INSULIN ANALOGS AND METHODS OF USING THE SAME

Abstract

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

Claims

1. A compound comprising a structure of:
VHH-L.sub.1-A-L.sub.2-B,
VHH-L.sub.1-B-L.sub.2-A,
A-L.sub.2-B-L.sub.1-VHH, or
B-L.sub.2-A-L.sub.1-VHH, wherein VHH comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:7, 8 and 9 or a sequence having at least 90% sequence similarity thereto, wherein A is an insulin A chain comprising an amino acid sequence of SEQ ID NO:3 or a sequence having at least 90% sequence similarity thereto, wherein B is an insulin B chain comprising an amino acid sequence of SEQ ID NO:4 or a sequence having at least 90% sequence similarity thereto, wherein L.sub.1 is a first linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22, and wherein n can be from 1 to 10, and wherein L.sub.2 is a second linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:34, 35 and 36.

2. The compound of claim 1, wherein A is SEQ ID NO:3.

3. The compound of claim 1, wherein B is SEQ ID NO:4.

4. The compound of claim 1, wherein A is SEQ ID NO:3 and B is SEQ ID NO:4.

5. The compound of claim 1, wherein A is SEQ ID NO:3 and includes at least one mutation selected from the group consisting of an E4Q mutation, a T8H mutation, a Y14E mutation and a N21G mutation.

6. The compound of claim 1, wherein B is SEQ ID NO:4 and includes at least one mutation selected from the group consisting of a N3D mutation, a N3K mutation, a N3S mutation, a S9A mutation, a Y16E mutation, a Y16F mutation, a Y16H mutation, a Y16R mutation, a Y16W mutation, a E21Q mutation or a F25H mutation.

7. The compound of claim 1, wherein A is SEQ ID NO:3 and includes at least one mutation selected from the group consisting of an E4Q mutation, a T8H mutation, a Y14E mutation and a N21G mutation, and wherein B is SEQ ID NO:4 and includes at least one mutation selected from the group consisting of a N3D mutation, a N3K mutation, a N3S mutation, a S9A mutation, a Y16E mutation, a Y16F mutation, a Y16H mutation, a Y16R mutation, a Y16W mutation, a E21Q mutation or a F25H mutation.

8. The compound of claim 1, wherein L.sub.1 is SEQ ID NO:23.

9. The compound of claim 1, wherein L.sub.1 is SEQ ID NO:24.

10. The compound of claim 1, wherein L.sub.1 is SEQ ID NO:25.

11. The compound of claim 1, wherein L.sub.1 is SEQ ID NO:26.

12. The compound of claim 1, wherein L.sub.1 is SEQ ID NO:27.

13. The compound of claim 1, wherein L.sub.1 is SEQ ID NO:28.

14. The compound of claim 1, wherein L.sub.1 is SEQ ID NO:29.

15. The compound of claim 1, wherein L.sub.1 is SEQ ID NO:30.

16. The compound of claim 1, wherein L.sub.1 is SEQ ID NO:31.

17. The compound of claim 1, wherein L.sub.1 is SEQ ID NO:32.

18. The compound of any claim 1, wherein L.sub.1 is SEQ ID NO:33.

19. The compound of claim 1, wherein L.sub.2 is SEQ ID NO:34.

20. A compound comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:37 to 81 or a sequence having at least 90% sequence similarity thereto.

21-22. (canceled)

23. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable buffer.

24. The pharmaceutical composition of claim 23 further comprising an additional therapeutic agent.

25. The pharmaceutical composition of claim 24, wherein the additional therapeutic agent is selected from the group consisting of a dipeptidyl peptidase 4 (DPP-IV) inhibitor, a native amylin or analog thereof, a short-acting (prandial) INS analog, a native incretin or analog thereof, a native insulin-like growth factor (IGF) or analog thereof, metformin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, a statin, a sulfonylurea (SU), a thiazolidinedione (TZD), and other anti-glycemic agents or other anti-obesity agents.

26. A method of treating cardiometabolic conditions, diseases and/or disorders in an individual, the method comprising the step of: administering to the individual an effective amount of the compound of claim 1 or the pharmaceutical composition of claim 22.

27. The method of claim 26 further comprising administering to the individual an effective amount of an additional therapeutic agent.

28. The method of claim 27, wherein the additional therapeutic agent is selected from the group consisting of a dipeptidyl peptidase 4 (DPP-IV) inhibitor, a native amylin or analog thereof, a short-acting (prandial) INS analog, a native incretin or analog thereof, a native insulin-like growth factor (IGF) or analog thereof, metformin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, a statin, a sulfonylurea (SU), a thiazolidinedione (TZD), and other anti-glycemic agents or other anti-obesity agents.

29-39. (canceled)

Description

EXAMPLES

[0202] The following non-limiting examples are offered for purposes of illustration, not limitation.

[0203] Polypeptide Expression

Example 1: Recombinant Expression of INS Analog 1

[0204] Example 1 is an INS analog having an amino acid sequence of:

TABLE-US-00046 (SEQ ID NO: 37) FVNQHLCGSHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITS RDANLYDYWGQGTLVTVSS.

[0205] Here, the INS analog of SEQ ID NO:37 is generated in a mammalian cell expression system using CHOK1 cell derivatives. A cDNA sequence encoding SEQ ID NO:37 is sub-cloned into GS-containing expression plasmid backbone (pEE12.4-based plasmids). The cDNA sequence is fused in frame with the coding sequence of a signal peptide sequence, METDTLLLWVLLLWVPGSTG (SEQ ID NO:82) to enhance secretion of the INS analog into the tissue culture medium. The expression is driven by the viral CMV promoter.

[0206] For generating the INS analog via transient transfection, CHOK1 cells are transfected with the recombinant expression plasmid using a PEI-based method. Briefly, the appropriate volume of CHOK1 suspension cells at a density of 4×10.sup.6 cells/ml is transferred in shake flasks, and both PEI and recombinant plasmid DNA are added to the cells. Cells are incubated in a suspension culture at 32° C. for 6 days. At the end of the incubation period, cells are removed by low speed centrifugation, and the INS analog is purified from the conditioned medium.

[0207] Alternatively, and for generating the INS analog via stable transfections, CHOK1 cells are stably transfected using electroporation and the appropriate amount of recombinant expression plasmid, and the transfected cells are maintained in suspension culture at an adequate cell density. Selection of the transfected cells is accomplished by growth in 25 μM MSX-containing serum-free medium and incubated at 35° C.-37° C. and 5%-7% CO.sub.2.

[0208] The INS analog secreted into the media from the CHO cells is purified by Protein A affinity chromatography followed by ion exchange, hydrophobic interaction, or size-exclusion chromatography. Specifically, the INS analog from harvested media is captured onto Mab Select Protein A resin (GE). The resin then is briefly washed with a running buffer, such as a phosphate-buffered saline (PBS; pH 7.4) or a running buffer containing Tris, to remove non-specifically bound material. The protein is eluted from the resin with a low pH solution, such as 10 mM citric acid, 150 mM NaCl pH 3. Fractions containing the INS analog are pooled 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 may be held at a low pH to inactivate potential viruses. The pH may be neutralized by adding a base such as 0.1 M Tris pH 8.0 for subsequent size-exclusion chromatography. The INS analog may be further purified by ion exchange chromatography using resins 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.

[0209] The INS analog may be further purified by hydrophobic interaction chromatography by using a Capto Phenyl ImpRes HIC Column (GE Healthcare). The purification is performed by adjusting the column charge solution to around 0.5 M Na.sub.2SO.sub.4 and eluting using a 10 column volume (CV) gradient going from 0.5 M to 0 M Na.sub.2SO.sub.4 in a 20 mM Tris pH 8 solution. After HIC, the INS analog may be even further purified by SEC by loading the concentrated Capto Phenyl ImpRes pool on a Superdex200 (GE Healthcare) with isocratic elution in PBS pH 7.4 or in 20 mM histidine, 50 mM NaCl pH 6.0.

[0210] Purified INS analog may be passed through a viral retention filter such as Planova 20N (Asahi Kasei Medical) followed by concentration/diafiltration into 20 mM histidine, 20 mM NaCl pH 6 using tangential flow ultrafiltration on a regenerated cellulose membrane (Millipore).

[0211] The INS analog therefore is prepared in this manner or in a similar manner that would be readily determined by one of skill in the art.

Example 2: Recombinant Expression of INS Analog 2

[0212] Example 2 is an INS analog having an amino acid sequence of:

TABLE-US-00047 (SEQ ID NO: 38) FVDQHLCGSHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITS RDANLYDYWGQGTLVTVSS.

[0213] Here, Example 2 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:38 is used in the expression plasmid.

Example 3: Recombinant Expression of INS Analog 3

[0214] Example 3 is an INS analog having an amino acid sequence of:

TABLE-US-00048 (SEQ ID NO: 39) FVNQHLCGAHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITS RDANLYDYWGQGTLVTVSS.

[0215] Here, Example 3 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:39 is used in the expression plasmid.

Example 4: Recombinant Expression of INS Analog 4

[0216] Example 4 is an INS analog having an amino acid sequence of:

TABLE-US-00049 (SEQ ID NO: 40) FVNQHLCGSHLVEALELVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITS RDANLYDYWGQGTLVTVSS.

[0217] Here, Example 4 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:40 is used in the expression plasmid.

Example 5: Recombinant Expression of INS Analog 5

[0218] Example 5 is an INS analog having an amino acid sequence of:

TABLE-US-00050 (SEQ ID NO: 41) FVNQHLCGSHLVEALHLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITS RDANLYDYWGQGTLVTVSS.

[0219] Here, Example 5 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:41 is used in the expression plasmid.

Example 6: Recombinant Expression of INS Analog 6

[0220] Example 6 is an INS analog having an amino acid sequence of:

TABLE-US-00051 (SEQ ID NO: 42) FVNQHLCGSHLVEALYLVCGERGFHYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITS RDANLYDYWGQGTLVTVSS.

[0221] Here, Example 6 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:42 is used in the expression plasmid.

Example 7: Recombinant Expression of INS Analog 7

[0222] Example 7 is an INS analog having an amino acid sequence of:

TABLE-US-00052 (SEQ ID NO: 43) FVSQHLCGSHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0223] Here, Example 7 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:43 is used in the expression plasmid.

Example 8: Recombinant Expression of INS Analog 8

[0224] Example 8 is an INS analog having an amino acid sequence of:

TABLE-US-00053 (SEQ ID NO: 44) FVSQHLCGSHLVEALYLVCGERGFHYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0225] Here, Example 8 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:44 is used in the expression plasmid.

Example 9: Recombinant Expression of INS Analog 9

[0226] Example 9 is an INS analog having an amino acid sequence of:

TABLE-US-00054 (SEQ ID NO: 45) FVSQHLCGSHLVEALYLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0227] Here, Example 9 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:45 is used in the expression plasmid.

Example 10: Recombinant Expression of INS Analog 10

[0228] Example 10 is an INS analog having an amino acid sequence of:

TABLE-US-00055 (SEQ ID NO: 46) FVSQHLCGSHLVEALHLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0229] Here, Example 10 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:46 is used in the expression plasmid.

Example 11: Recombinant Expression of INS Analog 11

[0230] Example 11 is an INS analog having an amino acid sequence of:

TABLE-US-00056 (SEQ ID NO: 47) FVSQHLCGSHLVEALHLVCGERGFFYTPKTGGGGGGGIVEQCCHSICSL EQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0231] Here, Example 11 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:47 is used in the expression plasmid.

Example 12: Recombinant Expression of INS Analog 12

[0232] Example 12 is an INS analog having an amino acid sequence of:

TABLE-US-00057 (SEQ ID NO: 48) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0233] Here, Example 12 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:48 is used in the expression plasmid.

Example 13: Recombinant Expression of INS Analog 13

[0234] Example 13 is an INS analog having an amino acid sequence of:

TABLE-US-00058 (SEQ ID NO: 49) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL EQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0235] Here, Example 13 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:49 is used in the expression plasmid.

Example 14: Recombinant Expression of INS Analog 14

[0236] Example 14 is an INS analog having an amino acid sequence of:

TABLE-US-00059 (SEQ ID NO: 50) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0237] Here, Example 14 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:50 is used in the expression plasmid.

Example 15: Recombinant Expression of INS Analog 15

[0238] Example 15 is an INS analog having an amino sequence of:

TABLE-US-00060 (SEQ ID NO: 51) FVSQHLCGSHLVEALFLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0239] Here, Example 15 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:51 is used in the expression plasmid.

Example 16: Recombinant Expression of INS Analog 16

[0240] Example 16 is an INS analog having an amino sequence of:

TABLE-US-00061 (SEQ ID NO: 52) FVSQHLCGSHLVEALWLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0241] Here, Example 16 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:52 is used in the expression plasmid.

Example 17: Recombinant Expression of INS Analog 17

[0242] Example 17 is an INS analog having an amino sequence of:

TABLE-US-00062 (SEQ ID NO: 53) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0243] Here, Example 17 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:53 is used in the expression plasmid.

Example 18: Recombinant Expression of INS Analog 18

[0244] Example 18 is an INS analog having an amino sequence of:

TABLE-US-00063 (SEQ ID NO: 54) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0245] Here, Example 18 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:54 is used in the expression plasmid.

Example 19: Recombinant Expression of INS Analog 19

[0246] Example 19 is an INS analog having an amino sequence of:

TABLE-US-00064 (SEQ ID NO: 55) FVSQHLCGSHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0247] Here, Example 19 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:55 is used in the expression plasmid.

Example 20: Recombinant Expression of INS Analog 20

[0248] Example 20 is an INS analog having an amino sequence of:

TABLE-US-00065 (SEQ ID NO: 56) FVSQHLCGSHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0249] Here, Example 20 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:56 is used in the expression plasmid.

Example 21: Recombinant Expression of INS Analog 21

[0250] Example 21 is an INS analog having an amino sequence of:

TABLE-US-00066 (SEQ ID NO: 57) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0251] Here, Example 21 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:57 is used in the expression plasmid.

Example 22: Recombinant Expression of INS Analog 22

[0252] Example 22 is an INS analog having an amino sequence of:

TABLE-US-00067 (SEQ ID NO: 58) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0253] Here, Example 22 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:58 is used in the expression plasmid.

Example 23: Recombinant Expression of INS Analog 23

[0254] Example 23 is an INS analog having an amino sequence of:

TABLE-US-00068 (SEQ ID NO: 59) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL EQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0255] Here, Example 23 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:59 is used in the expression plasmid.

Example 24: Recombinant Expression of INS Analog 24

[0256] Example 24 is an INS analog having an amino sequence of:

TABLE-US-00069 (SEQ ID NO: 60) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL EQLENYCGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0257] Here, Example 24 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:60 is used in the expression plasmid.

Example 25: Recombinant Expression of INS Analog 25

[0258] Example 25 is an INS analog having an amino sequence of:

TABLE-US-00070 (SEQ ID NO: 61) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0259] Here, Example 25 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:61 is used in the expression plasmid.

Example 26: Recombinant Expression of INS Analog 26

[0260] Example 26 is an INS analog having an amino sequence of:

TABLE-US-00071 (SEQ ID NO: 62) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSL YQLENYCGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0261] Here, Example 26 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:62 is used in the expression plasmid.

Example 27: Recombinant Expression of INS Analog 27

[0262] Example 27 is an INS analog having an amino sequence of:

TABLE-US-00072 (SEQ ID NO: 63) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP

[0263] Here, Example 27 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:63 is used in the expression plasmid.

Example 28: Recombinant Expression of INS Analog 28

[0264] Example 28 is an INS analog having an amino sequence of:

TABLE-US-00073 (SEQ ID NO: 64) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0265] Here, Example 28 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:64 is used in the expression plasmid.

Example 29: Recombinant Expression of INS Analog 29

[0266] Example 29 is an INS analog having an amino sequence of:

TABLE-US-00074 (SEQ ID NO: 65) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0267] Here, Example 29 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:65 is used in the expression plasmid.

Example 30: Recombinant Expression of INS Analog 30

[0268] Example 30 is an INS analog having an amino sequence of:

TABLE-US-00075 (SEQ ID NO: 66) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0269] Here, Example 30 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:66 is used in the expression plasmid.

Example 31: Recombinant Expression of INS Analog 31

[0270] Example 31 is an INS analog having an amino acid sequence of:

TABLE-US-00076 (SEQ ID NO: 67) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGEGGEGGEGGEGGEGGEGGEGGEVQLLESGGGLVQPGGSL RLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGR FTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYW GQGTLVTVSSPP.

[0271] Here, Example 31 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:67 is used in the expression plasmid.

Example 32: Recombinant Expression of INS Analog 32

[0272] Example 32 is an INS analog having an amino acid sequence of:

TABLE-US-00077 (SEQ ID NO: 68) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGEGGGGEGGGGEGGGGEGGGGEVQLLESGGGLVQPGGS LRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKG RFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPY WGQGTLVTVSSPP.

[0273] Here, Example 32 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:68 is used in the expression plasmid.

Example 33: Recombinant Expression of INS Analog 33

[0274] Example 33 is an INS analog having an amino acid sequence of:

TABLE-US-00078 (SEQ ID NO: 69) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGKGGGGKGGGGKGGGGKGGGGEVQLLESGGGLVQPGGS LRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKG RFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPY WGQGTLVTVSSPP.

[0275] Here, Example 33 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:69 is used in the expression plasmid.

Example 34: Recombinant Expression of INS Analog 34

[0276] Example 34 is an INS analog having an amino acid sequence of:

TABLE-US-00079 (SEQ ID NO: 70) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGAPAPAPAPAPAPAPAPAPAPGGGGEVQLLESGGGLVQ PGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYAD SVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVAD LYPYWGQGTLVTVSSPP.

[0277] Here, Example 34 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:70 is used in the expression plasmid.

Example 35: Recombinant Expression of INS Analog 35

[0278] Example 35 is an INS analog having an amino acid sequence of:

TABLE-US-00080 (SEQ ID NO: 71) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGEPEPEPEPEPEPEPEPEPEPGGGGEVQLLESGGGLVQ PGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYAD SVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVAD LYPYWGQGTLVTVSSPP.

[0279] Here, Example 35 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:71 is used in the expression plasmid.

Example 36: Recombinant Expression of INS Analog 36

[0280] Example 36 is an INS analog having an amino acid sequence of:

TABLE-US-00081 (SEQ ID NO: 72) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGKPKPKPKPKPKPKPKPKPKPGGGGEVQLLESGGGLVQ PGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYAD SVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVAD LYPYWGQGTLVTVSSPP.

[0281] Here, Example 36 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:72 is used in the expression plasmid.

Example 37: Recombinant Expression of INS Analog 37

[0282] Example 37 is an INS analog having an amino acid sequence of:

TABLE-US-00082 (SEQ ID NO: 73) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGPGPEPGPEPGPEPGPEPGPEPGPEPGPEPGPQEVQLLESGG GLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDIT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITS KVADLYPYWGQGTLVTVSSPP.

[0283] Here, Example 37 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:73 is used in the expression plasmid.

Example 38: Recombinant Expression of INS Analog 38

[0284] Example 38 is an INS analog having an amino acid sequence of:

TABLE-US-00083 (SEQ ID NO: 74) SFVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGPGPKPGPKPGPKPGPKPGPKPGPKPGPKPGPQEVQLLESGGG LVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYY ADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVA DLYPYWGQGTLVTVSSPP.

[0285] Here, Example 38 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:74 is used in the expression plasmid.

Example 39: Recombinant Expression of INS Analog 39

[0286] Example 39 is an INS analog having an amino acid sequence of:

TABLE-US-00084 (SEQ ID NO: 75) FVKQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0287] Here, Example 39 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:75 is used in the expression plasmid.

Example 40: Recombinant Expression of INS Analog 40

[0288] Example 40 is an INS analog having an amino acid sequence of:

TABLE-US-00085 (SEQ ID NO: 76) FVKQHLCGSHLVEALRLVCGQRGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0289] Here, Example 40 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:76 is used in the expression plasmid.

Example 41: Recombinant Expression of INS Analog 41

[0290] Example 41 is an INS analog having an amino acid sequence of:

TABLE-US-00086 (SEQ ID NO: 77) FVKQHLCGSHLVEALRLVCGQRGFHYTPKTGGGGGGGIVQQCCHSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0291] Here, Example 41 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:77 is used in the expression plasmid.

Example 42: Recombinant Expression of INS Analog 42

[0292] Example 42 is an INS analog having an amino acid sequence of:

TABLE-US-00087 (SEQ ID NO: 78) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0293] Here, Example 42 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:78 is used in the expression plasmid.

Example 43: Recombinant Expression of INS Analog 43

[0294] Example 43 is an INS analog having an amino acid sequence of:

TABLE-US-00088 (SEQ ID NO: 79) FVKQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0295] Here, Example 43 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:79 is used in the expression plasmid.

Example 44: Recombinant Expression of INS Analog 44

[0296] Example 44 is an INS analog having an amino acid sequence of:

TABLE-US-00089 (SEQ ID NO: 80) FVKQHLCGSHLVEALRLVCGQRGFHYTPKTGGGGGGGIVEQCCHSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0297] Here, Example 44 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:80 is used in the expression plasmid.

Example 45: Recombinant Expression of INS Analog 45

[0298] Example 45 is an INS analog having an amino acid sequence of:

TABLE-US-00090 (SEQ ID NO: 81) FVKQHLCGSHLVEALRLVCGQRGFHYTPKTGGGGGGGIVQQCCHSICSL YQLENYCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGG SLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVK GRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYP YWGQGTLVTVSSPP.

[0299] Here, Example 45 is generated essentially as described for Example 1 except that a cDNA sequence encoding SEQ ID NO:81 is used in the expression plasmid.

[0300] In Vitro Function

Example 46: INS Analog Albumin-Binding Studies Via SPR

[0301] In vitro binding of various INS analogs to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit serum albumin is determined by SPR. In particular, the affinity of Examples 23 to 30 to serum albumin of these species is summarized below in Tables 1-8.

[0302] Binding of the INS analogs of Examples 23 to 30 to various serum albumins is carried out on Biacore 8K instrument. Immobilization of serum albumin to a Series S Sensor Chip CM5 surface is performed according to the manufacturer's instructions (Amine Coupling Kit BR-1000-50). Briefly, carboxyl groups on the sensor chip surfaces (flow cell 1 and 2) are activated by injecting 70 μL of a mixture containing 75 mg/ml EDC and 11.5 mg/ml NHS at 10 μL/min. Human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit serum albumin are diluted in 10 mM NaOAc pH 4.0 (BR-1003-49) at 1, 1, 3, 1, 1, 1, 1 and 1 μg/mL and then injected over the activated chip surfaces (flow cell 2, channel 1 to 7) at 10 μL/min for 90 sec (human, mouse, rat, pig and cow serum albumin are obtained from Sigma Aldrich (St. Louis, Mo.); cynomolgus monkey serum albumin is obtained from Holzel Diagnostika (Cologne, Germany); dog serum albumin is obtained from Molecular Innovations (Novi, Mich.); and rabbit serum albumin is obtained from Fitzgerald Industries International (Acton, Mass.)). The various serum albumins are covalently immobilized through free amines onto a carboxymethyl dextran-coated sensor chip CM5 targeting a surface density average of about 77 (58-98) RU. Excess reactive groups on the surfaces (flow cell 1 and 2) are deactivated by injecting 70 μL of 1 M ETA HCl—NaOH pH 8.5 at 10 μL/min.

[0303] Examples 23 to 30 are 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 sample is individually injecting sequentially across the immobilized serum albumins surface and then dissociates for 600 sec at 50 μL/min flow rate at 25° C. The surface is regenerated by injecting 10 mM glycine-HCl pH 1.5 (BR-1003-54) at 50 μL/min for 100 sec. The resulting sensorgrams are analyzed using Biacore 8K Insight Evaluation Software (version 2.0.15.12933) 1:1 binding kinetics or steady-state affinity model fitting to calculate the binding kinetic parameter association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K.sub.D)

TABLE-US-00091 TABLE 1 Binding Kinetics of Example 23 to Human, Cynomolgus Monkey, Mouse, Rat, Pig, Dog, Cow and Rabbit Serum Albumin at 25° C. Name ka (1/Ms) kd (1/s) K.sub.D (M) Human SA 1.2E+05 9.0E−05 7.3E−10 Cyno SA 1.2E+05 8.4E−04 6.8E−09 Mouse SA 1.2E+05 9.3E−03 7.6E−08 Rat SA 1.2E+05 6.5E−03 5.3E−08 Pig SA 9.5E+04 1.2E−02 1.3E−07 Dog SA 1.4E+05 3.3E−03 2.3E−08 Cow SA n/a (steady state) 5.9E−07 Rabbit SA No binding

[0304] K.sub.D is determined as 0.73, 6.8, 76, 53, 130, 23 and 590 nM for human, cynomolgus monkey, mouse, rat, pig, dog and cow serum albumin binding with Example 23, respectively.

TABLE-US-00092 TABLE 2 Binding Kinetics of Example 24 to Human, Cynomolgus Monkey, Mouse, Rat, Pig, Dog, Cow and Rabbit Serum Albumin at 25° C. Name ka (1/Ms) kd (1/s) K.sub.D (M) Human SA 1.4E+05 1.2E−04 9.1E−10 Cyno SA 1.3E+05 7.5E−04 5.5E−09 Mouse SA 1.5E+05 7.3E−03 4.9E−08 Rat SA 1.4E+05 5.7E−03 4.0E−08 Pig SA 1.0E+05 1.1E−02 1.0E−07 Dog SA 1.5E+05 2.4E−03 1.6E−08 Cow SA n/a (steady state) 4.3E−07 Rabbit SA No binding

[0305] K.sub.D is determined as 0.91, 5.5, 49, 40, 100, 16 and 430 nM for human, cynomolgus monkey, mouse, rat, pig, dog and cow serum albumin binding with Example 24, respectively.

TABLE-US-00093 TABLE 3 Binding Kinetics of Example 25 to Human, Cynomolgus Monkey, Mouse, Rat, Pig, Dog, Cow and Rabbit Serum Albumin at 25° C. Name ka (1/Ms) kd (1/s) K.sub.D (M) Human SA 2.6E+05 8.4E−05 3.2E−10 Cyno SA 2.3E+05 7.9E−04 3.5E−09 Mouse SA 2.2E+05 9.2E−03 4.2E−08 Rat SA 2.0E+05 6.6E−03 3.4E−08 Pig SA 1.3E+05 1.1E−02 9.0E−08 Dog SA 2.0E+05 3.3E−03 1.6E−08 Cow SA n/a (steady state) 3.9E−07 Rabbit SA No binding

[0306] K.sub.D is determined as 0.32, 3.5, 42, 34, 90, 16 and 390 nM for human, cynomolgus monkey, mouse, rat, pig, dog and cow serum albumin binding with Example 25, respectively.

TABLE-US-00094 TABLE 4 Binding Kinetics of Example 26 to Human, Cynomolgus Monkey, Mouse, Rat, Pig, Dog, Cow and Rabbit Serum Albumin at 25° C. Name ka (1/Ms) kd (1/s) K.sub.D (M) Human SA 1.9E+05 9.4E−05 4.9E−10 Cyno SA 1.8E+05 6.9E−04 3.9E−09 Mouse SA 2.0E+05 6.9E−03 3.5E−08 Rat SA 1.7E+05 5.6E−03 3.2E−08 Pig SA 1.1E+05 9.8E−03 8.7E−08 Dog SA 1.7E+05 2.3E−03 1.3E−08 Bovine SA n/a (steady state) 4.0E−07 Rabbit SA No binding

[0307] K.sub.D is determined as 0.49, 3.9, 35, 32, 87, 13 and 400 nM for human, cynomolgus monkey, mouse, rat, pig, dog and cow serum albumin binding with Example 26, respectively.

TABLE-US-00095 TABLE 5 Binding Kinetics of Example 27 to Human, Cynomolgus Monkey, Mouse, Rat, Pig, Dog, Cow and Rabbit Serum Albumin at 25° C. Name ka (1/Ms) kd (1/s) K.sub.D (M) Human SA 1.7E+05 1.3E−04 7.5E−10 Cyno SA 1.7E+05 7.8E−04 4.7E−09 Mouse SA 1.7E+05 7.9E−03 4.5E−08 Rat SA 1.9E+05 6.1E−03 3.2E−08 Pig SA 1.2E+05 1.0E−02 9.0E−08 Dog SA 1.8E+05 3.0E−03 1.7E−08 Cow SA n/a (steady state) 3.9E−07 Rabbit SA No binding

[0308] K.sub.D is determined as 0.75, 4.7, 45, 32, 90, 17 and 390 nM for human, cynomolgus monkey, mouse, rat, pig, dog and cow serum albumin binding with Example 27, respectively.

TABLE-US-00096 TABLE 6 Binding Kinetics of Example 28 to Human, Cynomolgus Monkey, Mouse, Rat, Pig, Dog, Cow and Rabbit Serum Albumin at 25° C. Name ka (1/Ms) kd (1/s) K.sub.D (M) Human SA 1.6E+05 1.2E−04 7.3E−10 Cyno SA 1.7E+05 6.7E−04 4.1E−09 Mouse SA 1.6E+05 6.0E−03 3.7E−08 Rat SA 1.8E+05 4.8E−03 2.6E−08 Pig SA 1.1E+05 8.4E−03 7.5E−08 Dog SA 1.7E+05 2.1E−03 1.2E−08 Bovine SA n/a (steady state) 4.0E−07 Rabbit SA No binding

[0309] K.sub.D is determined as 0.73, 4.1, 37, 26, 75, 12 and 400 nM for human, cynomolgus monkey, mouse, rat, pig, dog and cow serum albumin binding with Example 28, respectively.

TABLE-US-00097 TABLE 7 Binding Kinetics of Example 29 to Human, Cynomolgus Monkey, Mouse, Rat, Pig, Dog, and Cow Serum Albumin at 25° C. Name ka (1/Ms) kd (1/s) K.sub.D (M) Human SA 1.8E+05 1.3E−04 7.4E−10 Cyno SA 1.8E+05 7.8E−04 4.4E−09 Mouse SA 1.8E+05 8.7E−03 4.8E−08 Rat SA 2.0E+05 6.5E−03 3.2E−08 Pig SA 1.5E+05 1.3E−02 8.6E−08 Dog SA 1.9E+05 3.2E−03 1.6E−08 Bovine SA n/a (steady state) 3.8E−07 Rabbit SA No binding

[0310] K.sub.D is determined as 0.74, 4.4, 48, 32, 86, 16, 380 nM for human, cynomolgus monkey, mouse, rat, pig, dog, and cow serum albumin binding with Example 29, respectively.

TABLE-US-00098 TABLE 8 Binding Kinetics of Example 30 to Human, Cynomolgus Monkey, Mouse, Rat, Pig, Dog, and Cow Serum Albumin at 25° C. Name ka (1/Ms) kd (1/s) K.sub.D (M) Human SA 1.6E+05 1.1E−04 7.3E−10 Cyno SA 1.6E+05 6.9E−04 4.3E−09 Mouse SA 1.9E+05 7.1E−03 3.9E−08 Rat SA 1.7E+05 5.1E−03 3.0E−08 Pig SA 1.1E+05 9.2E−03 8.0E−08 Dog SA 1.7E+05 2.2E−03 1.3E−08 Bovine SA n/a (steady state) 3.7E−07 Rabbit SA No binding

[0311] K.sub.D is determined as 0.73, 4.3, 39, 30, 80, 13 and 370 nM for human, cynomolgus monkey, mouse, rat, pig, dog and cow serum albumin binding with Example 30, respectively.

Example 47: INS Analog In Vitro Potency at IR-A and IR-B

[0312] Preparing Membranes: Cellular membranes are 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 are from cell passages 6 to 12, depending on the receptor. Frozen cell pellets are thawed in ice-cold homogenization/resuspension buffer (50 mM Tris-HCl, pH 7.5) containing one Complete® protease inhibitor tablet with EDTA (Roche Diagnostics) per 50 mL of buffer. The cells are homogenized with an overhead motor driven Teflon®-glass Potter-Elvehjem homogenizer using 15 to 20 strokes, followed by centrifugation at 1100×g for 10 min at 4° C. The supernatant is saved on ice and the pellets are re-homogenized as before and centrifuged at 1100×g for 10 min at 4° C. All supernatants are combined and subsequently centrifuged at 35,000×g for 60 min at 4° C. The pellet is resuspended in buffer (4 to 5 ml/g of starting cell paste) containing protease inhibitors and quick frozen in liquid nitrogen prior to storage at −80° C. Protein concentration is determined using a BCA kit (ThermoScientific) with bovine serum albumin (BSA) as standard.

[0313] Receptor Binding Assay Protocol: Receptor binding affinities (Ki) are determined from a competitive radioligand binding assay with either human recombinant (3-[.sup.125I]-iodotyrosyl-A14)-insulin (2200 Ci/mmol) or human recombinant [.sup.125I]-insulin-like growth factor-1 (1680 to 2800 Ci/mmol), both obtained from Perkin Elmer (Waltham, Mass.). The assays are performed with a SPA method using polyvinyltoluene (PVT) wheat germ agglutinin-coupled SPA beads (Perkin Elmer). Assay buffer contains 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and either 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). Ten-point concentration response curves using three-fold serial dilutions of test samples or controls are prepared in Assay Buffer using a Freedom/Evo robot (Tecan). Fifty L of compound dilution is added to 96-well white, clear-bottom microplates (Corning, 3632) with a TeMO robot (Tecan) followed by radioligand (50 μL, about 40 μM final), membranes (50 μL, 0.1 to 0.4 μg/well) and SPA beads (50 μL, 0.1 to 0.15 mg/well), which are added using a Multiflo F/X (Biotek) bulk dispensing instrument. The highest final assay concentrations for test compounds and controls are shown in the table below:

TABLE-US-00099 TABLE 9 Receptor Binding Assay Concentrations/Controls. 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

[0314] Following a ten-hour incubation and bead settling at room temperature, radioactivity is determined using a Microbeta™ Trilux scintillation counter (Perkin Elmer) and expressed as counts per minute (CPM).

[0315] Samples are tested in three independent assays run on three different days (n=3). For each run, samples are randomized with BHI (SEQ ID NOS:3 and 4), IGF-1 (PeproTech, Inc.; Rocky Hill, N.J.) and AspB10 INS (His10Asp of SEQ ID NO:4) controls included on each plate.

[0316] Data Analysis for IR Assays: Each compound is tested with a single replicate concentration response curve per experiment. The maximum binding response (MAX) is determined in an 8 well/plate using assay buffer only, and the minimum binding or nonspecific response (MIN) is determined in each well using 100 nM BHI. All test sample concentration responses are normalized to this control response and calculated as a percent specific inhibition after correcting for nonspecific binding as shown below:

% Specific Inhibition=100−[(CPM−MIN)/(MAX−MIN)×100].

[0317] Percent specific inhibition (y-axis) is plotted versus the log concentration of compound (x-axis). Concentrations resulting in 50% inhibition of binding (IC.sub.50) are determined from four-parameter logistic non-linear regression analysis (Analyzer, version 15, GeneData Screener). The affinity constant (Ki) is calculated from the IC.sub.50 value based upon the equation Ki=IC.sub.50/(1+L/Kd), where L equals the concentration of radioligand used in the experiment, and Kd equals the equilibrium binding affinity constant of the radioligand determined from saturation binding analysis. Reported Ki values are shown as the geometric mean with the standard error (Delta Method Standard Error) and the number of independent replicate determinations used to calculate the geometric mean indicated by n.

TABLE-US-00100 TABLE 10 In Vitro Potency of Ex.amples 1 to 30 to IR-A hIR-A in hIR=A in hIR-A in 0.001% 0.1% 0.1% NP-40 Ki BSA Ki HSA Ki Name (nM) SEM N (nM) SEM N (nM) SEM N BHI 0.182 0.042 3 0.278 0.032 3 0.298 0.031 3 Ex. 1 19.8 4.6 4 21.3 2.74 7 10.1 1.0 2 Ex. 2 45.1 n/a 1 31.4 7.2 3 31.9 n/a 1 Ex. 3 — — — 45.2 5.3 2 — — — Ex. 4 — — — 591 n/a 1 — — — Ex. 5 — — — 57.0 n/a 1 — — — Ex. 6 — — — 65.5 n/a 1 — — — Ex. 7 14.3 6.0 5 36.1 11.0 5 36.3 n/a 1 Ex. 8 47.3 n/a 1 139 n/a 1 — — — Ex. 9 6.67 n/a 1 11.7 n/a 1 — — — Ex. 10 20.1 n/a 1 146 n/a 1 — — — Ex. 11 20.5 10.7 4 36.0 12.6 4 31.8 n/a 1 Ex. 12 25.0 6.8 5 37.9 15.6 5 42.3 n/a 1 Ex. 13 23.1 11.0 4 60.3 19.6 4 34.6 n/a 1 Ex. 14 163 26 4 282 27 4 124 n/a 1 Ex. 15 13.1 n/a 1 39.2 n/a 1 — — — Ex. 16 43.1 n/a 1 46.0 13.8 3 — — — Ex. 17 31.3 2.8 4 81.8 13.4 4 31.3 n/a 1 Ex. 18 89.5 28.9 4 204 73 4 518 n/a 1 Ex. 19 18.5 n/a 1 41.8 n/a 1 — — — Ex. 20 18.5 n/a 1 51.3 n/a 1 — — — Ex. 21 8.3 n/a 1 28.0 n/a 1 — — — Ex. 22 17.1 n/a 1 59.7 n/a 1 — — — Ex. 23 18.9 n/a 1 55.1 n/a 1 — — — Ex. 24 21.8 n/a 1 56.8 n/a 1 — — — Ex. 25 174 28 3 630 90 3 125 45 3 Ex. 26 105 13 3 389 30 3 40.6 13.8 3 Ex. 27 50.5 n/a 1 103 n/a 1 — — — Ex. 28 32.7 n/a 1 71.0 n/a 1 — — — Ex. 29 34.0 4.3 3 179 8 3 28.2 2.3 3 Ex. 30 49.3 9.9 3 127 23 3 17.2 3.1 3

TABLE-US-00101 TABLE 11 In Vitro Potency of Ex.amples 1 to 30 to IR-B hIR-B in hIR−B in hIR-B in 0.001% 0.1% 0.1% NP-40 Ki BSA Ki HSA Ki Name (nM) SEM N (nM) SEM N (nM) SEM N BHI 0.331 0.054 3 0.167 0.028 3 0.165 0.019 3 Ex. 1 52.1 n/a 1 56.6 n/a 1 24.9 n/a 1 Ex. 2 39.2 n/a 1 56.0 n/a 1 29.7 n/a 1 Ex. 3 — — — — — — — — — Ex. 4 — — — — — — — — — Ex. 5 — — — — — — — — — Ex. 6 — — — — — — — — — Ex. 7 45.4 n/a 1 120 n/a 1 47.1 n/a 1 Ex. 8 — — — — — — — — — Ex. 9 — — — — — — — — — Ex. 10 — — — — — — — — — Ex. 11 81.1 n/a 1 164 n/a 1 95.4 n/a 1 Ex. 12 42.8 n/a 1 113 n/a 1 62.1 n/a 1 Ex. 13 39.2 n/a 1 131 n/a 1 89.0 n/a 1 Ex. 14 234 n/a 1 378 n/a 1 447 n/a 1 Ex. 15 — — — — — — — — — Ex. 16 — — — — — — — — — Ex. 17 44.3 n/a 1 110 n/a 1 102 n/a 1 Ex. 18 134 n/a 1 299 n/a 1 239 n/a 1 Ex. 19 — — — — — — — — — Ex. 20 — — — — — — — — — Ex. 21 — — — — — — — — — Ex. 22 — — — — — — — — — Ex. 23 — — — — — — — — — Ex. 24 — — — — — — — — — Ex. 25 641 19 3 790 86 3 279 19 3 Ex. 26 214 49 3 405 66 3 105 15 3 Ex. 27 — — — — — — — — — Ex. 28 — — — — — — — — — Ex. 29 62.5 4.6 3 124 13 3 38.1 3.7 3 Ex. 30 60.0 12.8 3 100 6 3 24.5 5.2 3

[0318] In Vivo Function

Example 48: INS Analog Glucose Lowering in Streptozotocin (STZ)-Treated Mice

[0319] STZ mice: 11-12 week-old, male, C57B1/6NHsd mice from Envigo RSM Inc. (Indianapolis, Ind.) are allowed to acclimate for a minimum of 3 days. The mice are individually housed in shoebox caging with corn cob bedding and mouse water lixits. Environmental conditions are as follows: photoperiod of 12 hours light and 12 hours dark (may be interrupted for study-related activities), temperature of 20° C. to 26° C., and relative humidity of 30% to 70%.

[0320] STZ is prepared as follows: add vehicle to pre-weighed STZ to achieve a dosing concentration of 16.67 mg/mL. Gently swirl to mix until powder is dissolved. Solution is kept on wet ice, protected from light and is used within 3 hr of preparation. On days 5 and 9 of pre-dose phase, prior to each STZ administration, following an overnight fast (not to exceed 16 hr), animals are dosed i.p. at a dose volume 6 mL/kg (100 mg/kg), based on most recent body weight. Any animal with a body weight below 19 g is not administered STZ.

[0321] Ten days after a second STZ treatment, animals are assigned to the study using a block randomization allocation tool (BRAT) designed to achieve glucometer value (250 mg/dL to 500 mg/dL inclusion criteria) and body weight balance. A single dose of pre-formulated test article is administered into the subcutaneous space between the shoulder blades (interscapular) at a dose volume of 10 mL/kg. Any possible dosing errors are noted.

[0322] Following test article treatment, body weight is monitored each morning. Food intake is monitored on days 1 (thrice: 0 to 4 hr, 4 to 12 hr and 12 to 24 hr), 2, 3, 4, 5, 6, 7 and 8. Glucose measurements are taken via tail clip using glucometers (in duplicate) 0, 4, 12, 24, 36, 48, 72, 96, 120, 144 and 168 hr post dose. At 4, 12, 24, 36, 48 and 72 hr post dose, an additional 40 μL of whole blood is collected for determining compound concentration.

[0323] All data is presented as mean±SEM of 5 animals per group. Percent change in glucose is calculated for each time point as follows: percent change at X hours post dose=((X time point glucose−animal's time 0 post dose glucose)×100)−100.

[0324] As shown below in Table 12, the INS analogs of Examples 1 to 6 demonstrate a sustained reduction in whole blood glucose levels after a single, 300 nmol/kg injection. Likewise, and as shown below in Tables 13 and 14, the INS analogs of Examples 7 to 18 demonstrate sustained reduction in whole blood glucose levels after a single, 200 nmol/kg injection.

TABLE-US-00102 TABLE 12 Glucose-Lowering Effect of 300 nmol/kg Dose of Examples 1 to 6 in STZ-Treated Mice Dose % Change in Glucose Name (nmol/kg) N 4 hr 12 hr 24 hr 36 hr 48 hr 72 hr 96 hr 120 hr 144 hr 168 hr Vehicle n/a 5 1.5 −9.9 1.8 −5.9 −0.1 4.2 −11.7 2.1 −15.2 −12.4 Ex. 1 300 5 −54.6 −26.2 −71.5 −75.8 −78.0 −77.0 −52.9 −33.9 −20.7 −19.1 Ex. 2 300 5 −66.6 −75.9 −80.0 −82.3 −82.6 −85.5 −52.7 −35.3 −24.0 −23.7 Ex. 3 300 5 −56.6 −61.7 −70.8 −82.9 −80.7 −80.1 −66.8 −45.9 −29.6 −25.0 Ex. 4 300 5 −33.5 −46.2 −63.6 −64.3 −73.3 −73.2 −63.6 −62.2 −50.4 −42.0 Ex. 5 300 5 −61.2 −71.7 −75.4 −81.8 −79.9 −84.8 −81.9 −75.4 −66.6 −52.5 Ex. 6 300 5 −64.5 −58.3 −74.9 −84.5 −79.4 −81.5 −62.2 −54.8 −41.8 −26.9 Vehicle n/a 5 5.8 18.9 4.9 6.2 2.0 3.0 4.2 3.0 4.2 8.0 Ex. 1 300 5 11.3 27.0 3.7 2.5 2.9 2.3 2.8 3.6 3.9 6.9 Ex. 2 300 5 1.7 1.8 1.4 0.6 2.0 1.5 6.4 4.4 4.9 7.2 Ex. 3 300 5 5.2 11.1 5.1 1.8 3.7 2.9 3.8 3.9 4.6 3.4 Ex. 4 300 5 7.3 6.8 6.7 8.8 3.4 4.5 3.9 2.2 2.2 2.9 Ex. 5 300 5 7.1 5.2 2.8 1.9 2.0 1.7 1.9 2.8 2.2 3.7 Ex. 6 300 5 6.7 8.9 4.9 0.8 2.9 4.3 4.7 6.8 4.9 3.6

TABLE-US-00103 TABLE 13 Glucose-Lowering Effect of 200 nmol/kg Dose of Ex.amples 9 and 11 to 16 in STZ-Treated Mice Dose % Change in Glucose Name (nmol/kg) N 4 hr 12 hr 24 hr 36 hr 48 hr 72 hr 96 hr 120 hr 144 hr 168 hr Vehicle n/a 5 24.4 18.2 19.2 27.0 20.9 24.2 13.1 17.0 16.4 19.0 Ex. 9 200 5 −70.9 −42.0 −73.0 −75.0 −84.0 −83.1 −54.6 −33.2 −11.7 −14.7 Ex. 11 200 5 −58.3 −42.4 −50.6 −46.9 −67.7 −73.2 −74.7 −69.3 −61.8 −49.5 Ex. 12 200 5 −57.9 −58.9 −75.2 −71.4 −81.5 −85.1 −84.4 −73.5 −54.2 −47.7 Ex. 13 200 5 −45.3 −44.7 −62.6 −69.0 −79.2 −79.8 −73.6 −66.4 −62.9 −54.9 Ex. 14 200 5 −35.9 −30.9 −66.1 −58.4 −79.9 −78.5 −77.7 −68.1 −60.3 −55.5 Ex. 15 200 5 −68.0 −71.3 −75.4 −80.1 −79.2 −76.4 −52.2 −23.9 −17.8 −16.3 Ex. 16 200 5 −59.5 −53.6 −64.7 −75.8 −75.4 −74.2 −43.2 −29.9 −21.5 −25.4 Vehicle n/a 5 7.8 4.7 5.9 6.1 4.7 7.5 7.1 8.2 8.1 8.3 Ex. 9 200 5 2.7 6.3 4.6 3.9 0.3 0.5 1.6 3.7 2.0 2.6 Ex. 11 200 5 3.9 9.2 9.9 11.1 6.4 3.0 1.3 2.8 2.0 6.7 Ex. 12 200 5 9.9 10.1 2.1 4.2 1.3 0.7 2.0 3.6 9.2 7.0 Ex. 13 200 5 9.0 12.0 7.1 9.4 2.6 1.3 2.8 4.3 3.7 7.2 Ex. 14 200 5 11.0 9.1 8.0 10.0 1.5 3.1 2.4 1.7 4.3 2.9 Ex. 15 200 5 5.3 2.9 4.0 1.4 3.6 4.6 4.7 6.3 5.5 4.2 Ex. 16 200 5 5.3 9.6 6.8 3.7 4.9 4.1 8.1 5.4 4.0 2.8

TABLE-US-00104 TABLE 14 Glucose Lowering Effect of 200 nmol/kg Dose of Ex.amples 7, 8, 10, 14, 17 and 18 in STZ-Treated Mice Dose % Change in Glucose Name (nmol/kg) N 4 hr 12 hr 24 hr 36 hr 48 hr 72 hr 96 hr 120 hr 144 hr 168 hr Vehicle n/a 5 3.85 7.49 2.56 18.83 10.33 3.84 0.08 1.81 1.08 −6.92 Ex. 7 200 5 −63.1 −41.3 −71.1 −67.2 −80.0 −80.2 −59.6 −37.8 −15.8 −17.1 Ex. 8 200 5 −46.1 −50.8 −71.5 −73.8 −78.0 −81.8 −65.7 −52.5 −43.1 −17.4 Ex. 10 200 5 −55.6 −56.1 −71.6 −58.8 −73.2 −79.6 −73.6 −66.7 −58.7 −47.3 Ex. 14 200 5 −30.2 −26.7 −66.7 −59.1 −71.3 −77.5 −74.6 −53.2 −55.6 −49.4 Ex. 17 200 5 −45.8 −22.3 −62.8 −49.6 −78.2 −78.4 −84.6 −70.5 −73.8 −63.7 Ex. 18 200 5 −50.1 −30.7 −67.0 −69.2 −81.0 −83.4 −81.4 −72.4 −62.0 −52.2 Vehicle n/a 5 2.3 4.6 5.0 4.3 7.2 5.3 4.1 3.8 6.5 7.2 Ex. 7 200 5 4.9 10.0 2.8 8.1 2.7 3.8 5.6 8.8 13.5 7.9 Ex. 8 200 5 9.8 7.0 2.0 2.6 1.1 1.1 5.2 7.1 6.4 6.6 Ex. 10 200 5 3.4 7.5 1.4 3.9 2.7 1.1 3.0 4.8 1.1 1.5 Ex. 14 200 5 6.2 8.8 3.1 9.2 2.8 2.1 1.3 3.0 5.4 5.7 Ex. 17 200 5 7.1 3.5 4.7 11.4 2.7 6.1 1.7 4.7 1.4 1.6 Ex. 18 200 5 4.6 3.5 3.6 6.5 1.9 0.5 2.2 2.9 3.8 7.2

Example 49: INS Analog Glucose Lowering in STZ-Treated Rats

[0325] 390-425 g, male Sprague Dawley rats from Envigo RMS Inc. (Indianapolis, Ind.) are allowed to acclimate for a minimum of 3 days. The rats are individually housed in shoebox caging with corn cob bedding and water lixits providing water ad libitum. Rats are fed Teklad Global Diets' Rodent 2014 feed. Environmental conditions as follows: photoperiod of 12 hr light and 12 hr dark (may be interrupted for study-related activities), temperature of 20° C. to 26° C., and relative humidity of 30% to 70%.

[0326] STZ is prepared as follows: 19 mL of cold sterile saline is added to a STZ vial (Zanosar®, Teva Parenteral Medicines, Inc., Irvine, Calif.) and is gently mixed until powder is dissolved. Repeat with a second vial of STZ, keep both on wet ice, and shield from light. These solutions are good for 3 hr under stated conditions. On day 8 of pre-dose phase, following a 6-hr fast, animals are dosed i.v. at a dose volume 0.8 mL/kg (40 mg/kg), based on most recent body weight. STZ dosing is done under anesthesia with Isoflurane. Rats are observed until fully awake.

[0327] Three days after STZ treatment, animals are assigned to the study using a BRAT designed to achieve glucometer value (450 mg/dL to 550 mg/dL inclusion criteria) and body weight balance. 50 out of the 60 rats are put on study. A single dose of pre-formulated test article (INS analogs at 50, 100, 200 and 400 nmol/kg in 20 mM histidine, 50 mM NaCl, pH 6.0) is administered into the subcutaneous space at a dose volume of 5 mL/kg.

[0328] Following test article treatment, body weight and food intake is monitored each morning (days 1-11 of dosing phase). Glucose measurements are taken via tail clip using glucometers (AccuChek® Aviva®, Roche, Indianapolis, Ind.) (in duplicate) at 0, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216 and 240 hr post dose.

[0329] All data is presented as mean±SEM of 4-5 animals per group. Percent change in glucose is calculated for each time point as follows: percent change at X hours post dose=((X time point glucose−animal's time 0 post dose glucose)×100)−100.

[0330] As shown below in Table 15, all four INS analogs demonstrate a dose-dependent (50, 100, 200 and 400 nmol/kg) reduction in whole blood glucose levels after a single injection.

TABLE-US-00105 TABLE 15 Glucose-Lowering Effect of Varied Doses of Ex.amples 25 and 26 in STZ-Treated Rats Dose % Change in Glucose (nmol/ 2 4 6 8 10 12 18 24 36 48 72 96 120 144 Name kg) N hr hr hr hr hr hr hr hr hr hr hr hr hr hr Vehicle n/a 5 −3.2 12.2 −8.6 −2.4 5.6 13.0 3.7 14.4 16.3 −1.1 1.7 0.9 −0.6 6.4 Ex. 25  50 5 −1.7 −4.5 12.5 −4.8 −8.3 12.1 5.7 −2.8 12.1 10.6 −6.6 11.1 −9.3 −2.5 Ex. 25 100 5 −2.6 −9.8 13.8 13.4 −7.3 20.5 11.0 −7.7 16.4 1.4 11.0 −6.1 −8.1 0.5 Ex. 25 200 5 13.7 26.5 29.9 31.0 14.9 16.3 2.9 38.0 10.0 26.2 41.1 45.1 23.3 11.5 Ex. 25 400 5 −6.3 24.1 40.4 54.1 43.3 −8.1 26.9 62.7 14.8 65.1 75.6 73.9 68.6 54.7 Ex. 26  50 5 6.6 −7.0 −3.5 −7.6 13.5 23.1 16.9 −6.9 17.3 1.5 11.5 19.6 19.0 −7.6 Ex. 26 100 5 1.1 −9.8 15.3 19.2 14.8 10.7 17.4 40.4 10.8 31.9 13.5 14.9 −9.7 1.9 Ex. 26 200 5 7.1 10.7 19.3 22.2 −5.0 9.5 −8.8 50.7 −8.2 43.9 53.8 55.7 51.4 28.2 Ex. 26 400 5 −4.0 18.4 44.0 48.7 46.3 27.2 22.5 72.9 37.8 81.8 85.8 82.6 73.9 61.8 Vehicle n/a 5 6.1 2.7 7.2 8.7 9.2 2.8 7.3 13.0 2.2 5.0 3.1 2.4 5.8 6.2 Ex. 25  50 5 3.4 5.2 5.7 9.8 8.9 4.2 2.4 7.7 4.3 7.3 3.4 3.9 4.3 4.8 Ex. 25 100 5 8.8 6.8 5.3 9.0 13.5 5.7 3.8 6.9 7.6 4.7 5.3 5.4 5.0 5.7 Ex. 25 200 5 8.5 3.8 9.4 12.5 16.5 2.3 6.1 12.6 4.9 4.0 6.0 12.0 4.9 6.2 Ex. 25 400 5 10.6 7.4 8.4 10.6 6.2 10.6 9.0 19.0 6.7 12.0 6.4 8.8 9.7 10.2 Ex. 26  50 5 9.3 7.2 7.7 8.2 13.2 8.6 8.5 7.3 8.9 6.3 23.1 21.5 21.0 23.7 Ex. 26 100 5 5.9 7.4 9.1 11.6 12.1 6.9 14.4 11.4 20.4 16.5 6.6 6.0 5.1 5.1 Ex. 26 200 5 1.7 6.1 2.8 5.6 6.2 1.7 4.3 9.4 2.2 6.0 4.5 10.4 8.8 7.6 Ex. 26 400 5 5.4 14.9 12.8 10.4 16.5 11.9 9.5 6.8 10.9 2.3 1.2 2.0 4.8 5.6 Glucose-Lowering Effect of Varied Doses of Ex.amples 25 and 26 in STZ-Treated Rats Dose % Change in Glucose (nmol/ 168 192 216 240 Name kg) hr hr hr hr Vehicle n/a 3.7 4.3 4.7 14.5 Ex. 25  50 3.8 2.6 0.0 6.4 Ex. 25 100 −1.8 1.9 7.1 13.9 Ex. 25 200 17.4 −2.5 6.3 18.1 Ex. 25 400 32.8 17.9 15.2 3.6 Ex. 26  50 13.5 15.0 10.1 −5.9 Ex. 26 100 7.1 7.0 15.3 16.7 Ex. 26 200 24.4 18.7 −9.0 10.0 Ex. 26 400 44.1 54.5 23.8 −5.5 Vehicle n/a 3.8 5.3 6.8 5.4 Ex. 25  50 7.3 4.7 6.4 6.5 Ex. 25 100 4.7 6.0 4.6 6.3 Ex. 25 200 7.4 7.9 4.0 3.7 Ex. 25 400 7.8 11.7 10.2 9.1 Ex. 26  50 22.2 22.9 23.7 25.2 Ex. 26 100 5.4 2.6 4.4 4.5 Ex. 26 200 2.9 1.0 3.1 2.2 Ex. 26 400 8.1 6.8 9.5 9.5

TABLE-US-00106 TABLE 16 Glucose Lowering Effect of Varied Doses of Examples 29 and 30 in STZ-Treated Rats % change in Glucose Dose 2 4 6 8 10 12 18 24 36 48 72 96 120 Name (nmol/kg) N hr hr hr hr hr hr hr hr hr hr hr hr hr Vehicle n/a 5 3.0 3.9 −9.7 −4.9 11.3 16.8 12.8 −3.0 18.6 6.9 7.0 11.7 7.0 Ex. 29 50 5 1.5 0.9 −3.9 11.5 2.7 15.4 5.0 15.8 12.6 10.3 10.1 10.0 −3.9 Ex. 29 100 5 6.1 2.1 14.1 19.4 −0.9 7.2 −5.4 32.8 3.2 26.8 40.2 27.5 21.8 Ex. 29 200 5 −1.1 −3.4 −8.1 21.7 13.0 5.7 −5.6 38.1 6.2 35.1 71.1 75.5 64.6 Ex. 29 400 5 11.8 36.0 63.8 74.6 67.7 41.3 35.2 70.3 38.8 83.8 84.7 84.7 86.8 Ex. 30 50 5 0.7 −0.4 11.4 −8.9 2.9 12.0 0.1 −5.2 11.4 16.6 15.0 19.0 15.8 Ex. 30 100 5 −4.3 −5.1 16.6 16.6 11.1 3.8 0.3 16.6 1.3 28.6 35.5 25.3 29.3 Ex. 30 200 5 7.3 −0.9 19.5 19.9 34.5 0.8 −8.3 49.7 1.2 45.6 67.2 72.2 66.7 Ex. 30 400 5 −1.8 18.0 38.5 66.9 42.0 11.8 26.1 62.7 38.0 74.8 83.9 85.1 84.2 Vehicle n/a 5 3.9 8.6 5.7 7.3 4.4 4.3 4.2 3.3 4.6 4.7 4.9 5.0 5.8 Ex. 29 50 5 2.2 3.4 3.3 3.2 8.4 4.8 2.2 13.1 3.1 4.4 2.9 3.0 4.3 Ex. 29 100 5 3.5 4.0 2.3 4.6 5.6 3.1 5.7 8.8 3.5 2.8 6.1 5.0 3.4 Ex. 29 200 5 4.8 4.1 7.7 7.8 13.8 6.4 4.7 11.4 2.9 6.4 3.6 6.4 6.0 Ex. 29 400 5 3.3 5.9 11.8 6.3 5.6 8.4 7.2 6.5 7.9 0.2 1.4 1.7 1.2 Ex. 30 50 5 4.0 5.9 6.0 6.8 7.3 4.1 3.2 5.5 4.2 7.1 8.2 5.6 4.3 Ex. 30 100 5 4.9 1.7 3.7 7.6 10.1 6.2 4.3 4.1 2.6 8.7 6.0 10.4 9.6 Ex. 30 200 5 4.6 6.6 3.4 14.1 16.4 5.8 2.3 11.6 1.8 9.1 5.5 3.1 2.5 Ex. 30 400 5 6.3 8.1 11.4 5.8 7.9 4.0 8.8 13.9 10.4 9.4 2.5 1.6 2.2 Glucose Lowering Effect of Varied Doses of Examples 29 and 30 in STZ-Treated Rats % change in Glucose Dose 144 168 192 216 240 Name (nmol/kg) hr hr hr hr hr Vehicle n/a 3.4 4.5 12.1 8.7 11.0 Ex. 29 50 −8.5 0.7 6.5 3.3 4.8 Ex. 29 100 18.8 17.6 −6.0 −8.7 −2.2 Ex. 29 200 53.0 41.1 23.7 14.6 −3.6 Ex. 29 400 85.5 84.0 70.8 64.5 44.9 Ex. 30 50 12.7 −4.1 2.7 4.0 0.2 Ex. 30 100 15.2 −9.4 −6.8 −4.2 −0.2 Ex. 30 200 65.6 39.7 25.0 21.7 16.0 Ex. 30 400 82.0 72.1 61.8 57.0 43.4 Vehicle n/a 5.6 4.3 6.3 5.4 3.6 Ex. 29 50 3.8 2.5 1.3 3.9 3.3 Ex. 29 100 4.0 3.5 4.6 5.2 3.4 Ex. 29 200 8.4 10.2 5.1 4.7 5.7 Ex. 29 400 1.1 1.8 3.4 8.7 7.6 Ex. 30 50 3.5 5.2 6.7 5.3 6.8 Ex. 30 100 3.8 6.9 5.1 4.0 1.8 Ex. 30 200 7.4 8.2 8.9 6.2 3.7 Ex. 30 400 3.8 8.0 6.8 6.9 8.0

Example 50: INS Analog Pharmacokinetics in STZ-Treated Rats

[0331] The pharmacokinetics of the INS analogs herein are tested in an STZ-induced diabetic rat model. Male STZ-treated rats are administered single, subcutaneous doses 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 is collected from each animal pre-dose and at 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216 and 240 hr post-dose. The blood samples are processed to K3EDTA plasma and are stored frozen at about −70° C. The INS analog concentrations in plasma are measured at Eli Lilly and Company (Indianapolis, Ind.), and the concentration-time data are used to calculate pharmacokinetic parameters (see, Table 12).

[0332] Plasma analog concentrations are measured from the animals by immunoaffinity-LC/MS using a Thermo orbitrap mass spectrometer (Q/Exactive or Fusion Lumos) coupled to a Dionex Ultimate 3000 UPLC system. The analogs are immunoprecipitated from K.sub.3EDTA 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). Following wash steps to remove non-specifically bound proteins, the variants are reduced (triethylphosphine, Aldrich 245275-5G), alkylated (2-iodoethanol, Aldrich 176850-25G) and are digested (Trypsin Gold, Promega E2019-12). The subsequent tryptic peptides from various regions of the variants are measured by LC/MS over the range of 0.293 nM to 150 nM as a surrogate measure of intact analog.

[0333] The pharmacokinetics of Examples 29 and 30 are about linear over the range of doses tested (50 to 400 nmol/kg SC). Apparent clearance for the two analogs ranges from 2.9 mL/hr/kg to 4.7 mL/hr/kg, and their elimination half-lives ranges from 25 hr to 42 hr (see, Table 17).

TABLE-US-00107 TABLE 17 Mean Pharmacokinetic Parameters of INS Analogs of Examples 29 and 30 Following Single 50, 100, 200 or 400 nmol/kg Subcutaneous Doses to Male STZ-Cmax/DoseTreated Rats. Dose (nmol/ AUC.sub.0-∞ (kg * nmol/ Tmax CL/F t½ Name kg) (hr * uM) L/nmol) (hr) (mL/hr/kg) (hr) Ex. 29 50 16.3 2.92 43 3.14 33.5 (2.7) (0.6) (H) (0.6) (3.8) 100 31.1 3.11 34 3.25 42.1 (3.2) (0.8) (13) (0.4) (5.6) 200 69.8 3.80 34 2.99 41.7 (16) (0.8) (10) (0.7) (3.0) 400 150 3.56 22 2.77 25.1 (30) (0.5) (15) (0.7) (2.9) Ex. 30 50 24.8 4.71 36 2.09 34.5 (5.5) (1.8) (8) (0.4) (5.2) 100 39.2 3.90 53 2.59 27.7 (5.1) (0.8) (25) (0.3) (3.3) 200 69.3 3.68 31 2.94 32.1 (10) (1.2) (H) (0.5) (3.3) 400 180 3.87 96 2.26 33.3 (24) (0.6) (0) (0.3) (1.5) Mean +/− (SD), N = 5 Abbreviations: AUC.sub.0-∞ = area under the curve from time 0 hours to infinity, CL/F = clearance/bioavailability, T.sub.max = time to maximal concentration, C.sub.max/Dose = maximum observed plasma concentration divided by the dose, t.sub.1/2 = half−life.

SEQUENCES

[0334] The following nucleic and/or amino acid sequences are referred to in the disclosure and are provided below for reference.

TABLE-US-00108 SEQ ID NO: 1-human preproinsulin (110 amino acids; NCBI Ref. No. NP_001278826.1) MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPK TRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENY CN SEQ ID NO: 2-human proinsulin (86 amino acids; 25-110 of NCBI Ref. No. NP_001278826.1) FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQ PLALEGSLQKRGIVEQCCTSICSLYQLENYCN SEQ ID NO: 3-human INS A chain (21 amino acids; 90-110 of NCBI Ref. No. NP_001278826.1) GIVEQCCTSICSLYQLENYCN SEQ ID NO: 4-human INS B chain (30 amino acids; 25-54 of NCBI Ref. No. NP_001278826.1) FVNQHLCGSHLVEALYLVCGERGFFYTPKT SEQ ID NO: 5-human INS receptor-A (1370 amino acids; NCBI Ref. No. NP_001073285.1) with C-terminal C9 tag MATGGRRGAAAAPLLVAVAALLLGAAGHLYPGEVCPGMDIRNNLTRLHELENC SVIEGHLQILLMFKTRPEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIR GSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDS VEDNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCP TICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHF QDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCT PCLGPCPKVCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLG LIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWS KHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCEN ELLKFSYIRTSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACG SNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTY GAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQA EDSELFELDYCLKGLKLPSRTWSPPFESEDSQKHNQSEYEDSAGECCSCPKTDSQI LKELEESSFRKTFEDYLHNVVFVPRPSRKRRSLGDVGNVTVAVPTVAAFPNTSST SVPTSPEEHRPFEKVVNKESLVISGLRHFTGYRIELQACNQDTPEERCSVAAYVSA RTMPEAKADDIVGPVTHEIFENNVVHLMWQEPKEPNGLIVLYEVSYRRYGDEEL HLCVSRKHFALERGCRLRGLSPGNYSVRIRATSLAGNGSWTEPTYFYVTDYLDVP SNIAKIIIGPLIFVFLFSVVIGSIYLFLRKRQPDGPLGPLYASSNPEYLSASDVFPCSV YVPDEWEVSREKITLLRELGQGSFGMVYEGNARDIIKGEAETRVAVKTVNESASL RERIEFLNEASVMKGFTCHHVVRLLGVVSKGQPTLVVMELMAHGDLKSYLRSLR PEAENNPGRPPPTLQEMIQMAAEIADGMAYLNAKKFVHRDLAARNCMVAHDFT VKIGDFGMTRDIYETDYYRKGGKGLLPVRWMAPESLKDGVFTTSSDMWSFGVV LWEITSLAEQPYQGLSNEQVLKFVMDGGYLDQPDNCPERVTDLMRMCWQFNPK MRPTFLEIVNLLKDDLHPSFPEVSFFHSEENKAPESEELEMEFEDMENVPLDRSSH CQREEAGGRDGGSSLGFKRSYEEHIPYTHMNGGKKNGRILTLPRSNPSAAAGTET SQVAPA SEQ ID NO: 6-human INS receptor-B (1382 amino acids; NCBI Ref. No. NP_000199.2) with C-terminal C9 tag MATGGRRGAAAAPLLVAVAALLLGAAGHLYPGEVCPGMDIRNNLTRLHELENC SVIEGHLQILLMFKTRPEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIR GSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDS VEDNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCP TICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHF QDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCT PCLGPCPKVCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLG LIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWS KHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCEN ELLKFSYIRTSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACG SNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTY GAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQA EDSELFELDYCLKGLKLPSRTWSPPFESEDSQKHNQSEYEDSAGECCSCPKTDSQI LKELEESSFRKTFEDYLHNVVFVPRKTSSGTGAEDPRPSRKRRSLGDVGNVTVAV PTVAAFPNTSSTSVPTSPEEHRPFEKVVNKESLVISGLRHFTGYRIELQACNQDTPE ERCSVAAYVSARTMPEAKADDIVGPVTHEIFENNVVHLMWQEPKEPNGLIVLYE VSYRRYGDEELHLCVSRKHFALERGCRLRGLSPGNYSVRIRATSLAGNGSWTEPT YFYVTDYLDVPSNIAKIIIGPLIFVFLFSVVIGSIYLFLRKRQPDGPLGPLYASSNPEY LSASDVFPCSVYVPDEWEVSREKITLLRELGQGSFGMVYEGNARDIIKGEAETRV AVKTVNESASLRERIEFLNEASVMKGFTCHHVVRLLGVVSKGQPTLVVMELMA HGDLKSYLRSLRPEAENNPGRPPPTLQEMIQMAAEIADGMAYLNAKKFVHRDLA ARNCMVAHDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMAPESLKDGVFT TSSDMWSFGVVLWEITSLAEQPYQGLSNEQVLKFVMDGGYLDQPDNCPERVTD LMRMCWQFNPKMRPTFLEIVNLLKDDLHPSFPEVSFFHSEENKAPESEELEMEFE DMENVPLDRS SHCQREEAGGRDGGS SLGFKRSYEEHIPYTHMNGGKKNGRILTL PRSNPSAAAGTETSQVAPA SEQ ID NO: 7-VHH moiety #1 (MC6.1C22.43) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGG VDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKV ADLYPYWGQGTLVTVSSPP SEQ ID NO: 8-VHH moiety #2 (MC6.1) EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGS VDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRD ANLYDYWGQGTLVTVSS SEQ ID NO: 9-VHH moiety #3 (MC6.1C80.43) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGG VDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKV ADLYPYWGQGTLVTVSSPP SEQ ID NO: 10-L.sub.1 (basic sequence of (GGGGQ).sub.n) (GGGGQ).sub.n SEQ ID NO: 11-L.sub.1 (basic sequence of (GGGQ).sub.n) (GGGQ).sub.n SEQ ID NO: 12-L.sub.1 (basic sequence of (GGGGS).sub.n) (GGGGS).sub.n SEQ ID NO: 13-L.sub.1 (basic sequence of (PGPQ).sub.n) (PGPQ).sub.n SEQ ID NO: 14-L.sub.1 (basic sequence of (PGPA).sub.n) (PGPA).sub.n SEQ ID NO: 15-L.sub.1 (basic sequence of (GGE).sub.nGG) (GGE).sub.nGG SEQ ID NO: 16-L.sub.1 (basic sequence of (GGGGE).sub.nGGGG) (GGGGE).sub.nGGGG SEQ ID NO: 17-L.sub.1 (basic sequence of (GGGGK).sub.nGGGG) (GGGGK).sub.nGGGG SEQ ID NO: 18-L.sub.1 (basic sequence of (GGGG(AP).sub.nGGGG) GGGG(AP)GGGG SEQ ID NO: 19-L.sub.1 (basic sequence of GGGG(EP).sub.n) GGGG(EP).sub.nGGGG SEQ ID NO: 20-L.sub.1 (basic sequence of GGGG(KP).sub.nGGGG) GGGG(KP)GGGG SEQ ID NO: 21-L.sub.1 (basic sequence of (PGPE).sub.nPGPQ) (PGPE).sub.nPGPQ SEQ ID NO: 22-L.sub.1 (basic sequence of (PGPK).sub.nPGPQ) (PGPK).sub.nPGPQ SEQ ID NO: 23-L.sub.1 #1 ((GGGGQ).sub.5) GGGGQGGGGQGGGGQGGGGQGGGGQ SEQ ID NO: 24-L.sub.1 #2 ((PGPQ)) PGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQ SEQ ID NO: 25-L.sub.1 #3 ((PGPA).sub.8) PGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPA SEQ ID NO: 26-L.sub.1 #4 (G.sub.2E).sub.7G.sub.2 GGEGGEGGEGGEGGEGGEGGEGG SEQ ID NO: 27-L.sub.1 #5 (G.sub.4E).sub.4G.sub.4 GGGGEGGGGEGGGGEGGGGEGGGG SEQ ID NO: 28-L.sub.1 #6 (G.sub.4K).sub.4G.sub.4 GGGGKGGGGKGGGGKGGGGKGGGG SEQ ID NO: 29-L.sub.1 #7 (G.sub.4(AP).sub.10G.sub.4) GGGGAPAPAPAPAPAPAPAPAPAPGGGG SEQ ID NO: 30-L.sub.1 #7 (G.sub.4(EP).sub.10G.sub.4) GGGGEPEPEPEPEPEPEPEPEPEPGGGG SEQ ID NO: 31-L.sub.1 #8 (G.sub.4(KP).sub.10G.sub.4) GGGGKPKPKPKPKPKPKPKPKPKPGGGG SEQ ID NO: 32-L.sub.1 #9 ((PGPE).sub.7PGPQ) PGPEPGPEPGPEPGPEPGPEPGPEPGPEPGPQ SEQ ID NO: 33-L.sub.1 #10 ((PGPK).sub.7PGPQ) PGPKPGPKPGPKPGPKPGPKPGPKPGPKPGPQ SEQ ID NO: 34-L.sub.2 #1 GGGGGG SEQ ID NO: 35-L.sub.2 #2 GGGSGGSGGG SEQ ID NO: 36-L.sub.2 #3 GGGSGGSGGSGGG SEQ ID NO: 37-INS Analog #1 (SCI(A21G)-(PGPA).sub.8-MC6.1) FVNQHLCGSHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTLVTVSS SEQ ID NO: 38-INS Analog #2 (SCI(B3D, A21G)-(PGPA).sub.8-MC6.1) FVDQHLCGSHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTLVTVSS SEQ ID NO: 39-INS Analog #3 (SCI(B9A, A21G)-(PGPA).sub.8-MC6.1) FVNQHLCGAHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLEN YCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRL SCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITYYADSVKGRFTISRDNS KNTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTLVTVSS SEQ ID NO: 40-INS Analog #4 (SCI(B16E, A21G)-(PGPA).sub.8-MC6.1) FVNQHLCGSHLVEALELVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTLVTVSS SEQ ID NO: 41-INS Analog #5 (SCI(B16H, A21G)-(PGPA).sub.8-MC6.1) FVNQHLCGSHLVEALHLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTLVTVSS SEQ ID NO: 42-INS Analog #6 (SCI(B25H, A21G)-(PGPA).sub.8-MC6.1) FVNQHLCGSHLVEALYLVCGERGFHYTPKTGGGGGGGIVEQCCTSICSLYQLEN YCGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRL SCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITYYADSVKGRFTISRDNS KNTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTLVTVSS SEQ ID NO: 43-INS Analog #7 (SCI(B3S, A21G)-(PGPA).sub.8-MC6.1C22.43) FVSQHLCGSHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 44-INS Analog #8 (SCI(B3S, B25H, A21G)-(PGPA).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALYLVCGERGFHYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 45-INS Analog #9 (SCI(B3S, B25H, A8H, A21G)-(PGPA).sub.3- MC6.1C22.43) FVSQHLCGSHLVEALYLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 46-INS Analog #10 (SCI(B3S, B16H, A21G)-(PGPA).sub.8- MC6.1C22.43) (SCIv5-(PGPA).sub.8-C22.43) FVSQHLCGSHLVEALHLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 47-INS Analog #11 (SCI(B3S, B16H, A8H, A14E, A21G)-(PGPA).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALHLVCGERGFFYTPKTGGGGGGGIVEQCCHSICSLEQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 48-INS Analog #12 (SCI(B3S, B16H, B25H, A8H, A21G)-(PGPA).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 49-INS Analog #13 (SCI(B3S, B16H, B25H, A8H, A14E, A21G)- (PGPA).sub.8-MC6.1C22.43) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLEQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 50-INS Analog #14 (SCI(B3S, B16R, A21G)-(PGPA).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 51-INS Analog #15 (SCI(B3S, B16F, A21G)-(PGPA).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALFLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 52-INS Analog #16 (SCI(B3S, B16W, A21G)-(PGPA).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALWLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 53-INS Analog #17 (SCI(B3S, B16R, A8H, A21G)-(PGPA).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 54-INS Analog #18 (SCI(B3S, B16R, B25H, A8H, A21G)-(PGPA)- MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGPGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPAEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 55-INS Analog #19 (SCI(B3S, A21G)-(G4Q).sub.5-MC6.1C22.43) FVSQHLCGSHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAAS GRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 56-INS Analog #20 (SCI(B3S, A21G)-(PGPQ).sub.8-MC6.1C22.43) FVSQHLCGSHLVEALYLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 57-INS Analog #21 (SCI(B3S, B16H, B25H, A8H, A21G)-(G4Q).sub.5- C22.43) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAAS GRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 58-INS Analog #22 (SCI(B3S, B16H, B25H, A8H, A21G)-(PGPQ).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 59-INS Analog #23 (SCI(B3S, B16H, B25H, A8H, A14E, A21G)- (G4Q).sub.5-MC6.1C22.43) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLEQLENY CGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAAS GRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 60-INS Analog #24 (SCI(B3S, B16H, B25H, A8H, A14E, A21G)- (PGPQ).sub.8-MC6.1C22.43) FVSQHLCGSHLVEALHLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLEQLENY CGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 61-INS Analog #25 SCI(B3S, B16R, A21G)-(G4Q).sub.5- MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAAS GRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 62-INS Analog #26 SCI(B3S, B16R, A21G)-(PGPQ).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCTSICSLYQLENY CGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 63-INS Analog #27 (SCI(B3S, B16R, A8H, A21G)-(G4Q).sub.5- MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAAS GRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 64-INS Analog #28 (SCI(B3S, B16R, A8H, A21G)-(PGPQ).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFFYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 65-INS Analog #29 (SCI(B3S, B16R, B25H, A8H, A21G)-(G4Q).sub.5- MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAAS GRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLY LQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 66-INS Analog #30 (SCI(B3S, B16R, B25H, A8H, A21G)-(PGPQ).sub.8- MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGPGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 67-INS Analog #31 (SCI(B3S, B16R, B25H, A8H, A21G)- (G2E)7G2-MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGGGEGGEGGEGGEGGEGGEGGEGGEVQLLESGGGLVQPGGSLRLSCAASGRYI DETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQM NSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 68-INS Analog #32 (SCI(B3S, B16R, B25H, A8H, A21G)- (G4E)4G4-MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGGGGGEGGGGEGGGGEGGGGEGGGGEVQLLESGGGLVQPGGSLRLSCAASGR YIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQ MNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 69-INS Analog #33 (SCI(B3S, B16R, B25H, A8H, A21G)- (G4K)4G4-MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGGGGGKGGGGKGGGGKGGGGKGGGGEVQLLESGGGLVQPGGSLRLSCAASG RYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYL QMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 70-INS Analog #34 (SCI(B3S, B16R, B25H, A8H, A21G)- G4(AP)10G4-MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGGGGGAPAPAPAPAPAPAPAPAPAPGGGGEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 71-INS Analog #35 (SCI(B3S, B16R, B25H, A8H, A21G)- G4(EP)10G4-MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGGGGGEPEPEPEPEPEPEPEPEPEPGGGGEVQLLESGGGLVQPGGSLRLSCAASG RYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYL QMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 72-INS Analog #36 (SCI(B3S, B16R, B25H, A8H, A21G)- G4(KP)10G4-MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGGGGGKPKPKPKPKPKPKPKPKPKPGGGGEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 73-INS Analog #37 (SCI(B3S, B16R, B25H, A8H, A21G)- (PGPE)7PGPQ-MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGPGPEPGPEPGPEPGPEPGPEPGPEPGPEPGPQEVQLLESGGGLVQPGGSLRLSCA ASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNT LYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 74-INS Analog #38 (SCI(B3S, B16R, B25H, A8H, A21G)- (PGPK)7PGPQ-MC6.1C22.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGPGPKPGPKPGPKPGPKPGPKPGPKPGPKPGPQEVQLLESGGGLVQPGGSLRLS CAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSK NTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 75-INS Analog #39 (SCI(B3K, B16R, B25H, A8H, A21G)-G4Q)5- MC6.1C22.43) FVKQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLEN YCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 76-INS Analog #40 (SCI(B3K, B16R, B21Q, B25H, A8H, A21G)- G4Q)5-MC6.1C22.43) FVKQHLCGSHLVEALRLVCGQRGFHYTPKTGGGGGGGTVEQCCHSICSLYQLEN YCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 77-INS Analog #41 (SCI(B3K, B16R, B21Q, B25H,  A4Q,  A8H, A21G)-G4Q)5-MC6.1C22.43) FVKQHLCGSHLVEALRLVCGQRGFHYTPKTGGGGGGGIVQQCCHSICSLYQLEN YCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 78-INS Analog #42 (SCI(B3S, B16R, B25H, A8H, A21G)-(G4Q)5-MC6.1C80.43) FVSQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLENY CGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAAS GRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 79-INS Analog #43 (SCI(B3K, B16R, B25H, A8H, A21G)-(G4Q)5-MC6.1C80.43) FVKQHLCGSHLVEALRLVCGERGFHYTPKTGGGGGGGIVEQCCHSICSLYQLEN YCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 80-INS Analog #44 (SCI(B3K, B16R, B21Q, B25H, A8H, A21G)-(G.sub.4Q).sub.5-MC6.1C80.43) FVKQHLCGSHLVEALRLVCGQRGFHYTPKTGGGGGGGIVEQCCHSICSLYQLEN YCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 81-INS Analog #45 (SCI(B3K, B16R, B21Q, B25H, A4Q, A8H, A21G)-(G4Q)5-MC6.1C80.43) FVKQHLCGSHLVEALRLVCGQRGFHYTPKTGGGGGGGIVQQCCHSICSLYQLEN YCGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 82-Signal Peptide METDTLLLWVLLLWVPGSTG SEQ ID NO: 83-C-terminal C9 tag TETSQVAPA SEQ ID NO: 84 (CDR1 #1) AASGRTVSSTAVA SEQ ID NO: 85 (CDR1 #2) AASGRYIDSTAVA SEQ ID NO: 86 (CDR1 #3) AASGRYIDETAVA SEQ ID NO: 87 (CDR2 #1) GIGGSVDITYYLDSVKG SEQ ID NO: 88 (CDR2 #2) GIGGSVDITYYADSVKG SEQ ID NO: 89 (CDR2 #3) GIGGGVDITYYADSVKG SEQ ID NO: 90 (CDR3 #1) AVRPGRPLITSRDANLYDY SEQ ID NO: 91 (CDR3 #2) AARPGRPLITSRVANLYPY SEQ ID NO: 92 (CDR3 #3) AARPGRPLITSKVADLYPY