA" means scintillation proximity assay; "SEC" means size exclusion chromatography; "SEM" means standard error of the mean; "SPR" means surface plasmon resonance; "SQ" means subcutaneous; "TFA" means trifluoroacetic acid; and "Trt" means trityl.

[0210] Insulin analog

[0211] The INS analogs herein have structural similarity to native human INS (SEQ ID NOs: 3 and 4), but have many structural differences. For example, when compared to native human INS (SEQ ID NOs: 3 and 4), the INS analogs herein include at least one variation compared to the amino acids present in native human INS, including a peptide linker between the A and B chains, and include pharmacokinetic enhancers such as albumin-binding VHH moieties. The INS analogs herein produce sufficient activity in the IR and thus have beneficial properties related to their developability as therapeutic treatments, including improved solubility in aqueous solution, improved chemical and physical formulation stability, extended pharmacokinetic properties (which can be adjusted based on the affinity of the VHH for serum albumin), and a minimized likelihood of immunogenicity.

[0212] In short, the INS analogs herein include amino acid sequences having one of the following structures from the amino terminus to the carboxyl terminus:

[0213] VHH-L1-A-L2-B,

[0214] VHH-L1-B-L2-A,

[0215] A-L2-B-L1-VHH, or

[0216] B-L2-A-L1-VHH,

[0217] wherein VHH is the part acting as a pharmacokinetic enhancer, A is the INS A chain, B is the INS B chain, L1 is the first peptide linker and L2 is the second peptide linker, and L1 and L2 are different from each other (i.e., each has a different amino acid sequence). In some cases, the INS analog has an amino acid sequence of B-L2-A-L1-VHH from the amino terminus to the carboxyl terminus.

[0218] Regarding the A chain, it can be the native INS A chain, such as the native human INS A chain (SEQ ID NO:3). Alternatively, the A chain can be a variant thereof. For example, the A chain variant can have an amino acid sequence that includes: an E4Q mutation, a T8H mutation, a Y14E mutation, an N21G mutation, or a combination thereof (e.g., T8H and N21G; or T8H, Y14E, and N21G). Alternatively, the A chain can be a truncation thereof. For example, the A chain can be the INS A chain lacking residues 1-3 of SEQ ID NO:3 (desA1-3) or lacking residue 21 (desA21).

[0219] Similarly, regarding the B chain, it can be the native INS chain, such as the native human INS B chain (SEQ ID NO:4). Alternatively, the B chain can be a variant thereof. For example, the B chain variant can have an amino acid sequence that includes: an N3D mutation, an N3K mutation, an N3S mutation, an S9A mutation, a Y16E mutation, a Y16F mutation, a Y16H mutation, a Y16R mutation, a Y16W mutation, an E21Q mutation, an F25H mutation, or a combination thereof (e.g., N3S and Y16F; N3S and Y16H; N3S and Y16R; N3S and Y16W; N3S and F25H; N3S and Y16R; N3S, Y16H, and F25H; or N3S, Y16R, and F25H). Alternatively, the B chain can be a truncation thereof. For example, the B chain can be the INS B chain lacking residues 1-3 of SEQ ID NO:4 (desB1-3) or lacking residues 27-30 (desB27-30).

[0220] In view of the above, the A chain can be the native human INS A chain (SEQ ID NO: 3) and the B chain can be the native human INS B chain (SEQ ID NO: 4). In other cases, the A chain can be a variant of SEQ ID NO: 3 and the B chain can be SEQ ID NO: 4. Alternatively, the A chain can be SEQ ID NO: 3 and the B chain can be a variant of SEQ ID NO: 4. Still alternatively, the A chain can be a variant of SEQ ID NO: 3 and the B chain can be a variant of SEQ ID NO: 4. In yet other cases, the A chain can be a truncation of SEQ ID NO: 3 and the B chain can be SEQ ID NO: 4. Alternatively, the A chain can be a truncation of SEQ ID NO: 3 and the B chain can be a variant of SEQ ID NO: 4 or a truncation of SEQ ID NO: 4. In yet other cases, the A chain can be SEQ ID NO: 3 and the B chain can be a truncation of SEQ ID NO: 4. Alternatively, the A chain can be a variant or a truncation of SEQ ID NO: 3 and the B chain can be a truncation of SEQ ID NO: 4.

[0221] Other A and B chains in INS analogs useful herein are described, for example, in International Patent Application Publication Nos. WO1996 / 034882, WO 2005 / 054291, WO 2006 / 097521, WO 2007 / 096332, WO 2007 / 104734, WO2007 / 104736, WO 2007 / 104737, WO 2007 / 104738, WO 2011 / 031622, WO 2011 / 159895, WO2014 / 071405, WO 2016 / 057529, WO 2017 / 052305, WO 2018 / 165290, WO 2018 / 217573 and WO2019 / 066570; see also, Glidden et al. (2018) J. Biol. Chem. 293:47-68; Hua et al. (2008) J. Biol. Chem. 283:14703-14716; Kaur et al. (2013) ACS Chem. Biol. 8:1822-1829; Mao et al. (2017) Appl. Microbiol. Biotechnol. 101:3259-3271; Mao et al. (2019) Appl. Microbiol. Biotechnol. 103:1-15; Sanlioglu et al. (2013) Islets 5:67-78.

[0222] Regarding L1, it can be a peptide of about 1 to about 50 amino acids. Alternatively, L1 can be about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 amino acids. Still alternatively, L1 can be about 5 to about 10 amino acids, about 10 to about 15 amino acids, about 15 to about 20 amino acids, about 20 to about 25 amino acids, about 25 to about 30 amino acids, about 30 to about 35 amino acids, about 35 to about 40 amino acids, about 40 to about 45 amino acids, or about 45 to about 50 amino acids. In some cases, L1 can be omitted such that the A-chain or B-chain is directly conjugated to the VHH moiety. In some cases, L1 can include the repeat sequence of (GGGGQ) n (SEQ ID NO:10), where n can be about 1 to about 10, especially 5 (i.e., (GGGGQ)5; SEQ ID NO:23). In other cases, L1 can include (PGPQ) n (SEQ ID NO:13), where n can be about 1 to about 10, especially 8 (i.e., (PGPQ)8; SEQ ID NO:24). In other cases, L1 can include (PGPA) n (SEQ ID NO:14), where n can be about 1 to about 10, especially 8 (i.e., (PGPA)8; SEQ ID NO:25). In other cases, L1 can include (GGE) n GG (SEQ IDNO:15), where n can be about 1 to about 10, especially 7 (i.e., (GGE)7GG; SEQ ID NO:26). In other cases, L1 can include (GGGGE) n GGGG (SEQ ID NO:16), where n can be about 1 to about 10, especially 4 (i.e., (GGGGE)4GGGG; SEQ ID NO:27). In other cases, L1 can include (GGGGK) n GGGG(SEQ ID NO:17), where n can be about 1 to about 10, especially 4 (i.e., (GGGGK)4GGGG; SEQ IDNO:28). In other cases, L1 can include GGGG(AP) n GGGG (SEQ ID NO:18), where n can be about 1 to about 10, especially 10 (i.e., GGGG(AP) 10 GGGG; SEQ ID NO:29). In other cases, L1 can include GGGG(EP) nRepeat sequences of GGGG (SEQ ID NO:19), where n can be from about 1 to about 10, especially 10 (i.e., GGGG(EP) 10 GGGG; SEQ ID NO:30). In other cases, L1 can include GGGG(KP) n Repeat sequences of GGGG (SEQ ID NO:20), where n can be from about 1 to about 10, especially 10 (i.e., GGGG(KP) 10 GGGG; SEQ ID NO:31). In other cases, L1 can include (PGPE) n Repeat sequences of PGPQ (SEQ ID NO:21), where n can be from about 1 to about 10, especially 7 (i.e., (PGPE)7PGPQ; SEQ ID NO:32). In other cases, L1 can include (PGPK) n Repeat sequences of PGPQ (SEQ IDNO:22), where n can be from about 1 to about 10, especially 7 (i.e., (PGPK)7PGPQ; SEQ ID NO:33).

[0223] Other linkers that can be used as L1 in INS analogs include, but are not limited to, (GGGQ) n (SEQ ID NO:11) or (GGGG S) n (SEQ ID NO:12).

[0224] Regarding L2, it can be a peptide of about 1 to about 15 amino acids. Alternatively, L2 can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 amino acids. Still alternatively, L2 can be about 1 to about 5 amino acids, about 5 to about 10 amino acids, about 10 to about 15 amino acids, especially 10 - 15 amino acids. In some cases, L2 can include a mixture of Ala / A, Gln / Q, Gly / G, Pro / P, and Ser / S residues. In other cases, L2 can be SEQ ID NO:34, 35, or 36.

[0225] Regarding VHH, it can be a polypeptide of about 50 to about 200 amino acids, especially about 125 to about 150 amino acids, which can bind to serum albumin or another serum protein with a long t½. In certain cases, the VHH can be any one of SEQ ID NO:7, 8, or 9. The structural features of these VHH moieties result in INS analogs with a longer t½ compared to native INS, especially native human INS (SEQ ID NO:3 and 4). Given that the VHH moieties herein target serum albumin, it can be expected that the t½ of the INS analogs herein will be similar to the t½ of the serum albumin of the species to which the INS analog is administered (considering any target-mediated drug disposition).

[0226] In addition to the variations described herein, the INS analogs can include one or more additional amino acid modifications, especially conservative substitutions, provided that the INS analogs are still capable of binding to and activating the IR.

[0227] Collectively, exemplary INS analogs are as follows:

[0228] INS analog 1, which includes the B chain of INS, L2 of 6 residues (bold), the A chain of INS with the N21G mutation, (PGPA)8 L1 (italic), and the VHH moiety (underlined) from the N-terminus to the C-terminus, and has the following amino acid sequence:

[0229]

[0230] INS analog 2, which includes the B chain of INS with the N3D mutation, L2 of 6 residues (bold), the A chain of INS with the N21G mutation, (PGPA)8 L1 (italic), and the VHH moiety (underlined) from the N-terminus to the C-terminus, and has the following amino acid sequence:

[0231]

[0232] INS analog 3, which includes the B chain of INS with the S9A mutation, L2 of 6 residues (bold), the A chain of INS with the N21G mutation, (PGPA)8 L1 (italic), and the VHH moiety (underlined) from the N-terminus to the C-terminus, and has the following amino acid sequence:

[0233]

[0234] INS analog 4, which includes the B chain of INS with the Y16E mutation, L2 of 6 residues (bold), the A chain of INS with the N21G mutation, (PGPA)8 L1 (italic), and the VHH moiety (underlined) from the N-terminus to the C-terminus, and has the following amino acid sequence:

[0235]

[0236] INS analog 5, which from the N-terminus to the C-terminus includes the B-chain of INS with a Y16H mutation, L2 of 6 residues (bold), the A-chain of INS with an N21G mutation, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0237]

[0238] INS analog 6, which from the N-terminus to the C-terminus includes the B-chain of INS with an F25H mutation, L2 of 6 residues (bold), the A-chain of INS with an N21G mutation, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0239]

[0240] INS analog 7, which from the N-terminus to the C-terminus includes the B-chain of INS with an N3S mutation, L2 of 6 residues (bold), the A-chain of INS with an N21G mutation, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0241]

[0242] INS analog 8, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with an N21G mutation, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0243]

[0244] INS analog 9, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0245]

[0246] INS analog 10, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S and Y16H mutations, L2 of 6 residues (bold), the A-chain of INS with N21G mutation, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0247]

[0248] INS analog 11, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S and Y16H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H, Y14E, and N21G mutations, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0249]

[0250] INS analog 12, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S, Y16H, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0251]

[0252] INS analog 13, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S, Y16H, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H, Y14E, and N21G mutations, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0253]

[0254] INS analog 14, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S and Y16R mutations, L2 of 6 residues (bold), the A-chain of INS with N21G mutation, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0255]

[0256] INS analog 15, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S and Y16F mutations, L2 of 6 residues (bold), the A-chain of INS with N21G mutation, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0257]

[0258] INS analog 16, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S and Y16W mutations, L2 of 6 residues (bold), the A-chain of INS with N21G mutation, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0259]

[0260] INS analog 17, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S and Y16R mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0261]

[0262] INS analog 18, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (PGPA)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0263]

[0264] INS analog 19, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S mutation, L2 of 6 residues (bold), the A-chain of INS with N21G mutation, (G4Q)5 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0265]

[0266] INS analog 20, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S mutation, L2 of 6 residues (bold), the A-chain of INS with N21G mutation, (PGPQ)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0267]

[0268] INS analog 21, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S, Y16H, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (G4Q)5 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0269]

[0270] INS analog 22, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S, Y16H, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (PGPQ)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0271]

[0272] INS analog 23, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S, Y16H, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with Y14E and N21G mutations, (G4Q)5 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0273]

[0274] INS analog 24, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S, Y16H, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with Y14E and N21G mutations, (PGPQ)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0275]

[0276] INS analog 25, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S and Y16R mutations, L2 of 6 residues (bold), the A-chain of INS with N21G mutations, (G4Q)5 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0277]

[0278] INS analog 26, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S and Y16R mutations, L2 of 6 residues (bold), the A-chain of INS with N21G mutations, (PGPQ)8 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0279]

[0280] INS analog 27, which from the N-terminus to the C-terminus includes a B-chain with N3S and Y16R mutations, a 6-residue L2 (bold), an A-chain of INS with T8H and N21G mutations, (G4Q)5 L1 (italic), and a VHH portion (underlined), having the following amino acid sequence:

[0281]

[0282] INS analog 28, which from the N-terminus to the C-terminus includes a B-chain of INS with N3S and Y16R mutations, a 6-residue L2 (bold), an A-chain of INS with T8H and N21G mutations, (PGPQ)8 L1 (italic), and a VHH portion (underlined), having the following amino acid sequence:

[0283]

[0284] INS analog 29, which from the N-terminus to the C-terminus includes a B-chain of INS with N3S, Y16R, and F25H mutations, a 6-residue L2 (bold), an A-chain of INS with T8H and N21G mutations, (G4Q)5 L1 (italic), and a VHH portion (underlined), having the following amino acid sequence:

[0285]

[0286] INS analog 30, which from the N-terminus to the C-terminus includes a B-chain of INS with N3S, Y16R, and F25H mutations, a 6-residue L2 (bold), an A-chain of INS with T8H and N21G mutations, (PGPQ)8 L1 (italic), and a VHH portion (underlined), having the following amino acid sequence:

[0287]

[0288] INS analog 31, which from the N-terminus to the C-terminus includes a B-chain of INS with N3S, Y16R, and F25H mutations, a 6-residue L2 (bold), an A-chain of INS with T8H and N21G mutations, (G2E)7G2 L1 (italic), and a VHH portion (underlined), having the following amino acid sequence:

[0289]

[0290] INS analog 32, which from the N-terminus to the C-terminus comprises the B-chain of INS with N3S, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (G4E)4G4 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0291]

[0292] INS analog 33, which from the N-terminus to the C-terminus comprises the B-chain of INS with N3S, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (G4K)4G4 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0293]

[0294] INS analog 34, which from the N-terminus to the C-terminus comprises the B-chain of INS with N3S, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, G4(AP) 10 G4 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0295]

[0296] INS analog 35, which from the N-terminus to the C-terminus comprises the B-chain of INS with N3S, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, G4(EP) 10 G4 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0297]

[0298] INS analog 36, which from the N-terminus to the C-terminus comprises the B-chain of INS with N3S, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, G4(KP) 10 G4 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0299]

[0300] INS analog 37, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (PGPE)7PGPQ L1 (italic), and the VHH portion (underlined), has the following amino acid sequence:

[0301]

[0302] INS analog 38, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (PGPK)7PGPQ L1 (italic), and the VHH portion (underlined), has the following amino acid sequence:

[0303]

[0304] INS analog 39, which from the N-terminus to the C-terminus includes the B-chain of INS with N3K, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (G4Q)5 L1 (italic), and the VHH portion (underlined), has the following amino acid sequence:

[0305]

[0306] INS analog 40, which from the N-terminus to the C-terminus includes the B-chain of INS with N3S, Y16R, E21Q, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (G4Q)5 L1 (italic), and the VHH portion (underlined), has the following amino acid sequence:

[0307]

[0308] INS analog 41, which from the N-terminus to the C-terminus includes the B-chain of INS with N3K, Y16R, E21Q, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (G4Q)5 L1 (italic), and the VHH portion (underlined), has the following amino acid sequence:

[0309]

[0310] INS analog 42, which from the N-terminus to the C-terminus comprises the B-chain of INS with N3S, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (G4Q)5 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0311]

[0312] INS analog 43, which from the N-terminus to the C-terminus comprises the B-chain of INS with N3K, Y16R, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (G4Q)5 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0313]

[0314] INS analog 44, which from the N-terminus to the C-terminus comprises the B-chain of INS with N3K, Y16R, E21Q, and F25H mutations, L2 of 6 residues (bold), the A-chain of INS with T8H and N21G mutations, (G4Q)5 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0315]

[0316] INS analog 45, which from the N-terminus to the C-terminus comprises the B-chain of INS with N3K, Y16R, and F25Q mutations, L2 of 6 residues (bold), the A-chain of INS with E4Q, T8H, and N21G mutations, (G4Q)5 L1 (italic), and the VHH portion (underlined), having the following amino acid sequence:

[0317]

[0318] (SEQ ID NO:81).

[0319] Using methods known in the art, including, for example, those described in the following examples, the half-life of the INS analogs herein can be measured. Similarly, using methods known in the art for measuring binding affinity, such as those described in the following examples, the affinity of the INS analogs herein for albumin of different species can be measured and is typically expressed as the equilibrium dissociation constant (K D ) value. In addition, using methods known in the art, including, for example, the in vitro activity assays described below, the activity of the INS analogs herein against IR or insulin-like growth factor 1 receptor (IGF-1R) can be measured and is typically expressed as EC50 Value

[0320] As a result of the above modifications, when administered to mammals, particularly humans, the INS analogs herein have a longer t½ than native INS, particularly native human INS (SEQ ID NO: 3 and 4). As pointed out above, the VHH portion herein targets serum albumin; thus, it can be expected that the t½ of the INS analogs herein will be similar to the t½ of the serum albumin of the species to which the INS analogs are administered. In some cases, the INS analogs can have a t½ of about 1 day to about 31 days, about 5 days to about 25 days, about 10 days to about 20 days, or even about 15 days. In other cases, when administered to humans, the INS analogs can have a t½ of about 1 to about 5 days, about 6 to about 10 days, about 11 to about 15 days, about 16 to about 20 days, about 21 to about 25 days, or even about 26 to about 31 days. In other cases, the INS analogs can have a t½ of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or even about 31 days or longer. In a particular case, when administered to humans, the INS analog can have a t½ of about 20 days.

[0321] Similarly, in some cases, when administered to humans, the INS analogs herein are from about 10-fold to about 1000-fold as potent as IR (such as native human IR-A or IR-B; SEQ ID NOs: 5 and 6, respectively) relative to, for example, native human INS (SEQ ID NOs: 3 and 4). In other cases, when administered to humans, the INS analogs herein are from about 25-fold to about 975-fold, about 50-fold to about 950-fold, about 75-fold to about 925-fold, about 100-fold to about 900-fold, about 125-fold to about 875-fold, 150-fold to about 850-fold, about 175-fold to about 825-fold, about 200-fold to about 800-fold, about 225-fold to about 775-fold, about 250-fold to about 750-fold, about 275-fold to about 725-fold, about 300-fold to about 700-fold, about 325-fold to about 675-fold, about 350-fold to about 650-fold, about 375-fold to about 625-fold, about 375-fold to about 600-fold, about 400-fold to about 575-fold, about 425-fold to about 550-fold, about 450-fold to about 500-fold, or about 475-fold as potent as IR (such as native human IR-A or IR-B; SEQ ID NOs: 5 and 6, respectively) relative to, for example, native human INS (SEQ ID NOs: 3 and 4). In other cases, when administered to humans, the INS analogs herein are from about 10-fold, about 25-fold, about 50-fold, about 75-fold, about 100-fold, about 125-fold, about 150-fold, about 175-fold, about 200-fold, about 225-fold, about 250-fold, about 275-fold, about 300-fold, about 325-fold, about 350-fold, about 375-fold, about 400-fold, about 425-fold, about 450-fold, about 475-fold, about 500-fold, about 525-fold, about 550-fold, about 575-fold, about 600-fold, about 625-fold, about 650-fold, about 675-fold, about 700-fold, about 725-fold, about 750-fold, about 775-fold, about 800-fold, about 825-fold, about 850-fold, about 875-fold, about 900-fold, about 925-fold, about 950-fold, about 975-fold, or about 1000-fold as potent as IR (such as native human IR-A or IR-B; SEQ ID NOs: 5 and 6, respectively) relative to, for example, native human INS (SEQ ID NOs: 3 and 4).

[0322] Drug compositions and kits

[0323] The INS analogs herein can be formulated into pharmaceutical compositions, which can be administered by parenteral routes (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or transdermal). Such pharmaceutical compositions and their preparation techniques are well known in the art. See, e.g., Remington, “The Science and Practice of Pharmacy” (ed. D.B. Troy, 21st ed., Lippincott, Williams & Wilkins, 2006). In certain cases, the INS analogs are administered subcutaneously or intravenously. However, alternatively, the INS analogs can be formulated in forms for other pharmaceutically acceptable routes, e.g., tablets or other solids for oral administration, timed-release capsules, and any other forms currently in use, including creams, lotions, inhalants, etc.

[0324] To improve their in vivo compatibility and effectiveness, the INS analogs herein can be reacted with any of a variety of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and the common techniques for preparing them are well known in the art (see, e.g., Stahl et al., “Handbook of Pharmaceutical Salts: Properties, Selection and Use” (2nd revised ed., Wiley-VCH, 2011)). Pharmaceutically acceptable salts for use herein include sodium salts, trifluoroacetates, hydrochlorides, and acetates.

[0325] The INS analogs herein can be administered by a physician or self-administered using an injection. It should be understood that those skilled in the art can readily determine the amounts of the gauge size and injection volume. However, the amount of the injection volume can be ≤ about 2 ml or even ≤ about 1 ml, and the needle gauge can be ≥ about 27 G or even ≥ about 29 G. Alternatively, the INS analogs herein can be administered by a pump system.

[0326] The present disclosure also provides and thus encompasses novel intermediates and methods that can be used to synthesize the INS analogs herein or their pharmaceutically acceptable salts. The intermediates and INS analogs can be prepared by a variety of techniques well known in the art. For example, methods using recombinant synthesis are illustrated in the following examples. The specific steps of each of the described techniques can be combined in different ways to prepare the INS analogs herein. The reagents and starting materials are readily available to those skilled in the art.

[0327] The INS analogs herein are generally effective over a wide dosage range. Exemplary dosages of the INS analogs or pharmaceutical compositions containing them can be in milligrams (mg), micrograms (µg), nanograms (ng), or picograms (pg) per kilogram (kg) of the individual. In this way, the daily dosage can be from about 1 µg to about 100 mg.

[0328] Here, the effective amount of the INS analog in the pharmaceutical composition can be a dosage from about 0.25 mg to about 5.0 mg. However, those skilled in the art understand that in certain cases, the effective amount (i.e., dosage / usage) may be less than the lower limit of the above range and still be sufficient, while in other cases, the effective amount may be a larger dosage and can be used with acceptable side effects.

[0329] In addition to the INS analogs, the pharmaceutical composition can further comprise at least one additional therapeutic agent, especially those commonly used as the standard of care for metabolic conditions, diseases, and disorders.

[0330] In this way, the pharmaceutical composition can comprise an effective amount of at least one INS analogue of SEQ ID NOs: 37 - 81, a pharmaceutically acceptable carrier, and optionally at least one additional therapeutic agent. For example, the pharmaceutical composition can comprise an effective amount of the INS analogue of SEQ ID NO: 37 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 38 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 39 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 40 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 41 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 42 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 43 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 44 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 45 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 46 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 47 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 48 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 49 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 50 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 51 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 52 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 53 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 54 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 55 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 56 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 57 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 58 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 59 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 60 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 61 and a pharmaceutically acceptable carrier, an effective amount of the INS analogue of SEQ ID NO: 62 and a pharmaceutically acceptable carrier, an effective amount of the SEQ IDThe INS analog of SEQ ID NO:63 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:64 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:65 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:66 and a pharmaceutically acceptable carrier, and an effective amount of the INS analog of SEQ ID NO:67 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:68 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:69 and a pharmaceutically acceptable carrier, and an effective amount of the INS analog of SEQ ID NO:70 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:71 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:72 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:73 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:74 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:75 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:76 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:77 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:78 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:79 and a pharmaceutically acceptable carrier, an effective amount of the INS analog of SEQ ID NO:80 and a pharmaceutically acceptable carrier, or an effective amount of the INS analog of SEQ ID NO:81 and a pharmaceutically acceptable carrier.

[0331] Alternatively, the INS analogs herein can be provided as part of a kit. In some cases, the kit includes means for administering to an individual at least one INS analog or a composition comprising the same (and optionally at least one additional therapeutic agent). In some cases, the kit includes a syringe and a needle for administering the INS analog or a composition comprising the same (and optionally at least one additional therapeutic agent). In certain cases, the INS analog or a composition comprising the same (and optionally at least one additional therapeutic agent) is pre-formulated in an aqueous solution within the syringe.

[0332] Methods for preparing and using insulin analogs

[0333] The INS analogs herein can be prepared by any number of standard recombinant DNA methods or standard chemical peptide synthesis methods known in the art. With respect to recombinant DNA methods, polynucleotides having nucleic acid sequences encoding the amino acid sequences of the INS analogs herein can be constructed using standard recombinant techniques, incorporated into recombinant expression vectors, and the vectors introduced into host cells such as bacteria, yeast, and mammalian cells to produce the INS analogs herein. See, e.g., Green and Sambrook, “Molecular Cloning: A Laboratory Manual” (4th ed., Cold Spring Harbor Laboratory Press, 2012).

[0334] With respect to recombinant DNA methods, the compounds herein can be prepared by generating protein or pre-protein molecules using recombinant DNA techniques. The DNA, including cDNA and synthetic DNA, can be double-stranded or single-stranded, and the coding sequences encoding the compounds herein may vary due to the redundancy or degeneracy of the genetic code. Briefly, the DNA sequences encoding the compounds herein are introduced into host cells to produce the compounds or their precursors. The host cells can be bacterial cells such as the K12 or B strains of Escherichia coli, fungal cells such as yeast cells, or mammalian cells such as Chinese hamster ovary (CHO) cells.

[0335] Appropriate host cells are transiently or stably transfected or transformed with an expression system such as an expression vector to produce the compounds or their precursors herein. Expression vectors generally replicate in host organisms either as episomes or as components of the host chromosomal DNA. Typically, the expression vectors will contain selectable markers such as tetracycline, neomycin, G418, and dihydrofolate reductase to permit selection of those cells transformed with the desired DNA sequence.

[0336] The specific biosynthetic or synthetic steps of each step described herein can be used, not used, or combined in different ways to prepare the compounds herein.

[0337] With respect to chemical peptide synthesis methods, standard manual or automated solid-phase synthesis procedures can be used. For example, automated peptide synthesizers are commercially available from, e.g., Applied Biosystems (Foster City, CA) and Protein Technologies Inc. (Tucson, AZ). Reagents for solid-phase synthesis are readily available from commercial sources. Solid-phase synthesizers can be used to block interfering groups, protect amino acids during the reaction, couple, deprotect, and cap unreacted amino acids according to the manufacturer's instructions. Additional details for the preparation of synthetic INS can be seen, e.g., Arai et al. (2018)Comm. Chem. 1:26; Belgi et al. (2011) Immun. Endoc. & Metab. Agents in Med. Chem. 11:40 - 47; Hossain and Wade (2017) Acc. Chem. Res. 50:2116 - 2127; and Liu et al. (2016) J. Pept. Sci. 22:260 - 270. See also, International Patent Application Publication No. WO 2011 / 031622.

[0338] One use of the INS analogs herein is for the treatment of metabolic conditions, diseases, and / or disorders. Exemplary conditions, diseases, and disorders include but are not limited to metabolic syndrome, diabetes, and obesity.

[0339] Another use of the INS analogs herein is for the treatment of cardiac and / or renal conditions, diseases, and / or disorders. Exemplary cardiac and / or renal conditions, diseases, and / or disorders include but are not limited to dyslipidemia, stroke, nephropathy, and retinopathy.

[0340] The method may include the steps described herein, and these steps may but need not be in the order described. However, other orders are conceivable. In addition, a single or multiple steps may be carried out in parallel and / or overlapping in time and / or individually or in steps repeated multiple times. In addition, the method may include additional, unspecified steps.

[0341] Thus, such a method may include selecting an individual having a metabolic condition, disease, or disorder or being predisposed thereto. Alternatively, the method may include selecting an individual having diabetes or being predisposed to diabetes. Alternatively, the method may include selecting an individual who is obese or prone to obesity. In some cases, the method may include selecting an individual having diabetes and obesity or being predisposed thereto.

[0342] The method may also include administering to the individual an effective amount of at least one INS analog herein, which may be in the form of a pharmaceutical composition as also described herein. In some cases, the INS analog / pharmaceutical composition may contain additional therapeutic agents such as DPP-IV inhibitors, native amylin or its analogs, short-acting (meal) INS analogs, native incretins or their analogs, native IGF or its analogs, metformin, SGLT2 inhibitors, inhibin, SU, TZD, and / or other antihyperglycemic agents or other anti-obesity agents, as well as other therapeutic agents for controlling comorbidities including but not limited to, high cholesterol, high triglycerides, hypertension, atrial fibrillation, and diabetes.

[0343] The concentrations / doses / usages of the INS analogs and optional additional therapeutic agents are discussed elsewhere herein.

[0344] With respect to the route of administration, the INS analog or a pharmaceutical composition comprising the same can be administered according to known methods, such as orally; by injection (i.e., intraarterial, intravenous, intraperitoneal, intracerebral, intraventricular, intramuscular, intraocular, intraportal or intralesional); by sustained release systems, or by implantable devices. In certain cases, the INS analog or a pharmaceutical composition comprising the same can be administered subcutaneously by bolus injection or continuously.

[0345] With respect to the dosing frequency, the INS analog or a pharmaceutical composition comprising the same can be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), every two weeks (i.e., every other week) or monthly. In certain cases, the INS analog or a pharmaceutical composition comprising the same is administered subcutaneously every other day, three times a week, twice a week, once a week, every other week or monthly. In certain specific cases, the INS analog or a pharmaceutical composition comprising the same is administered subcutaneously once a week (QW).

[0346] With respect to those cases in which the INS analog or a pharmaceutical composition comprising the same is administered in combination with an effective amount of at least one additional therapeutic agent, the additional therapeutic agent can be administered simultaneously with, separately from or sequentially to the INS analog or a pharmaceutical composition comprising the same.

[0347] Furthermore, the additional therapeutic agent can be administered at the same frequency as the INS analog or a pharmaceutical composition comprising the same (i.e., every other day, twice a week or even weekly). Alternatively, the additional therapeutic agent can be administered at a frequency different from that of the INS analog or a pharmaceutical composition comprising the same. In other cases, the additional therapeutic agent can be administered subcutaneously. In other cases, the additional therapeutic agent can be administered intravenously. In other cases, the additional therapeutic agent can be administered orally.

[0348] It is further contemplated that the method can be combined with diet and exercise, and / or can be combined with additional therapeutic agents other than those described above. Examples

[0349] The following non-limiting examples are provided for illustrative purposes and not for purposes of limitation.

[0350] Polypeptide expression

[0351] Example 1: Recombinant expression of INS analog 1

[0352] Example 1 is an INS analog having the following amino acid sequence:

[0353]

[0354] (SEQ ID NO:37).

[0355] Here, a CHOK1 cell derivative is used to produce the INS analogue of SEQ ID NO:37 in a mammalian cell expression system. The cDNA sequence encoding SEQ ID NO:37 is subcloned into an expression plasmid backbone containing GS (a plasmid based on pEE12.4). The cDNA sequence is in-frame fused with the coding sequence of the signal peptide sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO:82) to enhance the secretion of the INS analogue into the tissue culture medium. The expression is driven by the viral CMV promoter.

[0356] To generate the INS analogue by transient transfection, CHOK1 cells are transfected with the recombinant expression plasmid using a PEI-based method. Briefly, an appropriate volume of CHOK1 suspension cells at a density of 4 x 10 6 cells / ml are transferred to a shake flask, and PEI and the recombinant plasmid DNA are added to the cells. The cells are incubated in suspension culture at 32 °C for 6 days. At the end of the incubation period, the cells are removed by low-speed centrifugation, and the INS analogue is purified from the conditioned medium.

[0357] Alternatively, and to produce the INS analogue by stable transfection, CHOK1 cells are stably transfected using electroporation and an appropriate amount of the recombinant expression plasmid, and the transfected cells are maintained in suspension culture at an appropriate cell density. The selection of the transfected cells is accomplished by growing in serum-free medium containing 25 µM MSX and incubating at 35 °C - 37 °C and 5% - 7% CO2.

[0358] The INS analog secreted from CHO cells into the culture medium is purified by protein A affinity chromatography and subsequent ion exchange, hydrophobic interaction, or size exclusion chromatography. Specifically, the INS analog from the harvested culture medium is captured onto Mab Select Protein A resin (GE). The resin is then briefly washed with a running buffer such as phosphate buffered saline (PBS; pH 7.4) or a Tris-containing running buffer to remove nonspecifically bound materials. The protein is eluted from the resin using a low pH solution such as 10 mM citric acid, 150 mM NaCl pH 3. The fractions containing the INS analog are combined and then diluted 1:1 with 20 mM NaOAc pH 5. The final pH is adjusted to pH 5 using 1 M NaOH, and the solution can be kept at low pH to inactivate potential viruses. The pH can be neutralized by adding a base such as 0.1 M Tris pH 8.0 for subsequent size exclusion chromatography. The INS analog can be further purified by ion exchange chromatography using a resin such as POROS 50 HS (ThermoFisher). The INS analog is eluted from the column using a 0 mM to 500 mM NaCl gradient in 20 mM NaOAc (pH 5.0) over 15 column volumes.

[0359] The INS analog can be further purified by hydrophobic interaction chromatography using a Capto Phenyl ImpRes HIC column (GE Healthcare). Purification is carried out by adjusting the column loading solution to approximately 0.5 M Na2SO4 and eluting using a gradient of 0.5 M to 0 M Na2SO4 in 20 mM Tris pH 8 solution over 10 column volumes (CV). After HIC, the INS analog can even be further purified by SEC, where the concentrated Capto Phenyl ImpRes pool is loaded onto Superdex 200 (GE Healthcare) and eluted moderately in PBS pH 7.4 or in 20 mM histidine, 50 mM NaCl pH 6.0.

[0360] The purified INS analog can be passed through a virus retention filter such as Planova 20N (Asahi Kasei Medical), and then concentrated / diafiltered on a regenerated cellulose membrane (Millipore) by tangential flow ultrafiltration into 20 mM histidine, 20 mM NaCl pH 6.

[0361] Thus, the INS analog is prepared in this manner or in a similar manner readily determinable by those skilled in the art.

[0362] Example 2: Recombinant Expression of INS Analogue 2

[0363] Example 2 is an INS analogue having the following amino acid sequence:

[0364]

[0365] (SEQ ID NO:38).

[0366] Here, Example 2 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:38 was used in the expression plasmid.

[0367] Example 3: Recombinant Expression of INS Analogue 3

[0368] Example 3 is an INS analogue having the following amino acid sequence:

[0369]

[0370] (SEQ ID NO:39).

[0371] Here, Example 3 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:39 was used in the expression plasmid.

[0372] Example 4: Recombinant Expression of INS Analogue 4

[0373] Example 4 is an INS analogue having the following amino acid sequence:

[0374]

[0375] (SEQ ID NO:40).

[0376] Here, Example 4 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:40 was used in the expression plasmid.

[0377] Example 5: Recombinant Expression of INS Analogue 5

[0378] Example 5 is an INS analogue having the following amino acid sequence:

[0379]

[0380] (SEQ ID NO:41).

[0381] Here, Example 5 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:41 was used in the expression plasmid.

[0382] Example 6: Recombinant Expression of INS Analogue 6

[0383] Example 6 is an INS analogue having the following amino acid sequence:

[0384]

[0385] (SEQ ID NO:42).

[0386] Here, Example 6 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:42 was used in the expression plasmid.

[0387] Example 7: Recombinant Expression of INS Analogue 7

[0388] Example 7 is an INS analogue having the following amino acid sequence:

[0389]

[0390] (SEQ ID NO:43).

[0391] Here, Example 7 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:43 was used in the expression plasmid.

[0392] Example 8: Recombinant Expression of INS Analogue 8

[0393] Example 8 is an INS analogue having the following amino acid sequence:

[0394]

[0395] (SEQ ID NO:44).

[0396] Here, Example 8 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:44 was used in the expression plasmid.

[0397] Example 9: Recombinant Expression of INS Analogue 9

[0398] Example 9 is an INS analogue having the following amino acid sequence:

[0399]

[0400] (SEQ ID NO:45).

[0401] Here, Example 9 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:45 was used in the expression plasmid.

[0402] Example 10: Recombinant Expression of INS Analogue 10

[0403] Example 10 is an INS analogue having the following amino acid sequence:

[0404]

[0405] (SEQ ID NO:46).

[0406] Here, Example 10 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:46 was used in the expression plasmid.

[0407] Example 11: Recombinant Expression of INS Analogue 11

[0408] Example 11 is an INS analogue having the following amino acid sequence:

[0409]

[0410] (SEQ ID NO:47).

[0411] Here, Example 11 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:47 was used in the expression plasmid.

[0412] Example 12: Recombinant Expression of INS Analogue 12

[0413] Example 12 is an INS analogue having the following amino acid sequence:

[0414]

[0415] (SEQ ID NO:48).

[0416] Here, Example 12 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:48 was used in the expression plasmid.

[0417] Example 13: Recombinant Expression of INS Analogue 13

[0418] Example 13 is an INS analogue having the following amino acid sequence:

[0419]

[0420] (SEQ ID NO:49).

[0421] Here, Example 13 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:49 was used in the expression plasmid.

[0422] Example 14: Recombinant Expression of INS Analogue 14

[0423] Example 14 is an INS analogue having the following amino acid sequence:

[0424]

[0425] (SEQ ID NO:50).

[0426] Here, Example 14 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:50 was used in the expression plasmid.

[0427] Example 15: Recombinant Expression of INS Analogue 15

[0428] Example 15 is an INS analogue having the following amino acid sequence:

[0429]

[0430] (SEQ ID NO:51).

[0431] Here, Example 15 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:51 was used in the expression plasmid.

[0432] Example 16: Recombinant Expression of INS Analogue 16

[0433] Example 16 is an INS analogue having the following amino acid sequence:

[0434]

[0435] (SEQ ID NO:52).

[0436] Here, Example 16 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:52 was used in the expression plasmid.

[0437] Example 17: Recombinant Expression of INS Analogue 17

[0438] Example 17 is an INS analogue having the following amino acid sequence:

[0439]

[0440] (SEQ ID NO:53).

[0441] Here, Example 17 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:53 was used in the expression plasmid.

[0442] Example 18: Recombinant expression of INS analogue 18

[0443] Example 18 is an INS analogue having the following amino acid sequence:

[0444]

[0445] (SEQ ID NO:54).

[0446] Here, Example 18 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:54 was used in the expression plasmid.

[0447] Example 19: Recombinant expression of INS analogue 19

[0448] Example 19 is an INS analogue having the following amino acid sequence:

[0449]

[0450] (SEQ ID NO:55).

[0451] Here, Example 19 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:55 was used in the expression plasmid.

[0452] Example 20: Recombinant expression of INS analogue 20

[0453] Example 20 is an INS analogue having the following amino acid sequence:

[0454]

[0455] (SEQ ID NO:56).

[0456] Here, Example 20 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:56 was used in the expression plasmid.

[0457] Example 21: Recombinant expression of INS analogue 21

[0458] Example 21 is an INS analogue having the following amino acid sequence:

[0459]

[0460] (SEQ ID NO:57).

[0461] Here, Example 21 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:57 was used in the expression plasmid.

[0462] Example 22: Recombinant expression of INS analogue 22

[0463] Example 22 is an INS analogue having the following amino acid sequence:

[0464]

[0465] (SEQ ID NO:58).

[0466] Here, Example 22 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:58 was used in the expression plasmid.

[0467] Example 23: Recombinant expression of INS analogue 23

[0468] Example 23 is an INS analogue having the following amino acid sequence:

[0469]

[0470] (SEQ ID NO:59).

[0471] Here, Example 23 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:59 was used in the expression plasmid.

[0472] Example 24: Recombinant expression of INS analogue 24

[0473] Example 24 is an INS analogue having the following amino acid sequence:

[0474]

[0475] (SEQ ID NO:60).

[0476] Here, Example 24 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:60 was used in the expression plasmid.

[0477] Example 25: Recombinant expression of INS analogue 25

[0478] Example 25 is an INS analogue having the following amino acid sequence:

[0479]

[0480] (SEQ ID NO:61).

[0481] Here, Example 25 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:61 was used in the expression plasmid.

[0482] Example 26: Recombinant Expression of INS Analogue 26

[0483] Example 26 is an INS analogue having the following amino acid sequence:

[0484]

[0485] (SEQ ID NO:62).

[0486] Here, Example 26 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:62 was used in the expression plasmid.

[0487] Example 27: Recombinant Expression of INS Analogue 27

[0488] Example 27 is an INS analogue having the following amino acid sequence:

[0489]

[0490] (SEQ ID NO:63)

[0491] Here, Example 27 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:63 was used in the expression plasmid.

[0492] Example 28: Recombinant Expression of INS Analogue 28

[0493] Example 28 is an INS analogue having the following amino acid sequence:

[0494]

[0495] (SEQ ID NO:64).

[0496] Here, Example 28 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:64 was used in the expression plasmid.

[0497] Example 29: Recombinant Expression of INS Analogue 29

[0498] Example 29 is an INS analogue having the following amino acid sequence:

[0499]

[0500] (SEQ ID NO:65).

[0501] Here, Example 29 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:65 was used in the expression plasmid.

[0502] Example 30: Recombinant expression of INS analogue 30

[0503] Example 30 is an INS analogue having the following amino acid sequence:

[0504]

[0505] (SEQ ID NO:66).

[0506] Here, Example 30 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:66 was used in the expression plasmid.

[0507] Example 31: Recombinant expression of INS analogue 31

[0508] Example 31 is an INS analogue having the following amino acid sequence:

[0509]

[0510] (SEQ ID NO:67).

[0511] Here, Example 31 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:67 was used in the expression plasmid.

[0512] Example 32: Recombinant expression of INS analogue 32

[0513] Example 32 is an INS analogue having the following amino acid sequence:

[0514]

[0515] (SEQ ID NO:68).

[0516] Here, Example 32 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:68 was used in the expression plasmid.

[0517] Example 33: Recombinant expression of INS analogue 33

[0518] Example 33 is an INS analog having the following amino acid sequence:

[0519]

[0520] (SEQ ID NO:69).

[0521] Here, Example 33 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:69 was used in the expression plasmid.

[0522] Example 34: Recombinant Expression of INS Analog 34

[0523] Example 34 is an INS analog having the following amino acid sequence:

[0524]

[0525] (SEQ ID NO:70).

[0526] Here, Example 34 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:70 was used in the expression plasmid.

[0527] Example 35: Recombinant Expression of INS Analog 35

[0528] Example 35 is an INS analog having the following amino acid sequence:

[0529]

[0530] (SEQ ID NO:71).

[0531] Here, Example 35 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:71 was used in the expression plasmid.

[0532] Example 36: Recombinant Expression of INS Analog 36

[0533] Example 36 is an INS analog having the following amino acid sequence:

[0534]

[0535] (SEQ ID NO:72).

[0536] Here, Example 36 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:72 was used in the expression plasmid.

[0537] Example 37: Recombinant Expression of INS Analogue 37

[0538] Example 37 is an INS analogue having the following amino acid sequence:

[0539]

[0540] (SEQ ID NO:73).

[0541] Here, Example 37 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:73 was used in the expression plasmid.

[0542] Example 38: Recombinant Expression of INS Analogue 38

[0543] Example 38 is an INS analogue having the following amino acid sequence:

[0544]

[0545] (SEQ ID NO:74).

[0546] Here, Example 38 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:74 was used in the expression plasmid.

[0547] Example 39: Recombinant Expression of INS Analogue 39

[0548] Example 39 is an INS analogue having the following amino acid sequence:

[0549]

[0550] (SEQ ID NO:75).

[0551] Here, Example 39 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:75 was used in the expression plasmid.

[0552] Example 40: Recombinant Expression of INS Analogue 40

[0553] Example 40 is an INS analogue having the following amino acid sequence:

[0554]

[0555] (SEQ ID NO:76).

[0556] Here, Example 40 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:76 was used in the expression plasmid.

[0557] Example 41: Recombinant Expression of INS Analogue 41

[0558] Example 41 is an INS analogue having the following amino acid sequence:

[0559]

[0560] (SEQ ID NO:77).

[0561] Here, Example 41 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:77 was used in the expression plasmid.

[0562] Example 42: Recombinant Expression of INS Analogue 42

[0563] Example 42 is an INS analogue having the following amino acid sequence:

[0564]

[0565] (SEQ ID NO:78).

[0566] Here, Example 42 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:78 was used in the expression plasmid.

[0567] Example 43: Recombinant Expression of INS Analogue 43

[0568] Example 43 is an INS analogue having the following amino acid sequence:

[0569]

[0570] (SEQ ID NO:79).

[0571] Here, Example 43 was produced substantially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:79 was used in the expression plasmid.

[0572] Example 44: Recombinant Expression of INS Analogue 44

[0573] Example 44 is an INS analogue having the following amino acid sequence:

[0574]

[0575] (SEQ ID NO:80).

[0576] Here, Example 44 was generated essentially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:80 was used in the expression plasmid.

[0577] Example 45: Recombinant Expression of INS Analogue 45

[0578] Example 45 is an INS analogue having the following amino acid sequence:

[0579]

[0580] (SEQ ID NO:81).

[0581] Here, Example 45 was generated essentially as described for Example 1, except that the cDNA sequence encoding SEQ ID NO:81 was used in the expression plasmid.

[0582] In Vitro Function

[0583] Example 46: INS Analogue Albumin Binding Study by SPR

[0584] The in vitro binding of various INS analogues to human, cynomolgus monkey, mouse, rat, pig, dog, bovine, and rabbit serum albumin was determined by SPR. In particular, the affinities of Examples 23 to 30 for the serum albumin of these species are summarized in Table 1-8 below.

[0585] The binding of the INS analogs of Examples 23 to 30 to various serum albumins was carried out on a Biacore 8K instrument. Serum albumin was immobilized on the surface of the S series sensor chip CM5 according to the manufacturer's instructions (Amine Coupling Kit BR-1000-50). Briefly, carboxyl groups on the surface of the sensor chip (flow cells 1 and 2) were activated by injecting a 70 μL mixture containing 75 mg / ml EDC and 11.5 mg / ml NHS at 10 μL / min. Human, cynomolgus monkey, mouse, rat, pig, dog, bovine, and rabbit serum albumins were diluted in 10 mM NaOAc pH 4.0 (BR-1003-49) at 1, 1, 3, 1, 1, 1, 1, and 1 μg / mL, respectively, and then injected onto the activated chip surface (flow cell 2, channels 1-7) at 10 μL / min for 90 seconds (human, mouse, rat, pig, and bovine serum albumins were obtained from Sigma Aldrich (St. Louis, MO); cynomolgus monkey serum albumin was obtained from Hölzel Diagnostika (Cologne, Germany); dog serum albumin was obtained from Molecular Innovations (Novi, MI); and rabbit serum albumin was obtained from Fitzgerald Industries International (Acton, MA)). The various serum albumins were covalently immobilized to the carboxymethyl dextran-coated sensor chip CM5 via free amines, with a target average surface density of approximately 77 (58-98) RU. The excess reactive groups on the surface (flow cells 1 and 2) were inactivated by injecting 70 μL of 1 M ETA HCl-NaOH pH 8.5 at 10 μL / min.

[0586] Examples 23 - 30 were diluted in HBS - EP+ buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20) at concentrations of 1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.457, 0.152, 0.051, and 0.017 nM. 150 μL of the sample was sequentially injected individually over the immobilized serum albumin surface and then dissociated for 600 seconds at a flow rate of 50 μL / min at 25°C. The surface was regenerated by injecting 10 mM glycine - HCl pH 1.5 (BR - 1003 - 54) for 100 seconds at 50 μL / min. The resulting sensorgrams were analyzed by fitting to a 1:1 binding kinetics or steady - state affinity model using Biacore 8K Insight evaluation software (version 2.0.15.12933) to calculate the binding kinetic parameters association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K D ).

[0587] Table 1: Binding kinetics of Example 23 with human, cynomolgus monkey, mouse, rat, pig, dog, bovine, and rabbit serum albumin at 25°C

[0588]

[0589] For the binding of human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin to Example 23, K D was determined to be 0.73, 6.8, 76, 53, 130, 23, and 590 nM, respectively.

[0590] Table 2: Binding kinetics of Example 24 with human, cynomolgus monkey, mouse, rat, pig, dog, bovine, and rabbit serum albumin at 25°C

[0591]

[0592] For the binding of human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin to Example 24, K D was determined to be 0.91, 5.5, 49, 40, 100, 16, and 430 nM, respectively.

[0593] Table 3: Binding kinetics of Example 25 with human, cynomolgus monkey, mouse, rat, pig, dog, bovine, and rabbit serum albumin at 25°C

[0594]

[0595] For the binding of human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin to Example 25, K DThey were determined to be 0.32, 3.5, 42, 34, 90, 16, and 390 nM, respectively.

[0596] Table 4: Binding kinetics of Example 26 with human, cynomolgus monkey, mouse, rat, pig, dog, bovine, and rabbit serum albumins at 25 °C

[0597]

[0598] For the binding of human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumins to Example 26, K D They were determined to be 0.49, 3.9, 35, 32, 87, 13, and 400 nM, respectively.

[0599] Table 5: Binding kinetics of Example 27 with human, cynomolgus monkey, mouse, rat, pig, dog, bovine, and rabbit serum albumins at 25 °C

[0600]

[0601] For the binding of human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumins to Example 27, K D They were determined to be 0.75, 4.7, 45, 32, 90, 17, and 390 nM, respectively.

[0602] Table 6: Binding kinetics of Example 28 with human, cynomolgus monkey, mouse, rat, pig, dog, bovine, and rabbit serum albumins at 25 °C

[0603]

[0604] For the binding of human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumins to Example 28, K D They were determined to be 0.73, 4.1, 37, 26, 75, 12, and 400 nM, respectively.

[0605] Table 7: Binding kinetics of Example 29 with human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumins at 25 °C

[0606]

[0607] For the binding of human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumins to Example 29, K D They were determined to be 0.74, 4.4, 48, 32, 86, 16, 380 nM.

[0608] Table 8: Binding kinetics of Example 30 with human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumins at 25 °C

[0609]

[0610] For the binding of human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumins to Example 30, the K D was determined to be 0.73, 4.3, 39, 30, 80, 13, and 370 nM, respectively.

[0611] Example 47: In Vitro Potency of INS Analogs on IR-A and IR-B

[0612] Preparation of membranes: Cell membranes were prepared from HEK293 cells stably transfected with human IR-A (hIR-A; SEQ ID NO:5) and human IR-B (hIR-B; SEQ ID NO:6) containing a C-terminal C9 tag (TETSQVAPA; SEQ ID NO:83). Typically, cell pellets were from passages 6 to 12, depending on the receptor. The frozen cell pellets were thawed in ice-cold homogenization / resuspension buffer (50 mM Tris-HCl, pH 7.5) containing 1 tablet of Complete® protease inhibitor and EDTA (Roche Diagnostics) per 50 mL of buffer. The cells were homogenized using a Teflon®-glass Potter-Elvehjem homogenizer driven by a high-speed motor, using 15 to 20 strokes, and then centrifuged at 1100 x g for 10 min at 4°C. The supernatant was kept on ice, and the pellet was re-homogenized as before and centrifuged at 1100 x g for 10 min at 4°C. All supernatants were combined and then centrifuged at 35,000 x g for 60 min at 4°C. The pellet was resuspended in buffer containing protease inhibitor (4 - 5 ml / g of starting cell paste) and flash-frozen in liquid nitrogen before storage at -80°C. Protein concentration was determined using a BCA kit (ThermoScientific) with bovine serum albumin (BSA) as the standard.

[0613] Receptor binding assay protocol: By competitive radioligand binding assay using human recombinant (3- 125 I]-iodotyrosyl-A14)-insulin (2200 Ci / mmol) or human recombinant 125I]-Insulin-like growth factor-1 (1680-2800 Ci / mmol) was used to determine the receptor binding affinity (Ki). The SPA method was used for the assay using polyvinyltoluene (PVT) wheat germ agglutinin-conjugated SPA beads (Perkin Elmer). The assay buffer contained 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and any one of the following: A) 0.1% w / v fatty acid-free BSA; B) 0.1% w / v fatty acid-free human serum albumin (HSA); C) 0.1% w / v rat serum albumin (RSA); or D) 0.001% Nonidet P-40 Substitute (NP-40, Roche Diagnostics). A ten-point concentration-response curve of triplicate serial dilutions of the test sample or control was prepared in the assay buffer using a Freedom / Evo robot (Tecan). 50 μL of the compound diluent was added to a 96-well white clear-bottom microplate (Corning, 3632) using a TeMO robot (Tecan), and then the radioligand (50 μL, final ~40 pM), membrane (50 μL, 0.1-0.4 μg / well), and SPA beads (50 μL, 0.1-0.15 mg / well) were added using a Multiflo F / X (Biotek) bulk dispensing instrument. The highest final assay concentrations of the test compounds and controls are shown in the table below:

[0614] Table 9: Receptor Binding Assay Concentrations / Controls

[0615] Highest final assay concentration (nM) Compound or control IR-A / IR-B binding Compound X 10000 - 15000 BHI 100 IGF-1 1000 AspB10 INS 40

[0616] After incubation at room temperature for 10 hours and sedimentation of the beads, radioactivity was measured using a Microbeta™ Trilux scintillation counter (PerkinElmer) and expressed as counts per minute (CPM).

[0617] Samples (n = 3) were tested in three independent assays run on three different days. For each run, the samples were randomized with the BHI (SEQ ID NO:3 and 4), IGF-1 (PeproTech, Inc.; Rocky Hill, NJ), and AspB10 INS (His10Asp of SEQ ID NO:4) controls included on each plate.

[0618] Data analysis of IR assay: Each experiment tested each compound with a single replicate concentration-response curve. The maximum binding response (MAX) was determined using only the assay buffer at 8 wells / plate, and the minimum binding or non-specific response (MIN) was determined in each well using 100 nM BHI. All test sample concentration-responses were normalized to this control response and calculated as the percentage of specific inhibition after correcting for non-specific binding as follows:

[0619] % Specific inhibition = 100 - [(CPM - MIN) / (MAX - MIN) x 100].

[0620] The percentage of specific inhibition (y-axis) was plotted against the log concentration of the compound (x-axis). The concentration resulting in 50% binding inhibition (IC 50 ) was determined by four-parameter logistic non-linear regression analysis (Analyzer, version 15, GeneData Screener). The affinity constant (Ki) was calculated from the IC 50 value based on the equation Ki = IC 50 / (1 + L / Kd), where L is equal to the concentration of the radioligand used in the experiment and Kd is equal to the equilibrium binding affinity constant of the radioligand determined from saturation binding analysis. The reported Ki values were shown as geometric means and standard errors (standard error by the Delta method), and the number of independent replicate assays was used to calculate the geometric mean indicated by n.

[0621] Table 10: In vitro potency of Examples 1 - 30 against IR-A

[0622]

[0623] Table 11: In vitro potency of Examples 1 - 30 against IR-B

[0624]

[0625] In vivo function

[0626] Example 48: INS analogs lower glucose in streptozotocin (STZ)-treated mice

[0627] STZ mice: Male C57Bl / 6NHsd mice, 11 - 12 weeks old, from Envigo RSM Inc. (Indianapolis, IN) were allowed to acclimatize for at least 3 days. The mice were housed individually in shoebox cages with corncob bedding and a mouse waterer. Environmental conditions were as follows: a 12-hour light and 12-hour dark photoperiod (which may be interrupted due to study-related activities), a temperature of 20°C to 26°C, and a relative humidity of 30% to 70%.

[0628] STZ was prepared as follows: A vehicle was added to pre-weighed STZ to achieve a dosing concentration of 16.67 mg / mL. It was gently vortexed to mix until the powder dissolved. The solution was kept on wet ice, protected from light and used within 3 hours after preparation. On days 5 and 9 of the pre-dosing phase, before each STZ administration, after an overnight fast (not exceeding 16 hours), the animals were administered intraperitoneally at a dose volume of 6 mL / kg (100 mg / kg) based on the most recent body weight. Any animal with a body weight below 19 grams was not administered STZ.

[0629] Ten days after the second STZ treatment, animals were assigned to the study using a Block Randomization Allocation Tool (BRAT) designed to achieve a blood glucose meter value (inclusion criteria of 250 mg / dL to 500 mg / dL) and body weight balance. A single dose of the pre-formulated test article was administered subcutaneously (interscapular) into the space between the scapulae at a dose volume of 10 mL / kg. Any possible dosing errors were recorded.

[0630] After treatment with the test article, body weight was monitored every morning. Food intake was monitored on day 1 (three times: 0 to 4 hours, 4 to 12 hours, and 12 to 24 hours), days 2, 3, 4, 5, 6, 7, and 8. Glucose was measured using a blood glucose meter (in duplicate) by tail clip at 0, 4, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours after dosing. Another 40 µL of whole blood was collected at 4, 12, 24, 36, 48, and 72 hours after dosing for determination of compound concentration.

[0631] All data were expressed as mean ± SEM for 5 animals per group. The percentage change in glucose at each time point was calculated as follows: Percentage change at X hours after dosing = (((Glucose at X time point - Glucose of the animal at time 0 after dosing) x 100) - 100).

[0632] As shown in Table 12 below, after a single injection of 300 nmol / kg, the INS analogs of Examples 1 to 6 showed a sustained decrease in whole blood glucose levels. Similarly, as shown in Tables 13 and 14 below, after a single injection of 200 nmol / kg, the INS analogs of Examples 7 to 18 showed a sustained decrease in whole blood glucose levels.

[0633]

[0634]

[0635]

[0636] Example 49: INS Analogue Reduces Glucose in STZ-Treated Rats

[0637] Male Sprague-Dawley rats weighing 390 - 425 g from Envigo RMS Inc. (Indianapolis, IN) were allowed to acclimate for at least 3 days. The rats were individually housed in shoebox cages with corncob bedding and a water bottle with free access to water. The rats were fed Teklad Global Diets' Rodent 2014 diet. Environmental conditions were as follows: a 12-hour light and 12-hour dark photoperiod (which may be interrupted for study-related activities), a temperature of 20°C to 26°C, and a relative humidity of 30% to 70%.

[0638] STZ was prepared as follows: 19 mL of cold sterile saline was added to the STZ vial (Zanosar®, Teva Parenteral Medicines, Inc., Irvine, CA) and gently mixed until the powder dissolved. The second vial of STZ was repeated, and both were placed on wet ice and protected from light. These solutions remained good for 3 hours under these conditions. On day 8 of the pre-dose phase, after a 6-hour fast, the animals were dosed intravenously at a dose volume of 0.8 mL / kg (40 mg / kg) based on the most recent body weight. STZ administration was performed under isoflurane anesthesia. The rats were observed until fully awake.

[0639] Three days after STZ treatment, animals were assigned to the study using BRAT aimed at achieving blood glucose meter values (450 mg / dL to 550 mg / dL inclusion criteria) and body weight balance. 50 out of 60 rats were included in the study. A single dose of the pre-formulated test article (50, 100, 200, and 400 nmol / kg of the INS analogue in 20 mM histidine, 50 mM NaCl pH 6.0) was administered into the subcutaneous space at a dose volume of 5 mL / kg.

[0640] After test article treatment, body weight and food intake were monitored every morning (days 1 - 11 of the dosing phase). Glucose was measured by tail bleeding using a blood glucose meter (AccuChek® Aviva®, Roche, Indianapolis, IN) (in duplicate) at 0, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, and 240 hours post-dose.

[0641] All data were expressed as mean ± SEM per group of 4 - 5 animals. The percent change in glucose was calculated at each time point as follows: percent change at X hours post - dosing = (((glucose at X time point - glucose of the animal at time 0 post - dosing) x 100) - 100).

[0642] As shown in Table 15 below, after a single injection, all four INS analogs exhibited a dose - dependent (50, 100, 200, and 400 nmol / kg) decrease in whole - blood glucose levels.

[0643]

[0644]

[0645]

[0646]

[0647] Example 50: Pharmacokinetics of INS Analogs in STZ - Treated Rats

[0648] The pharmacokinetics of the INS analogs herein were tested in a STZ - induced diabetic rat model. Male rats treated with STZ were administered a single subcutaneous dose of various INS analogs at 50, 100, 200, or 400 nmol / kg (5 mL / kg dose, in 20 mM histidine, 50 mM NaCl pH 6.0). Blood was collected from each animal before dosing and at 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, and 240 hours post - dosing. Blood samples were processed into K3EDTA plasma and stored frozen at approximately - 70°C. The INS analog concentration in plasma was measured at Eli Lilly and Company (Indianapolis, IN), and pharmacokinetic parameters were calculated using the concentration - time data (see, Table 12).

[0649] The plasma analogue concentration of animals was measured by immunoaffinity-LC / MS using a Thermo orbitrap mass spectrometer (Q / Exactive or Fusion Lumos) coupled with a Dionex Ultimate 3000 UPLC system. The analogue was immunoprecipitated from K3EDTA rat plasma using an anti-camelid-VHH-biotin monoclonal antibody (Eli Lilly and Company, clone 96A3F5) immobilized to streptavidin-coated magnetic beads (Dynal M-280, Thermo E2017-02). After a washing step to remove nonspecifically bound proteins, the variant was reduced (triethylphosphine, Aldrich 245275-5G), alkylated (2-iodoethanol, Aldrich 176850-25G), and digested (Trypsin Gold, Promega E2019-12). The subsequent tryptic peptides from different regions of the variant were measured by LC / MS in the range of 0.293 nM to 150 nM as an alternative measurement of the intact analogue.

[0650] The pharmacokinetics of Examples 29 and 30 were approximately linear within the tested dose range (50 - 400 nmol / kg subcutaneous). The apparent clearance rates of the two analogues ranged from 2.9 mL / hr / kg to 4.7 mL / hr / kg, and their elimination half-lives ranged from 25 hours to 42 hours (see, Table 17).

[0651] Table 17: Mean pharmacokinetic parameters of the INS analogues of Examples 29 and 30 after a single 50, 100, 200, or 400 nmol / kg subcutaneous dose in STZ-treated male rats

[0652]

[0653] Mean ± (SD), N = 5

[0654] Abbreviation: AUC 0-∞ = Area under the curve from time 0 hours to infinity, CL / F = Clearance / Bioavailability, T max = Time to reach maximum concentration, C max / Dose = Observed maximum plasma concentration divided by dose, t 1 / 2 = Half-life.

[0655] Sequence

[0656] The following nucleic acid and / or amino acid sequences are mentioned in this disclosure and are provided below for reference.

[0657] SEQ ID NO:1 - Preproinsulin (110 amino acids; NCBI Ref. No. NP_001278826.1)

[0658]

[0659] SEQ ID NO:2 - Proinsulin (86 amino acids; 25 - 110 of NCBI Ref. No. NP_001278826.1)

[0660]

[0661] SEQ ID NO:3 - Human INS A chain (21 amino acids; 90 - 110 of NCBI Ref. No. NP_001278826.1)

[0662]

[0663] SEQ ID NO:4 - Human INS B chain (30 amino acids; 25 - 54 of NCBI Ref. No. NP_001278826.1)

[0664]

[0665] SEQ ID NO:5–Human INS receptor - A with C - terminal C9 tag (1370 amino acids; NCBI Ref. No. NP_001073285.1)

[0666]

[0667]

[0668] SEQ ID NO:6–Human INS receptor - B with C - terminal C9 tag (1382 amino acids; NCBI Ref. No. NP_000199.2)

[0669]

[0670]

[0671] SEQ ID NO:7 - VHH portion #1 (MC6.1C22.43)

[0672]

[0673] SEQ ID NO:8 - VHH portion #2 (MC6.1)

[0674]

[0675] SEQ ID NO:9 - VHH portion #3 (MC6.1C80.43)

[0676]

[0677] SEQ ID NO:10 - L1 ((GGGGQ) n basic sequence)

[0678]

[0679] SEQ ID NO:11 - L1 ((GGGQ) n basic sequence)

[0680]

[0681] SEQ ID NO:12 - L1 ((GGGGS) n basic sequence)

[0682]

[0683] SEQ ID NO:13 - L1 ((PGPQ) n basic sequence)

[0684]

[0685] SEQ ID NO:14 - L1 ((PGPA) n basic sequence)

[0686]

[0687] SEQ ID NO:15 - L1 ((GGE) n basic sequence of GG)

[0688]

[0689] SEQ ID NO:16 - L1 ((GGGGE) n basic sequence of GGGG)

[0690]

[0691] SEQ ID NO:17 - L1 ((GGGGK) n basic sequence of GGGG)

[0692]

[0693] SEQ ID NO:18 - L1 ((GGGG(AP) n (basic sequence of GGGG)

[0694]

[0695] SEQ ID NO:19 - L1 (GGGG(EP) n (basic sequence of

[0696]

[0697] SEQ ID NO:20 - L1 (GGGG(KP) n (basic sequence of GGGG)

[0698]

[0699] SEQ ID NO:21 - L1 ((PGPE) n (basic sequence of PGPQ)

[0700]

[0701] SEQ ID NO:22 - L1 ((PGPK) n (basic sequence of PGPQ)

[0702]

[0703] SEQ ID NO:23 - L1 #1 ((GGGGQ)5)

[0704]

[0705] SEQ ID NO:24 - L1 #2 ((PGPQ)8)

[0706]

[0707] SEQ ID NO:25 - L1 #3 ((PGPA)8)

[0708]

[0709] SEQ ID NO:26 - L1 #4 (G2E)7G2

[0710]

[0711] SEQ ID NO:27 - L1 #5 (G4E)4G4

[0712]

[0713] SEQ ID NO:28 - L1 #6 (G4K)4G4

[0714]

[0715] SEQ ID NO:29 - L1 #7 (G4(AP) 10 G4)

[0716]

[0717] SEQ ID NO:30 - L1 #7 (G4(EP) 10 G4)

[0718]

[0719] SEQ ID NO:31 - L1 #8 (G4(KP) 10 G4)

[0720]

[0721] SEQ ID NO:32 - L1 #9 ((PGPE)7PGPQ)

[0722]

[0723] SEQ ID NO:33 - L1 #10 ((PGPK)7PGPQ)

[0724]

[0725] SEQ ID NO:34 - L2 #1

[0726]

[0727] SEQ ID NO:35 - L2 #2

[0728]

[0729] SEQ ID NO:36 - L2 #3

[0730]

[0731] SEQ ID NO:37 - INS Analogue #1 (SCI(A21G)-(PGPA)8-MC6.1)

[0732]

[0733] SEQ ID NO:38 - INS Analogue #2 (SCI(B3D,A21G)-(PGPA)8-MC6.1)

[0734]

[0735] SEQ ID NO:39 - INS Analogue #3 (SCI(B9A,A21G)-(PGPA)8-MC6.1)

[0736]

[0737] SEQ ID NO:40 - INS Analogue #4 (SCI(B16E,A21G)-(PGPA)8-MC6.1)

[0738]

[0739] SEQ ID NO:41 - INS Analogue #5 (SCI(B16H,A21G)-(PGPA)8-MC6.1)

[0740]

[0741] SEQ ID NO:42 - INS Analogue #6 (SCI(B25H,A21G)-(PGPA)8-MC6.1)

[0742]

[0743] SEQ ID NO:43 - INS Analogue #7 (SCI(B3S,A21G)-(PGPA)8-MC6.1C22.43)

[0744]

[0745] SEQ ID NO:44 - INS Analogue #8 (SCI(B3S,B25H,A21G)-(PGPA)8-MC6.1C22.43)

[0746]

[0747] SEQ ID NO:45 - INS analog #9 (SCI(B3S,B25H,A8H,A21G)-(PGPA)8-MC6.1C22.43)

[0748]

[0749] SEQ ID NO:46 - INS analog #10 (SCI(B3S,B16H,A21G)-(PGPA)8-MC6.1C22.43)(SCIv5-(PGPA)8-C22.43)

[0750]

[0751] SEQ ID NO:47 - INS analog #11 (SCI(B3S,B16H,A8H,A14E,A21G)-(PGPA)8-MC6.1C22.43)

[0752]

[0753] SEQ ID NO:48 - INS analog #12 (SCI(B3S,B16H,B25H,A8H,A21G)-(PGPA)8-MC6.1C22.43)

[0754]

[0755] SEQ ID NO:49 - INS analog #13 (SCI(B3S,B16H,B25H,A8H,A14E,A21G)-(PGPA)8-MC6.1C22.43)

[0756]

[0757] SEQ ID NO:50 - INS analog #14 (SCI(B3S,B16R,A21G)-(PGPA)8-MC6.1C22.43)

[0758]

[0759] SEQ ID NO:51 - INS analog #15 (SCI(B3S,B16F,A21G)-(PGPA)8-MC6.1C22.43)

[0760]

[0761] SEQ ID NO:52 - INS Analogue #16 (SCI(B3S,B16W,A21G)-(PGPA)8-MC6.1C22.43)

[0762]

[0763] SEQ ID NO:53 - INS Analogue #17 (SCI(B3S,B16R,A8H,A21G)-(PGPA)8-MC6.1C22.43)

[0764]

[0765] SEQ ID NO:54 - INS Analogue #18 (SCI(B3S,B16R,B25H,A8H,A21G)-(PGPA)8-MC6.1C22.43)

[0766]

[0767] SEQ ID NO:55 - INS Analogue #19 (SCI(B3S,A21G)-(G4Q)5-MC6.1C22.43)

[0768]

[0769] SEQ ID NO:56 - INS Analogue #20 (SCI(B3S,A21G)-(PGPQ)8-MC6.1C22.43)

[0770]

[0771] SEQ ID NO:57 - INS Analogue #21 (SCI(B3S,B16H,B25H,A8H,A21G)-(G4Q)5-C22.43)

[0772]

[0773] SEQ ID NO:58 - INS Analogue #22 (SCI(B3S,B16H,B25H,A8H,A21G)-(PGPQ)8-MC6.1C22.43)

[0774]

[0775] SEQ ID NO:59 - INS analog #23 (SCI(B3S,B16H,B25H,A8H,A14E,A21G)-(G4Q)5-MC6.1C22.43)

[0776]

[0777] SEQ ID NO:60 - INS analog #24 (SCI(B3S,B16H,B25H,A8H,A14E,A21G)-(PGPQ)8-MC6.1C22.43)

[0778]

[0779] SEQ ID NO:61 - INS analog #25 SCI(B3S,B16R,A21G)-(G4Q)5-MC6.1C22.43)

[0780]

[0781] SEQ ID NO:62 - INS analog #26 SCI(B3S,B16R,A21G)-(PGPQ)8-MC6.1C22.43)

[0782]

[0783] SEQ ID NO:63 - INS analog #27 (SCI(B3S,B16R,A8H,A21G)-(G4Q)5-MC6.1C22.43)

[0784]

[0785] SEQ ID NO:64 - INS analog #28 (SCI(B3S,B16R,A8H,A21G)-(PGPQ)8-MC6.1C22.43)

[0786]

[0787] SEQ ID NO:65 - INS analog #29 (SCI(B3S,B16R,B25H,A8H,A21G)-(G4Q)5-MC6.1C22.43)

[0788]

[0789] SEQ ID NO:66 - INS Analogue #30 (SCI(B3S,B16R,B25H,A8H,A21G)-(PGPQ)8-MC6.1C22.43)

[0790]

[0791] SEQ ID NO:67 - INS Analogue #31 (SCI(B3S,B16R,B25H,A8H,A21G)-(G2E)7G2-MC6.1C22.43)

[0792]

[0793] SEQ ID NO:68 - INS Analogue #32 (SCI(B3S,B16R,B25H,A8H,A21G)-(G4E)4G4-MC6.1C22.43)

[0794]

[0795] SEQ ID NO:69 - INS Analogue #33 (SCI(B3S,B16R,B25H,A8H,A21G)-(G4K)4G4-MC6.1C22.43)

[0796]

[0797] SEQ ID NO:70 - INS Analogue #34 (SCI(B3S,B16R,B25H,A8H,A21G)-G4(AP)10G4-MC6.1C22.43)

[0798]

[0799] SEQ ID NO:71 - INS Analogue #35 (SCI(B3S,B16R,B25H,A8H,A21G)-G4(EP)10G4-MC6.1C22.43)

[0800]

[0801] SEQ ID NO:72 - INS Analogue #36 (SCI(B3S,B16R,B25H,A8H,A21G)-G4(KP)10G4-MC6.1C22.43)

[0802]

[0803] SEQ ID NO:73 - INS analog #37 (SCI(B3S,B16R,B25H,A8H,A21G)-(PGPE)7PGPQ-MC6.1C22.43)

[0804]

[0805] SEQ ID NO:74 - INS analog #38 (SCI(B3S,B16R,B25H,A8H,A21G)-(PGPK)7PGPQ-MC6.1C22.43)

[0806]

[0807] SEQ ID NO:75 - INS analog #39 (SCI(B3K,B16R,B25H,A8H,A21G)-G4Q)5-MC6.1C22.43)

[0808]

[0809] SEQ ID NO:76 - INS analog #40 (SCI(B3K,B16R,B21Q,B25H,A8H,A21G)-G4Q)5-MC6.1C22.43)

[0810]

[0811] SEQ ID NO:77 - INS analog #41

[0812] (SCI(B3K,B16R,B21Q,B25H,A4Q,A8H,A21G)-G4Q)5- MC6.1C22.43)

[0813]

[0814] SEQ ID NO:78 - INS analog #42

[0815] (SCI(B3S,B16R,B25H,A8H,A21G)-(G4Q)5-MC6.1C80.43)

[0816]

[0817] SEQ ID NO:79 - INS analog #43

[0818] (SCI(B3K,B16R,B25H,A8H,A21G)-(G4Q)5-MC6.1C80.43)

[0819]

[0820] SEQ ID NO:80 - INS analog #44

[0821] (SCI(B3K,B16R,B21Q,B25H,A8H,A21G)-(G4Q)5-MC6.1C80.43)

[0822]

[0823] SEQ ID NO:81 - INS analog #45

[0824] (SCI(B3K,B16R,B21Q,B25H,A4Q,A8H,A21G)-(G4Q)5-MC6.1C80.43)

[0825]

[0826] SEQ ID NO:82 - Signal peptide

[0827]

[0828] SEQ ID NO:83 - C-terminal C9 tag

[0829]

[0830] SEQ ID NO:84 (CDR1 #1)

[0831]

[0832] SEQ ID NO:85 (CDR1 #2)

[0833]

[0834] SEQ ID NO:86 (CDR1 #3)

[0835]

[0836] SEQ ID NO:87 (CDR2 #1)

[0837]

[0838] SEQ ID NO:88 (CDR2 #2)

[0839]

[0840] SEQ ID NO:89 (CDR2 #3)

[0841]

[0842] SEQ ID NO:90 (CDR3 #1)

[0843]

[0844] SEQ ID NO:91 (CDR3 #2)

[0845]

[0846] SEQ ID NO:92 (CDR3 #3)

[0847] Sequence Listing <110> Eli Lilly and Company <120> Insulin Analogs and Methods of Use Thereof <130> X22580 <160> 92 <170> PatentIn version 3.5 <210> 1 <211> 110 <212> PRT <213> Homo sapiens <400> 1 Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu 1 5 10 15 Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly 20 25 30 Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe 35 40 45 Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly 50 55 60 Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu 65 70 75 80 Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys 85 90 95 Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 100 105 110 <210> 2 <211> 86 <212> PRT <213> Homo sapiens <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 Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 3 <211> 21 <212> PRT <213> Homo sapiens <400> 3 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> 4 <211> 30 <212> PRT <213> Homo sapiens <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 Thr 20 25 30 <210> 5 <211> 1383 <212> PRT <213> Homo sapiens <400> 5 Met Ala Thr Gly Gly Arg Arg Gly Ala Ala Ala Ala Pro Leu Leu Val 1 5 10 15 Ala Val Ala Ala Leu Leu Leu Gly Ala Ala Gly His Leu Tyr Pro Gly 20 25 30 Glu Val Cys Pro Gly Met Asp Ile Arg Asn Asn Leu Thr Arg Leu His 35 40 45 Glu Leu Glu Asn Cys Ser Val Ile Glu Gly His Leu Gln Ile Leu Leu 50 55 60 Met Phe Lys Thr Arg Pro Glu Asp Phe Arg Asp Leu Ser Phe Pro Lys 65 70 75 80 Leu Ile Met Ile Thr Asp Tyr Leu Leu Leu Phe Arg Val Tyr Gly Leu 85 90 95 Glu Ser Leu Lys Asp Leu Phe Pro Asn Leu Thr Val Ile Arg Gly Ser 100 105 110 Arg Leu Phe Phe Asn Tyr Ala Leu Val Ile Phe Glu Met Val His Leu 115 120 125 Lys Glu Leu Gly Leu Tyr Asn Leu Met Asn Ile Thr Arg Gly Ser Val 130 135 140 Arg Ile Glu Lys Asn Asn Glu Leu Cys Tyr Leu Ala Thr Ile Asp Trp 145 150 155 160 Ser Arg Ile Leu Asp Ser Val Glu Asp Asn Tyr Ile Val Leu Asn Lys 165 170 175 Asp Asp Asn Glu Glu Cys Gly Asp Ile Cys Pro Gly Thr Ala Lys Gly 180 185 190 Lys Thr Asn Cys Pro Ala Thr Val Ile Asn Gly Gln Phe Val Glu Arg 195 200 205 Cys Trp Thr His Ser His Cys Gln Lys Val Cys Pro Thr Ile Cys Lys 210 215 220 Ser His Gly Cys Thr Ala Glu Gly Leu Cys Cys His Ser Glu Cys Leu 225 230 235 240 Gly Asn Cys Ser Gln Pro Asp Asp Pro Thr Lys Cys Val Ala Cys Arg 245 250 255 Asn Phe Tyr Leu Asp Gly Arg Cys Val Glu Thr Cys Pro Pro Pro Tyr 260 265 270 Tyr His Phe Gln Asp Trp Arg Cys Val Asn Phe Ser Phe Cys Gln Asp 275 280 285 Leu His His Lys Cys Lys Asn Ser Arg Arg Gln Gly Cys His Gln Tyr 290 295 300 Val Ile His Asn Asn Lys Cys Ile Pro Glu Cys Pro Ser Gly Tyr Thr 305 310 315 320 Met Asn Ser Ser Asn Leu Leu Cys Thr Pro Cys Leu Gly Pro Cys Pro 325 330 335 Lys Val Cys His Leu Leu Glu Gly Glu Lys Thr Ile Asp Ser Val Thr 340 345 350 Ser Ala Gln Glu Leu Arg Gly Cys Thr Val Ile Asn Gly Ser Leu Ile 355 360 365 Ile Asn Ile Arg Gly Gly Asn Asn Leu Ala Ala Glu Leu Glu Ala Asn 370 375 380 Leu Gly Leu Ile Glu Glu Ile Ser Gly Tyr Leu Lys Ile Arg Arg Ser 385 390 395 400 Tyr Ala Leu Val Ser Leu Ser Phe Phe Arg Lys Leu Arg Leu Ile Arg 405 410 415 Gly Glu Thr Leu Glu Ile Gly Asn Tyr Ser Phe Tyr Ala Leu Asp Asn 420 425 430 Gln Asn Leu Arg Gln Leu Trp Asp Trp Ser Lys His Asn Leu Thr Ile 435 440 445 Thr Gln Gly Lys Leu Phe Phe His Tyr Asn Pro Lys Leu Cys Leu Ser 450 455 460 Glu Ile His Lys Met Glu Glu Val Ser Gly Thr Lys Gly Arg Gln Glu 465 470 475 480 Arg Asn Asp Ile Ala Leu Lys Thr Asn Gly Asp Gln Ala Ser Cys Glu 485 490 495 Asn Glu Leu Leu Lys Phe Ser Tyr Ile Arg Thr Ser Phe Asp Lys Ile 500 505 510 Leu Leu Arg Trp Glu Pro Tyr Trp Pro Pro Asp Phe Arg Asp Leu Leu 515 520 525 Gly Phe Met Leu Phe Tyr Lys Glu Ala Pro Tyr Gln Asn Val Thr Glu 530 535 540 Phe Asp Gly Gln Asp Ala Cys Gly Ser Asn Ser Trp Thr Val Val Asp 545 550 555 560 Ile Asp Pro Pro Leu Arg Ser Asn Asp Pro Lys Ser Gln Asn His Pro 565 570 575 Gly Trp Leu Met Arg Gly Leu Lys Pro Trp Thr Gln Tyr Ala Ile Phe 580 585 590 Val Lys Thr Leu Val Thr Phe Ser Asp Glu Arg Arg Thr Tyr Gly Ala 595 600 605 Lys Ser Asp Ile Ile Tyr Val Gln Thr Asp Ala Thr Asn Pro Ser Val 610 615 620 Pro Leu Asp Pro Ile Ser Val Ser Asn Ser Ser Ser Gln Ile Ile Leu 625 630 635 640 Lys Trp Lys Pro Pro Ser Asp Pro Asn Gly Asn Ile Thr His Tyr Leu 645 650 655 Val Phe Trp Glu Arg Gln Ala Glu Asp Ser Glu Leu Phe Glu Leu Asp 660 665 670 Tyr Cys Leu Lys Gly Leu Lys Leu Pro Ser Arg Thr Trp Ser Pro Pro 675 680 685 Phe Glu Ser Glu Asp Ser Gln Lys His Asn Gln Ser Glu Tyr Glu Asp 690 695 700 Ser Ala Gly Glu Cys Cys Ser Cys Pro Lys Thr Asp Ser Gln Ile Leu 705 710 715 720 Lys Glu Leu Glu Glu Ser Ser Phe Arg Lys Thr Phe Glu Asp Tyr Leu 725 730 735 His Asn Val Val Phe Val Pro Arg Pro Ser Arg Lys Arg Arg Ser Leu 740 745 750 Gly Asp Val Gly Asn Val Thr Val Ala Val Pro Thr Val Ala Ala Phe 755 760 765 Pro Asn Thr Ser Ser Thr Ser Val Pro Thr Ser Pro Glu Glu His Arg 770 775 780 Pro Phe Glu Lys Val Val Asn Lys Glu Ser Leu Val Ile Ser Gly Leu 785 790 795 800 Arg His Phe Thr Gly Tyr Arg Ile Glu Leu Gln Ala Cys Asn Gln Asp 805 810 815 Thr Pro Glu Glu Arg Cys Ser Val Ala Ala Tyr Val Ser Ala Arg Thr 820 825 830 Met Pro Glu Ala Lys Ala Asp Asp Ile Val Gly Pro Val Thr His Glu 835 840 845 Ile Phe Glu Asn Asn Val Val His Leu Met Trp Gln Glu Pro Lys Glu 850 855 860 Pro Asn Gly Leu Ile Val Leu Tyr Glu Val Ser Tyr Arg Arg Tyr Gly 865 870 875 880 Asp Glu Glu Leu His Leu Cys Val Ser Arg Lys His Phe Ala Leu Glu 885 890 895 Arg Gly Cys Arg Leu Arg Gly Leu Ser Pro Gly Asn Tyr Ser Val Arg 900 905 910 Ile Arg Ala Thr Ser Leu Ala Gly Asn Gly Ser Trp Thr Glu Pro Thr 915 920 925 Tyr Phe Tyr Val Thr Asp Tyr Leu Asp Val Pro Ser Asn Ile Ala Lys 930 935 940 Ile Ile Ile Gly Pro Leu Ile Phe Val Phe Leu Phe Ser Val Val Ile 945 950 955 960 Gly Ser Ile Tyr Leu Phe Leu Arg Lys Arg Gln Pro Asp Gly Pro Leu 965 970 975 Gly Pro Leu Tyr Ala Ser Ser Asn Pro Glu Tyr Leu Ser Ala Ser Asp 980 985 990 Val Phe Pro Cys Ser Val Tyr Val Pro Asp Glu Trp Glu Val Ser Arg 995 1000 1005 Glu Lys Ile Thr Leu Leu Arg Glu Leu Gly Gln Gly Ser Phe Gly 1010 1015 1020 Met Val Tyr Glu Gly Asn Ala Arg Asp Ile Ile Lys Gly Glu Ala 1025 1030 1035 Glu Thr Arg Val Ala Val Lys Thr Val Asn Glu Ser Ala Ser Leu 1040 1045 1050 Arg Glu Arg Ile Glu Phe Leu Asn Glu Ala Ser Val Met Lys Gly 1055 1060 1065 Phe Thr Cys His His Val Val Arg Leu Leu Gly Val Val Ser Lys 1070 1075 1080 Gly Gln Pro Thr Leu Val Val Met Glu Leu Met Ala His Gly Asp 1085 1090 1095 Leu Lys Ser Tyr Leu Arg Ser Leu Arg Pro Glu Ala Glu Asn Asn 1100 1105 1110 Pro Gly Arg Pro Pro Pro Thr Leu Gln Glu Met Ile Gln Met Ala 1115 1120 1125 Ala Glu Ile Ala Asp Gly Met Ala Tyr Leu Asn Ala Lys Lys Phe 1130 1135 1140 Val His Arg Asp Leu Ala Ala Arg Asn Cys Met Val Ala His Asp 1145 1150 1155 Phe Thr Val Lys Ile Gly Asp Phe Gly Met Thr Arg Asp Ile Tyr 1160 1165 1170 Glu Thr Asp Tyr Tyr Arg Lys Gly Gly Lys Gly Leu Leu Pro Val 1175 1180 1185 Arg Trp Met Ala Pro Glu Ser Leu Lys Asp Gly Val Phe Thr Thr 1190 1195 1200 Ser Ser Asp Met Trp Ser Phe Gly Val Val Leu Trp Glu Ile Thr 1205 1210 1215 Ser Leu Ala Glu Gln Pro Tyr Gln Gly Leu Ser Asn Glu Gln Val 1220 1225 1230 Leu Lys Phe Val Met Asp Gly Gly Tyr Leu Asp Gln Pro Asp Asn 1235 1240 1245 Cys Pro Glu Arg Val Thr Asp Leu Met Arg Met Cys Trp Gln Phe 1250 1255 1260 Asn Pro Lys Met Arg Pro Thr Phe Leu Glu Ile Val Asn Leu Leu 1265 1270 1275 Lys Asp Asp Leu His Pro Ser Phe Pro Glu Val Ser Phe Phe His 1280 1285 1290 Ser Glu Glu Asn Lys Ala Pro Glu Ser Glu Glu Leu Glu Met Glu 1295 1300 1305 Phe Glu Asp Met Glu Asn Val Pro Leu Asp Arg Ser Ser His Cys 1310 1315 1320 Gln Arg Glu Glu Ala Gly Gly Arg Asp Gly Gly Ser Ser Leu Gly 1325 1330 1335 Phe Lys Arg Ser Tyr Glu Glu His Ile Pro Tyr Thr His Met Asn 1340 1345 1350 Gly Gly Lys Lys Asn Gly Arg Ile Leu Thr Leu Pro Arg Ser Asn 1355 1360 1365 Pro Ser Ala Ala Ala Gly Thr Glu Thr Ser Gln Val Ala Pro Ala 1370 1375 1380 <210> 6 <211> 1395 <212> PRT <213> Homo sapiens <400> 6 Met Ala Thr Gly Gly Arg Arg Gly Ala Ala Ala Ala Pro Leu Leu Val 1 5 10 15 Ala Val Ala Ala Leu Leu Leu Gly Ala Ala Gly His Leu Tyr Pro Gly 20 25 30 Glu Val Cys Pro Gly Met Asp Ile Arg Asn Asn Leu Thr Arg Leu His 35 40 45 Glu Leu Glu Asn Cys Ser Val Ile Glu Gly His Leu Gln Ile Leu Leu 50 55 60 Met Phe Lys Thr Arg Pro Glu Asp Phe Arg Asp Leu Ser Phe Pro Lys 65 70 75 80 Leu Ile Met Ile Thr Asp Tyr Leu Leu Leu Phe Arg Val Tyr Gly Leu 85 90 95 Glu Ser Leu Lys Asp Leu Phe Pro Asn Leu Thr Val Ile Arg Gly Ser 100 105 110 Arg Leu Phe Phe Asn Tyr Ala Leu Val Ile Phe Glu Met Val His Leu 115 120 125 Lys Glu Leu Gly Leu Tyr Asn Leu Met Asn Ile Thr Arg Gly Ser Val 130 135 140 Arg Ile Glu Lys Asn Asn Glu Leu Cys Tyr Leu Ala Thr Ile Asp Trp 145 150 155 160 Ser Arg Ile Leu Asp Ser Val Glu Asp Asn Tyr Ile Val Leu Asn Lys 165 170 175 Asp Asp Asn Glu Glu Cys Gly Asp Ile Cys Pro Gly Thr Ala Lys Gly 180 185 190 Lys Thr Asn Cys Pro Ala Thr Val Ile Asn Gly Gln Phe Val Glu Arg 195 200 205 Cys Trp Thr His Ser His Cys Gln Lys Val Cys Pro Thr Ile Cys Lys 210 215 220 Ser His Gly Cys Thr Ala Glu Gly Leu Cys Cys His Ser Glu Cys Leu 225 230 235 240 Gly Asn Cys Ser Gln Pro Asp Asp Pro Thr Lys Cys Val Ala Cys Arg 245 250 255 Asn Phe Tyr Leu Asp Gly Arg Cys Val Glu Thr Cys Pro Pro Pro Tyr 260 265 270 Tyr His Phe Gln Asp Trp Arg Cys Val Asn Phe Ser Phe Cys Gln Asp 275 280 285 Leu His His Lys Cys Lys Asn Ser Arg Arg Gln Gly Cys His Gln Tyr 290 295 300 Val Ile His Asn Asn Lys Cys Ile Pro Glu Cys Pro Ser Gly Tyr Thr 305 310 315 320 Met Asn Ser Ser Asn Leu Leu Cys Thr Pro Cys Leu Gly Pro Cys Pro 325 330 335 Lys Val Cys His Leu Leu Glu Gly Glu Lys Thr Ile Asp Ser Val Thr 340 345 350 Ser Ala Gln Glu Leu Arg Gly Cys Thr Val Ile Asn Gly Ser Leu Ile 355 360 365 Ile Asn Ile Arg Gly Gly Asn Asn Leu Ala Ala Glu Leu Glu Ala Asn 370 375 380 Leu Gly Leu Ile Glu Glu Ile Ser Gly Tyr Leu Lys Ile Arg Arg Ser 385 390 395 400 Tyr Ala Leu Val Ser Leu Ser Phe Phe Arg Lys Leu Arg Leu Ile Arg 405 410 415 Gly Glu Thr Leu Glu Ile Gly Asn Tyr Ser Phe Tyr Ala Leu Asp Asn 420 425 430 Gln Asn Leu Arg Gln Leu Trp Asp Trp Ser Lys His Asn Leu Thr Ile 435 440 445 Thr Gln Gly Lys Leu Phe Phe His Tyr Asn Pro Lys Leu Cys Leu Ser 450 455 460 Glu Ile His Lys Met Glu Glu Val Ser Gly Thr Lys Gly Arg Gln Glu 465 470 475 480 Arg Asn Asp Ile Ala Leu Lys Thr Asn Gly Asp Gln Ala Ser Cys Glu 485 490 495 Asn Glu Leu Leu Lys Phe Ser Tyr Ile Arg Thr Ser Phe Asp Lys Ile 500 505 510 Leu Leu Arg Trp Glu Pro Tyr Trp Pro Pro Asp Phe Arg Asp Leu Leu 515 520 525 Gly Phe Met Leu Phe Tyr Lys Glu Ala Pro Tyr Gln Asn Val Thr Glu 530 535 540 Phe Asp Gly Gln Asp Ala Cys Gly Ser Asn Ser Trp Thr Val Val Asp 545 550 555 560 Ile Asp Pro Pro Leu Arg Ser Asn Asp Pro Lys Ser Gln Asn His Pro 565 570 575 Gly Trp Leu Met Arg Gly Leu Lys Pro Trp Thr Gln Tyr Ala Ile Phe 580 585 590 Val Lys Thr Leu Val Thr Phe Ser Asp Glu Arg Arg Thr Tyr Gly Ala 595 600 605 Lys Ser Asp Ile Ile Tyr Val Gln Thr Asp Ala Thr Asn Pro Ser Val 610 615 620 Pro Leu Asp Pro Ile Ser Val Ser Asn Ser Ser Ser Gln Ile Ile Leu 625 630 635 640 Lys Trp Lys Pro Pro Ser Asp Pro Asn Gly Asn Ile Thr His Tyr Leu 645 650 655 Val Phe Trp Glu Arg Gln Ala Glu Asp Ser Glu Leu Phe Glu Leu Asp 660 665 670 Tyr Cys Leu Lys Gly Leu Lys Leu Pro Ser Arg Thr Trp Ser Pro Pro 675 680 685 Phe Glu Ser Glu Asp Ser Gln Lys His Asn Gln Ser Glu Tyr Glu Asp 690 695 700 Ser Ala Gly Glu Cys Cys Ser Cys Pro Lys Thr Asp Ser Gln Ile Leu 705 710 715 720 Lys Glu Leu Glu Glu Ser Ser Phe Arg Lys Thr Phe Glu Asp Tyr Leu 725 730 735 His Asn Val Val Phe Val Pro Arg Lys Thr Ser Ser Gly Thr Gly Ala 740 745 750 Glu Asp Pro Arg Pro Ser Arg Lys Arg Arg Ser Leu Gly Asp Val Gly 755 760 765 Asn Val Thr Val Ala Val Pro Thr Val Ala Ala Phe Pro Asn Thr Ser 770 775 780 Ser Thr Ser Val Pro Thr Ser Pro Glu Glu His Arg Pro Phe Glu Lys 785 790 795 800 Val Val Asn Lys Glu Ser Leu Val Ile Ser Gly Leu Arg His Phe Thr 805 810 815 Gly Tyr Arg Ile Glu Leu Gln Ala Cys Asn Gln Asp Thr Pro Glu Glu 820 825 830 Arg Cys Ser Val Ala Ala Tyr Val Ser Ala Arg Thr Met Pro Glu Ala 835 840 845 Lys Ala Asp Asp Ile Val Gly Pro Val Thr His Glu Ile Phe Glu Asn 850 855 860 Asn Val Val His Leu Met Trp Gln Glu Pro Lys Glu Pro Asn Gly Leu 865 870 875 880 Ile Val Leu Tyr Glu Val Ser Tyr Arg Arg Tyr Gly Asp Glu Glu Leu 885 890 895 His Leu Cys Val Ser Arg Lys His Phe Ala Leu Glu Arg Gly Cys Arg 900 905 910 Leu Arg Gly Leu Ser Pro Gly Asn Tyr Ser Val Arg Ile Arg Ala Thr 915 920 925 Ser Leu Ala Gly Asn Gly Ser Trp Thr Glu Pro Thr Tyr Phe Tyr Val 930 935 940 Thr Asp Tyr Leu Asp Val Pro Ser Asn Ile Ala Lys Ile Ile Ile Gly 945 950 955 960 Pro Leu Ile Phe Val Phe Leu Phe Ser Val Val Ile Gly Ser Ile Tyr 965 970 975 Leu Phe Leu Arg Lys Arg Gln Pro Asp Gly Pro Leu Gly Pro Leu Tyr 980 985 990 Ala Ser Ser Asn Pro Glu Tyr Leu Ser Ala Ser Asp Val Phe Pro Cys 995 1000 1005 Ser Val Tyr Val Pro Asp Glu Trp Glu Val Ser Arg Glu Lys Ile 1010 1015 1020 Thr Leu Leu Arg Glu Leu Gly Gln Gly Ser Phe Gly Met Val Tyr 1025 1030 1035 Glu Gly Asn Ala Arg Asp Ile Ile Lys Gly Glu Ala Glu Thr Arg 1040 1045 1050 Val Ala Val Lys Thr Val Asn Glu Ser Ala Ser Leu Arg Glu Arg 1055 1060 1065 Ile Glu Phe Leu Asn Glu Ala Ser Val Met Lys Gly Phe Thr Cys 1070 1075 1080 His His Val Val Arg Leu Leu Gly Val Val Ser Lys Gly Gln Pro 1085 1090 1095 Thr Leu Val Val Met Glu Leu Met Ala His Gly Asp Leu Lys Ser 1100 1105 1110 Tyr Leu Arg Ser Leu Arg Pro Glu Ala Glu Asn Asn Pro Gly Arg 1115 1120 1125 Pro Pro Pro Thr Leu Gln Glu Met Ile Gln Met Ala Ala Glu Ile 1130 1135 1140 Ala Asp Gly Met Ala Tyr Leu Asn Ala Lys Lys Phe Val His Arg 1145 1150 1155 Asp Leu Ala Ala Arg Asn Cys Met Val Ala His Asp Phe Thr Val 1160 1165 1170 Lys Ile Gly Asp Phe Gly Met Thr Arg Asp Ile Tyr Glu Thr Asp 1175 1180 1185 Tyr Tyr Arg Lys Gly Gly Lys Gly Leu Leu Pro Val Arg Trp Met 1190 1195 1200 Ala Pro Glu Ser Leu Lys Asp Gly Val Phe Thr Thr Ser Ser Asp 1205 1210 1215 Met Trp Ser Phe Gly Val Val Leu Trp Glu Ile Thr Ser Leu Ala 1220 1225 1230 Glu Gln Pro Tyr Gln Gly Leu Ser Asn Glu Gln Val Leu Lys Phe 1235 1240 1245 Val Met Asp Gly Gly Tyr Leu Asp Gln Pro Asp Asn Cys Pro Glu 1250 1255 1260 Arg Val Thr Asp Leu Met Arg Met Cys Trp Gln Phe Asn Pro Lys 1265 1270 1275 Met Arg Pro Thr Phe Leu Glu Ile Val Asn Leu Leu Lys Asp Asp 1280 1285 1290 Leu His Pro Ser Phe Pro Glu Val Ser Phe Phe His Ser Glu Glu 1295 1300 1305 Asn Lys Ala Pro Glu Ser Glu Glu Leu Glu Met Glu Phe Glu Asp 1310 1315 1320 Met Glu Asn Val Pro Leu Asp Arg Ser Ser His Cys Gln Arg Glu 1325 1330 1335 Glu Ala Gly Gly Arg Asp Gly Gly Ser Ser Leu Gly Phe Lys Arg 1340 1345 1350 Ser Tyr Glu Glu His Ile Pro Tyr Thr His Met Asn Gly Gly Lys 1355 1360 1365 Lys Asn Gly Arg Ile Leu Thr Leu Pro Arg Ser Asn Pro Ser Ala 1370 1375 1380 Ala Ala Gly Thr Glu Thr Ser Gln Val Ala Pro Ala 1385 1390 1395 <210> 7 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp Glu Thr 20 25 30 Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala Asp Leu 100 105 110 Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Pro Pro 115 120 125 <210> 8 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 8 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Val Ser Ser Thr 20 25 30 Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Gly Gly Ser Val Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Arg Pro Gly Arg Pro Leu Ile Thr Ser Arg Asp Ala Asn Leu 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 9 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp Glu Thr 20 25 30 Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val 35 40 45 Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala Asp Leu 100 105 110 Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Pro Pro 115 120 125 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 10 Gly Gly Gly Gly Gln 1 5 <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 11 Gly Gly Gly Gln 1 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 12 Gly Gly Gly Gly Ser 1 5 <210> 13 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 13 Pro Gly Pro Gln 1 <210> 14 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 14 Pro Gly Pro Ala 1 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 15 Gly Gly Glu Gly Gly 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 16 Gly Gly Gly Gly Glu Gly Gly Gly Gly 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 17 Gly Gly Gly Gly Lys Gly Gly Gly Gly 1 5 <210> 18 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 18 Gly Gly Gly Gly Ala Pro Gly Gly Gly Gly 1 5 10 <210> 19 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 19 Gly Gly Gly Gly Glu Pro Gly Gly Gly Gly 1 5 10 <210> 20 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 20 Gly Gly Gly Gly Lys Pro Gly Gly Gly Gly 1 5 10 <210> 21 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 21 Pro Gly Pro Glu Pro Gly Pro Gln 1 5 <210> 22 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 22 Pro Gly Pro Lys Pro Gly Pro Gln 1 5 <210> 23 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 23 Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly 1 5 10 15 Gly Gly Gly Gln Gly Gly Gly Gly Gln 20 25 <210> 24 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 24 Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro Gln 1 5 10 15 Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro Gln 20 25 30 <210> 25 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 25 Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala 1 5 10 15 Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala 20 25 30 <210> 26 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 26 Gly Gly Glu Gly Gly Glu Gly Gly Glu Gly Gly Glu Gly Gly Glu Gly 1 5 10 15 Gly Glu Gly Gly Glu Gly Gly 20 <210> 27 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 27 Gly Gly Gly Gly Glu Gly Gly Gly Gly Glu Gly Gly Gly Gly Glu Gly 1 5 10 15 Gly Gly Gly Glu Gly Gly Gly Gly 20 <210> 28 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 28 Gly Gly Gly Gly Lys Gly Gly Gly Gly Lys Gly Gly Gly Gly Lys Gly 1 5 10 15 Gly Gly Gly Lys Gly Gly Gly Gly 20 <210> 29 <211> 28 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 29 Gly Gly Gly Gly Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro 1 5 10 15 Ala Pro Ala Pro Ala Pro Ala Pro Gly Gly Gly Gly 20 25 <210> 30 <211> 28 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 30 Gly Gly Gly Gly Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro 1 5 10 15 Glu Pro Glu Pro Glu Pro Glu Pro Gly Gly Gly Gly 20 25 <210> 31 <211> 28 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 31 Gly Gly Gly Gly Lys Pro Lys Pro Lys Pro Lys Pro Lys Pro Lys Pro 1 5 10 15 Lys Pro Lys Pro Lys Pro Lys Pro Gly Gly Gly Gly 20 25 <210> 32 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 32 Pro Gly Pro Glu Pro Gly Pro Glu Pro Gly Pro Glu Pro Gly Pro Glu 1 5 10 15 Pro Gly Pro Glu Pro Gly Pro Glu Pro Gly Pro Glu Pro Gly Pro Gln 20 25 30 <210> 33 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 33 Pro Gly Pro Lys Pro Gly Pro Lys Pro Gly Pro Lys Pro Gly Pro Lys 1 5 10 15 Pro Gly Pro Lys Pro Gly Pro Lys Pro Gly Pro Lys Pro Gly Pro Gln 20 25 30 <210> 34 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 34 Gly Gly Gly Gly Gly Gly 1 5 <210> 35 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 35 Gly Gly Gly Ser Gly Gly Ser Gly Gly Gly 1 5 10 <210> 36 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 36 Gly Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly 1 5 10 <210> 37 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 37 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 Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Thr Val Ser Ser Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Ser Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Arg Asp Ala Asn Leu Tyr Asp Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser 210 215 <210> 38 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 38 Phe Val Asp 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 Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Thr Val Ser Ser Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Ser Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Arg Asp Ala Asn Leu Tyr Asp Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser 210 215 <210> 39 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 39 Phe Val Asn Gln His Leu Cys Gly Ala 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 Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Thr Val Ser Ser Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Ser Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Arg Asp Ala Asn Leu Tyr Asp Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser 210 215 <210> 40 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 40 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 Phe Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Thr Val Ser Ser Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Ser Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Arg Asp Ala Asn Leu Tyr Asp Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser 210 215 <210> 41 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 41 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Thr Val Ser Ser Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Ser Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Arg Asp Ala Asn Leu Tyr Asp Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser 210 215 <210> 42 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Thr Val Ser Ser Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Ser Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Arg Asp Ala Asn Leu Tyr Asp Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser 210 215 <210> 43 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 43 Phe Val Ser 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 Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 44 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 44 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 45 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 45 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 46 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 46 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 47 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 47 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Glu Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 48 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 48 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 49 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 49 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Glu Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 50 <211> 217 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 50 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 51 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 51 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Phe 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 52 <211> 217 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 52 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 53 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 53 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 54 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 54 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Ala Pro Gly Pro 50 55 60 Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro Ala Pro Gly Pro 65 70 75 80 Ala Pro Gly Pro Ala Pro Gly Pro Ala Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 55 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 55 Phe Val Ser 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 Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 56 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 56 Phe Val Ser 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 Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Gln Pro Gly Pro 50 55 60 Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro 65 70 75 80 Gln Pro Gly Pro Gln Pro Gly Pro Gln Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 57 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 57 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 58 <211> 217 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 58 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Gln Pro Gly Pro 50 55 60 Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro 65 70 75 80 Gln Pro Gly Pro Gln Pro Gly Pro Gln Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 59 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 59 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Glu Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 60 <211> 217 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 60 Phe Val Ser 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 Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Glu Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Gln Pro Gly Pro 50 55 60 Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro 65 70 75 80 Gln Pro Gly Pro Gln Pro Gly Pro Gln Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 61 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 61 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 62 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 62 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Gln Pro Gly Pro 50 55 60 Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro 65 70 75 80 Gln Pro Gly Pro Gln Pro Gly Pro Gln Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 63 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 63 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 64 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 64 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Gln Pro Gly Pro 50 55 60 Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro 65 70 75 80 Gln Pro Gly Pro Gln Pro Gly Pro Gln Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 65 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 65 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 66 <211> 217 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 66 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Gln Pro Gly Pro 50 55 60 Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro Gln Pro Gly Pro 65 70 75 80 Gln Pro Gly Pro Gln Pro Gly Pro Gln Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 67 <211> 208 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 67 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Glu Gly Gly Glu Gly 50 55 60 Gly Glu Gly Gly Glu Gly Gly Glu Gly Gly Glu Gly Gly Glu Gly Gly 65 70 75 80 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 85 90 95 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp Glu Thr 100 105 110 Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 115 120 125 Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp Ser Val 130 135 140 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 145 150 155 160 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 165 170 175 Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala Asp Leu 180 185 190 Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Pro Pro 195 200 205 <210> 68 <211> 209 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 68 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Glu Gly Gly 50 55 60 Gly Gly Glu Gly Gly Gly Gly Glu Gly Gly Gly Gly Glu Gly Gly Gly 65 70 75 80 Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 85 90 95 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp Glu 100 105 110 Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 115 120 125 Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp Ser 130 135 140 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 145 150 155 160 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 165 170 175 Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala Asp 180 185 190 Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Pro 195 200 205 Pro <210> 69 <211> 209 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 69 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Lys Gly Gly 50 55 60 Gly Gly Lys Gly Gly Gly Gly Lys Gly Gly Gly Gly Lys Gly Gly Gly 65 70 75 80 Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 85 90 95 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp Glu 100 105 110 Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 115 120 125 Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp Ser 130 135 140 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 145 150 155 160 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 165 170 175 Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala Asp 180 185 190 Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Pro 195 200 205 Pro <210> 70 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 70 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Ala Pro Ala 50 55 60 Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala 65 70 75 80 Pro Gly Gly Gly Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 85 90 95 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 100 105 110 Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys 115 120 125 Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr 130 135 140 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 145 150 155 160 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 165 170 175 Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser 180 185 190 Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr 195 200 205 Val Ser Ser Pro Pro 210 <210> 71 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 71 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Glu Pro Glu 50 55 60 Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu 65 70 75 80 Pro Gly Gly Gly Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 85 90 95 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 100 105 110 Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys 115 120 125 Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr 130 135 140 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 145 150 155 160 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 165 170 175 Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser 180 185 190 Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr 195 200 205 Val Ser Ser Pro Pro 210 <210> 72 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 72 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Lys Pro Lys 50 55 60 Pro Lys Pro Lys Pro Lys Pro Lys Pro Lys Pro Lys Pro Lys Pro Lys Pro Lys 65 70 75 80 Pro Gly Gly Gly Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 85 90 95 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 100 105 110 Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys 115 120 125 Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr 130 135 140 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 145 150 155 160 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 165 170 175 Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser 180 185 190 Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr 195 200 205 Val Ser Ser Pro Pro 210 <210> 73 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 73 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Glu Pro Gly Pro 50 55 60 Glu Pro Gly Pro Glu Pro Gly Pro Glu Pro Gly Pro Glu Pro Gly Pro 65 70 75 80 Glu Pro Gly Pro Glu Pro Gly Pro Gln Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 74 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 74 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Pro Gly Pro Lys Pro Gly Pro 50 55 60 Lys Pro Gly Pro Lys Pro Gly Pro Lys Pro Gly Pro Lys Pro Gly Pro 65 70 75 80 Lys Pro Gly Pro Lys Pro Gly Pro Gln Glu Val Gln Leu Leu Glu Ser 85 90 95 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 100 105 110 Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala Trp Phe Arg Gln 115 120 125 Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Gly Gly Gly Val 130 135 140 Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 145 150 155 160 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 165 170 175 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro Gly Arg Pro 180 185 190 Leu Ile Thr Ser Lys Val Ala Asp Leu Tyr Pro Tyr Trp Gly Gln Gly 195 200 205 Thr Leu Val Thr Val Ser Ser Pro Pro 210 215 <210> 75 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 75 Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 76 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 76 Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Gln Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 77 <211> 210 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 77 Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Gln Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Gln Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 78 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 78 Phe Val Ser Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 79 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 79 Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 80 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 80 Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Gln Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 81 <211> 210 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 81 Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Arg 1 5 10 15 Leu Val Cys Gly Gln Arg Gly Phe His Tyr Thr Pro Lys Thr Gly Gly 20 25 30 Gly Gly Gly Gly Gly Ile Val Gln Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Gly Gly Gly Gly Gly Gln Gly Gly 50 55 60 Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly 65 70 75 80 Gly Gln Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 85 90 95 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Ile Asp 100 105 110 Glu Thr Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu 115 120 125 Phe Val Ala Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp 130 135 140 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 145 150 155 160 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 165 170 175 Tyr Cys Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala 180 185 190 Asp Leu Tyr Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 195 200 205 Pro Pro 210 <210> 82 <211> 20 <212> PRT <213> Mus musculus <400> 82 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 <210> 83 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 83 Thr Glu Thr Ser Gln Val Ala Pro Ala 1 5 <210> 84 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 84 Ala Ala Ser Gly Arg Thr Val Ser Ser Thr Ala Val Ala 1 5 10 <210> 85 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 85 Ala Ala Ser Gly Arg Tyr Ile Asp Ser Thr Ala Val Ala 1 5 10 <210> 86 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 86 Ala Ala Ser Gly Arg Tyr Ile Asp Glu Thr Ala Val Ala 1 5 10 <210> 87 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 87 Gly Ile Gly Gly Ser Val Asp Ile Thr Tyr Tyr Leu Asp Ser Val Lys 1 5 10 15 Gly <210> 88 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 88 Gly Ile Gly Gly Ser Val Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 89 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 89 Gly Ile Gly Gly Gly Val Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 90 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 90 Ala Val Arg Pro Gly Arg Pro Leu Ile Thr Ser Arg Asp Ala Asn Leu 1 5 10 15 Tyr Asp Tyr <210> 91 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 91 Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Arg Val Ala Asn Leu 1 5 10 15 Tyr Pro Tyr <210> 92 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 92 Ala Ala Arg Pro Gly Arg Pro Leu Ile Thr Ser Lys Val Ala Asp Leu 1 5 10 15 Tyr Pro Tyr

Claims

1. A compound comprising the following structure: B-L2-A-L1-VHH, wherein VHH is an amino acid sequence selected from SEQ ID NO:7 and 8, wherein A is the insulin A chain, which is SEQ ID NO:3 and has a mutation selected from the following: N21G mutation; T8H mutation and N21G mutation; and T8H mutation, Y14E mutation and N21G mutation, wherein B is the insulin B chain, which is SEQ ID NO:4, or SEQ ID NO:4 and has a mutation selected from the following: N3D mutation; N3S mutation; S9A mutation; Y16E mutation; Y16H mutation; F25H mutation; N3S mutation and F25H mutation; N3S mutation and Y16H mutation; N3S mutation, Y16H mutation and F25H mutation; N3S mutation and Y16R mutation; N3S mutation and Y16F mutation; N3S mutation and Y16W mutation; and N3S mutation, Y16R mutation and F25H mutation, wherein L1 is the first linker, which is an amino acid sequence selected from SEQ ID NO:23 and 25, and wherein L2 is the second linker, which is the amino acid sequence of SEQ ID NO:

34.

2. The compound according to claim 1, wherein B is SEQ ID NO:

4.

3. The compound according to claim 1, wherein B is SEQ ID NO:4 and has a mutation selected from the following: N3D mutation; N3S mutation; S9A mutation; Y16E mutation; Y16H mutation; F25H mutation; N3S mutation and F25H mutation; N3S mutation and Y16H mutation; N3S mutation, Y16H mutation and F25H mutation; N3S mutation and Y16R mutation; N3S mutation and Y16F mutation; N3S mutation and Y16W mutation; and N3S mutation, Y16R mutation and F25H mutation.

4. The compound according to claim 1, wherein A is SEQ ID NO:3 and has a mutation selected from the following: N21G mutation; T8H mutation and N21G mutation; and T8H mutation, Y14E mutation and N21G mutation, and wherein B is SEQ ID NO:4 and has a mutation selected from the following: N3D mutation; N3S mutation; S9A mutation; Y16E mutation; Y16H mutation; F25H mutation; N3S mutation and F25H mutation; N3S mutation and Y16H mutation; N3S mutation, Y16H mutation and F25H mutation; N3S mutation and Y16R mutation; N3S mutation and Y16F mutation; N3S mutation and Y16W mutation; and N3S mutation, Y16R mutation and F25H mutation.

5. The compound according to any one of claims 1-4, wherein L1 is SEQ ID NO:

23.

6. The compound according to any one of claims 1-4, wherein L1 is SEQ ID NO:

25.

7. A compound comprising an amino acid sequence selected from SEQ ID NO:37 to 54, 61 to 62 and 65 to 66.

8. A compound consisting of an amino acid sequence selected from SEQ ID NO: 37 to 54, 61 to 62, and 65 to 66.

9. A pharmaceutical composition comprising the compound according to any one of claims 1 - 8 and a pharmaceutically acceptable buffer.

10. The pharmaceutical composition according to claim 9, further comprising an additional therapeutic agent.

11. The pharmaceutical composition according to claim 10, wherein the additional therapeutic agent is selected from: dipeptidyl peptidase 4 (DPP-IV) inhibitors, native amylin or its analogs, short-acting INS analogs, native incretins or their analogs, native insulin-like growth factor (IGF) or its analogs, metformin, sodium-glucose cotransporter-2 (SGLT2) inhibitors, inhibin, sulfonylurea (SU), thiazolidinedione (TZD), and other anti-hyperglycemic agents or other anti-obesity agents.

12. Use of an effective amount of the compound according to any one of claims 1 - 8 or the pharmaceutical composition according to claim 9 in the manufacture of a medicament for treating diabetes in an individual.

13. The use according to claim 12, wherein the compound or pharmaceutical composition is further administered in combination with an effective amount of an additional therapeutic agent.

14. The use according to claim 13, wherein the additional therapeutic agent is selected from: dipeptidyl peptidase 4 (DPP-IV) inhibitors, native amylin or its analogs, short-acting INS analogs, native incretins or their analogs, native insulin-like growth factor (IGF) or its analogs, metformin, sodium-glucose cotransporter-2 (SGLT2) inhibitors, inhibin, sulfonylurea (SU), thiazolidinedione (TZD), and other anti-hyperglycemic agents or other anti-obesity agents.

15. The compound according to any one of claims 1 - 8 for use in therapy.

16. The compound according to any one of claims 1 - 8 for use in treating diabetes.

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