RECOMBINANT INTERLEUKIN-15 ANALOG
20220348624 · 2022-11-03
Assignee
Inventors
- Yao Zhao (Beijing, CN)
- Jianjun Zhang (Beijing, CN)
- Xiaoting ZHU (Beijing, CN)
- Yanling XU (Beijing, CN)
- Lujia PENG (Beijing, CN)
- Jishu WANG (Beijing, CN)
- Wei Zhang (Beijing, CN)
Cpc classification
C07K2319/35
CHEMISTRY; METALLURGY
C07K2319/31
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
A61K47/542
HUMAN NECESSITIES
C12N15/63
CHEMISTRY; METALLURGY
International classification
Abstract
The amino acid sequence of the present IL-15 analog includes the amino acid sequence of IL-15 and an amino acid sequence including at least one positively charged amino acid added to the C-terminal of the amino acid sequence of IL-15. The present IL-15 analog is highly expressed in Escherichia Coli, wherein the expression level is about 20-fold higher than that of the wild-type IL-15, and there is no significant difference in cell activity in vitro. In addition, a conjugate of the IL-15 analog improves the half-life and the long-term efficacy of the IL-15 analog by coupling with the fatty acid chain. These improvements lay a foundation for the industrialization of IL-15 protein drugs.
Claims
1. An IL-15 analog, wherein an amino acid sequence of the IL-15 analog comprises an amino acid sequence of IL-15, and an amino acid sequence comprising at least one amino acid added to a C-terminal of the amino acid sequence of IL-15.
2. The IL-15 analog of claim 1, wherein the amino acid sequence added to the C-terminal of the amino acid sequence of IL-15 comprises at least one positively charged amino acid.
3. The IL-15 analog of claim 2, wherein the amino acid sequence of the IL-15 analog is characterized by:
IL-15-X.sub.a-Y.sub.b-Z.sub.c wherein X, Y and Z each represent an amino acid sequence added at the C-terminal of the amino acid sequence of IL-15, and a, b and c each represent a number of the amino acids; wherein X and Z each are any amino acid or a combination of any amino acids, and a and c each are 0 to 20; and wherein Y is the positively charged amino acid or a combination of any positively charged amino acids, or a combination of the positively charged amino acid and any other amino acids, and b is 1 to 7.
4. The IL-15 analog of claim 3, wherein the positively charged amino acid is H, R or K.
5. The IL-15 analog of claim 4, wherein X comprises at least one amino acid selected from the group consisting of V, I, P, L, E, A, S, C, T, and G.
6. The IL-15 analog of claim 3, wherein the amino acid sequence of the IL-15 analog is further characterized by:
IL-15-linker-X.sub.a-Y.sub.b-Z.sub.c wherein the linker represents a linker sequence between the amino acid sequence of IL-15 and the amino acid sequence added to the C-terminal thereof.
7. The IL-15 analog of claim 6, wherein the linker is (GGGGS).sub.n, (GS).sub.n or (GAPQ).sub.n, with n being 0 to 10.
8. The IL-15 analog of claim 7, wherein X.sub.a comprises LPBTG with B being any amino acid, and the linker is (GS).sub.n.
9. The IL-15 analog of claim 1, wherein the amino acid sequence added to the C-terminal of the amino acid sequence of IL-15 is selected from the group consisting of: SEQ ID NOS: 122-150, KKK, KKC, K, and KK.
10. The IL-15 analog of claim 1, wherein the amino acid sequence of IL-15 is selected from the group consisting of: 1) the amino acid sequence shown in SEQ ID NO. 1; 2) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 1 through substitution, deletion, or addition of one or more amino acids; and 3) an amino acid being at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO. 1.
11. A nucleotide sequence encoding the IL-15 analog according to claim 1.
12. A method for preparing the IL-15 analog of claim 1, comprising expressing the IL-15 analog in a prokaryotic system.
13. The method of claim 12, comprising linking a nucleotide sequence encoding the IL-15 analog to a prokaryotic expression vector, and transferring a resulting vector into a prokaryotic expression host bacterium, and performing an induction to express the IL-15 analog.
14. A recombinant expression vector, comprising a nucleotide sequence encoding the IL-15 analog of claim 1.
15. A host bacterium transformed with a nucleotide sequence encoding the IL-15 analog of claim 1.
16. A conjugate of an IL-15 analog, comprising an amino acid sequence of the IL-15 analog linked with a fatty acid chain, wherein the amino acid sequence of the IL-15 analog comprises an amino acid sequence of IL-15, and an amino acid sequence comprising at least one amino acid added to a C-terminal of the amino acid sequence of IL-15.
17. The conjugate of the IL-15 analog of claim 16, wherein the amino acid sequence of the IL-15 analog is characterized by either one of: 1) IL-15-X.sub.a-Y.sub.b-Z.sub.c wherein X, Y and Z each represent an amino acid sequence added at the C-terminal of the amino acid sequence of IL-15, and a, b and c each represent a number of the amino acids; and wherein X and Z each are any amino acid or a combination of any amino acids, and a and c each are 0 to 20; and wherein Y is a positively charged amino acid or a combination of any positively charged amino acids, or a combination of a positively charged amino acid and any other amino acids, and b is 1 to 7; and 2) IL-15-linker-X.sub.a-Y.sub.b-Z.sub.c wherein the linker represents a linker sequence between the amino acid sequence of IL-15 and the amino acid sequence added to the C-terminal thereof.
18. The conjugate of the IL-15 analog of claim 17, wherein X.sub.a comprises LPBTG with B being any amino acid, and the linker is (GS).sub.n with n being 0 to 10.
19. The conjugate of the IL-15 analog of claim 17, wherein the fatty acid chain is —(CH.sub.2).sub.m—COOH, wherein m is 12 to 19.
20. The conjugate of the IL-15 analog of claim 17, wherein the IL-15 analog and the fatty acid chain are linked by in vitro coupling.
21. The conjugate of the IL-15 analog of claim 20, wherein the in vitro coupling is performed with one of the following methods: 1) coupling through an enzymatic reaction, wherein the fatty acid chain in the enzymatic reaction has GGG at an N-terminal; 2) coupling with free cysteine residues introduced into the amino acid sequence of the IL-15 analog; and 3) coupling with an amino group at an N-terminal of the amino acid sequence of the IL-15 analog.
22. The conjugate of the IL-15 analog of claim 21, wherein when coupling through the enzymatic reaction, the fatty acid chain has 3 glycine residues at a terminal of the fatty acid chain; when coupling with the free cysteine residues introduced into the amino acid sequence of the IL-15 analog, the fatty acid chain has a maleimide ester or a halogenated reactive group; and when coupling with the amino group at the N-terminal of the amino acid sequence of the IL-15 analog, the fatty acid chain has an aldehyde group or a succinimidyl ester functional group.
23. The conjugate of the IL-15 analog of claim 16, wherein the IL-15 analog is obtained by adding a sequence comprising -GS-LPETG, as set forth in SEQ ID NO: 152, to the C-terminal of the amino acid sequence of IL-15.
24. The conjugate of the IL-15 analog of claim 16, wherein the fatty acid chain is selected from the group consisting of: TABLE-US-00015 GGG-PEG2-Lys-(CH.sub.2).sub.16-COOH, NHS-PEG2-PEG2-γ-Glu-(CH.sub.2).sub.17-COOH, GGG-PEG4-PEG4-PEG4-Lys-(CH.sub.2).sub.17-COOH, GGG-PEG4-γ-Glu-γ-Glu-Lys-(CH.sub.2).sub.17-COOH, HOOC-(CH.sub.2).sub.16-γ-Glu-γ-Glu-Lys-GGG, HOOC-(CH.sub.2).sub.16-CONH-γ-Glu-γ-Glu-PEG2-Lys-Br, GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH.sub.2).sub.17-COOH, GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH.sub.2).sub.19-COOH, GGG-OEG-C2DA-2OEG-γ-Glu-(CH.sub.2).sub.19-COOH, GGG-OEG-C2DA-2OEG-γ-Glu-Trx-(CH.sub.2).sub.19-COOH, CHO-PEG2-PEG2-γ-Glu-(CH.sub.2).sub.17-COOH, and Mal-C2DA-2OEG-γ-Glu-Tn-(CH.sub.2).sub.19-COOH.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0054]
[0055]
[0056]
[0057]
[0058]
[0059]
[0060]
[0061]
[0062]
[0063]
[0064]
[0065]
[0066]
[0067]
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0068] Hereinafter, the present invention will be further described in conjunction with examples. It should be understood that these examples are used for illustrative purposes only and are not intended to limit the protection scope of the present invention.
[0069] In the following examples, the experimental methods without special instructions were usually carried out in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). Unless otherwise specified, the reagents used are commercially available or publicly available reagents.
[0070] In particular embodiments according to the present disclosure, the positively charged amino acids were leucine (Lys, K), arginine (Arg, R) and histidine (His, H).
[0071] As the basis for modification, the amino acid sequence of IL-15 was selected from the group of:
[0072] 1) the amino acid sequence shown in SEQ ID NO: 1;
[0073] 2) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1 through substitution, deletion or addition of one or more amino acids; and
[0074] 3) an amino acid being at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1.
[0075] In particular embodiments according to the present disclosure, the expression of IL-15, especially in prokaryotic expression systems, could be enhanced by adding one or more amino acids, especially positively charged amino acids, to the C-terminal of IL-15.
[0076] In some particular embodiments, the amino acid sequence of the IL-15 analog could be represented as the following general formula (that is, the amino acid sequence X.sub.a-Y.sub.b-Z.sub.c was added to the C-terminal of IL-15):
IL-15-X.sub.a-Y.sub.b-Z.sub.c
[0077] wherein X, Y and Z each represented an amino acid sequence added at the C-terminal, and a, b and c each represented the number of the amino acids; and
[0078] wherein X and Z each were any amino acid or a combination of any amino acids, and a and c each were 0 to 20; and
[0079] wherein Y was a positively charged amino acid or a combination of any positively charged amino acids, or a combination of a positively charged amino acid and any other amino acids, and b was 1 to 7.
[0080] In some particular embodiments, the amino acid sequence of the IL-15 analog could be represented as the following general formula (that is, a linker sequence and the amino acid sequence X.sub.a-Y.sub.b-Z.sub.c were added to the C-terminal of IL-15):
IL-15-linker-X.sub.a-Y.sub.b-Z.sub.c
[0081] wherein X, Y and Z each represented an amino acid sequence added at the C-terminal, and a, b and c each represented the number of the amino acids; and
[0082] wherein X and Z each were any amino acid or a combination of any amino acids, and a and c each were 0 to 20; and
[0083] wherein Y was a positively charged amino acid or a combination of any positively charged amino acids, or a combination of a positively charged amino acid and any other amino acids, and b was 1 to 7.
[0084] In some optional particular embodiments, the linker could be (GGGGS).sub.n, (GS).sub.n or (GAPQ).sub.n, with n being 1 to 5.
[0085] In some particular embodiments, in the above general formula of the amino acid sequence of IL-15 analogs, X.sub.a comprised LPBTG (SEQ ID NO: 151), wherein B was any amino acid and the linker was (GS).sub.n, with n being 0 to 10. In a particular embodiment, the IL-15 analog was obtained by adding a sequence comprising -GS-LPETG (SEQ ID NO: 152) to the terminal of the amino acid sequence of IL-15.
[0086] In some particular embodiments, the IL-15 analogs were coupled with fatty acid chains in vitro to improve the long-term efficacy of the IL-15 analogs. The way of in vitro coupling of fatty acid chains could be selected from the group of:
[0087] 1) coupling with the amino group at the N-terminal, for example, through an aldehyde group or a succinimide ester;
[0088] 2) site-directed coupling with the introduced free cysteine residue (for example, introducing a free cysteine residue at the C-terminal of the IL-15 analog), through a maleimide group or a halogenated group; and
[0089] 3) utilizing an enzymatic reaction, for example, the enzyme Sortase A could utilize the small peptide LPETG (SEQ ID NO: 153) at the C-terminal of IL-15 to couple the IL-15 with the fatty acid chain with GGG at the N-terminal (see M. L. Bentley et al., J. Biol. Chem. 2008, 283: 14762-14771). The fatty acid chains could comprise the structure as shown in Table 2.
TABLE-US-00003 TABLE 2 The structure of fatty acid chains Name Fatty acid chain Reaction with IL-15 816366 GGG-PEG2-Lys-(CH.sub.2).sub.16—COOH (IL-15)-LPET-816366 823479 GGG-PEG4-PEG4-PEG4-Lys-(CH.sub.2).sub.17—COOH (IL-15)-LPET-823479 823480 GGG-PEG4-γ-Glu-γ-Glu-Lys-(CH.sub.2).sub.17—COOH (IL-15)-LPET-823480 818389 HOOC—(CH.sub.2).sub.16-γ-Glu-γ-Glu-Lys-GGG (IL-15)-LPET-818389 817549 HOOC—(CH.sub.2).sub.16—CONH-γ-Glu-γ-Glu-PEG2-Lys-Br (IL-15)-Cysteine-817549 823848 GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH.sub.2).sub.17—COOH (IL-15)-LPET-823 848 823853 GGG-γ-Glu-C2DA-2OEG-γ-Glu-(CH.sub.2).sub.19—COOH (IL-15)-LPET-823 853 823854 GGG-OEG-C2DA-2OEG-γ-Glu-(CH.sub.2).sub.19—COOH (IL-15)-LPET-823 854 823856 GGG-OEG-C2DA-2OEG-γ-Glu-Trx-(CH.sub.2).sub.19—COOH (IL-15)-LPET-823 856 820044 NHS-PEG2-PEG2-γ-Glu-(CH.sub.2).sub.17—COOH 820044-(IL-15) 820045 CHO-PEG2-PEG2-γ-G1u-(CH.sub.2).sub.17—COOH 820045-(IL-15) 823855 Mal-C2DA-2OEG-γ-Glu-Tn-(CH.sub.2).sub.19—COOH (IL-15)-Cysteine-823855 *In the above table, LPET represents that the amino acid sequence added to the C-terminal of IL-15 includes LPET.
Example 1: Expression of Wild-Type IL-15 and IL-15 Analogs in Escherichia Coli
[0090] 1.1 Construction of Expression Vector
[0091] The wild-type IL-15 nucleotide sequence was synthesized by Sangon Biotech (Shanghai).
[0092] (1) Design of Primers
[0093] The sequence of the C-terminal of IL-15 was altered by introducing a base sequence of different amino acids to be added into the reverse primer. The amino acid sequence, the nucleotide sequence and the primer sequence (used in the construction of IL-15 analogs) of the wild-type IL-15 and constructed IL-15 analogs are shown in Table 3.
TABLE-US-00004 TABLE 3 Amino acid sequence, nucleotide sequence and primer sequence (used in the construction of IL-15 analogs) of the wild-type IL-15 and IL-15 analogs Sequence SEQ ID NO: IL-15 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMK 1 CFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGC KECEELEEKNIKEFLQSFVHIVQMFINTS IL-15 ATGAACTGGGTGAACGTTATCAGCGACCTGAAGAAAATCGA 2 GGATCTGATTCAGAGCATGCACATTGACGCGACCCTGTACA CCGAAAGCGATGTGCACCCGAGCTGCAAGGTTACCGCGATG AAATGCTTCCTGCTGGAGCTGCAAGTGATCAGCCTGGAAAG CGGTGACGCGAGCATTCACGATACCGTTGAGAACCTGATCA TTCTGGCGAACAACAGCCTGAGCAGCAACGGTAACGTGAC CGAGAGCGGCTGCAAGGAATGCGAGGAACTGGAGGAAAA GAACATCAAAGAATTCCTGCAGAGCTTTGTGCACATCGTTC AAATGTTTATTAACACCAGC Analog IL-15-(GS).sub.3-HHHHHH 154 1 IL-15-ggttctggttctggttctcaccaccaccaccaccac 155 Forward primer for Analog 1: 23 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 1: 24 gtggtggtggtggtgagaaccagaaccagaaccGCTGGTGTTAATAAACATTT GAACG Reverse primer 2 for Analog 1: 25 gtggtggtggtggtgctcgagTTAGTGGTGGTGGTGGTGGTGAGAACC Analog IL-15-(GS).sub.1-HHHHHH 156 2 IL-15-ggttctcaccaccaccaccaccac 157 Forward primer for Analog 2: 26 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 2: 27 gtggtggtggtggtggtgagaaccGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 2: 28 gtggtggtggtggtgctcgagTTAGTGGTGGTGGTGGTGGTGAG Analog IL-15-PLASTKKR 158 3 IL-15-ccacttgctagcaccaaaaagcgt 159 Forward primer for Analog 3: 29 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 3: 30 acgctttttggtgctagcaagtggGCT GGT GTTAATAAAC ATTT GAACG Reverse primer 2 for Analog 3: 31 gtggtggtggtggtgctcgagTTAACGCTTTTTGGTGCTAGCAAG Analog IL-15-LPKSAKKK 160 4 IL-15-cttccaaagtctgctaaaaagaag 161 Forward primer for Analog 4: 32 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 4: 33 cttctttttagcagactttggaagGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 4: 34 gtggtggtggtggtgctcgagTTACTTCTTTTTAGCAGACTTTGGAAGG Analog IL-15-KKKKKKK 162 5 IL-15-aaaaagaagaaaaaaaagaag 163 Forward primer for Analog 5: 35 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 5: 36 cttcttttttttcttctttttGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 5: 37 gtggtggtggtggtgctcgagTTACTTCTTTTTTTTCTTCTTTTTGCTGG Analog IL-15-(GAPQGAPQ)-LVESAHHH 164 6 IL-15-ggtgctccacagggtgctccacagcttgtggaatctgcacaccatcat 165 Forward primer for Analog 6: 38 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog6: 39 attccacaagctgtggagcaccctgtggagcaccGCTGGTGTTAATAAACATTT GAACG Reverse primer 2 for Analog 6: 40 gtggtggtggtggtgctcgagTTAatgatggtgtgcagATTCCACAAGCTGTGG AGCAC Analog IL-15-GS-LVSSAHHK 166 7 IL-15-ggtagccttgtatctagcgctcaccacaaa 167 Forward primer for Analog 7: 41 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 7: 42 tttgtggtgagcgctagatacaaggctaccGCTGGTGTTAATAAACATTTGAA CG Reverse primer 2 for Analog 7: 43 gtggtggtggtggtgctcgagTTATTTGTGGTGAGCGCTAGATACAA Analog IL-15-GS-LIEHHRRK 168 8 IL-15-ggtagccttatcgaacaccaccgtcgcaaa 169 Forward primer for Analog 8: 44 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 8: 45 tttgcgacggtggtgttcgataaggctaccGCTGGTGTTAATAAACATTTGAA CG Reverse primer 2 for Analog 8: 46 gtggtggtggtggtgctcgagTTATTTGCGACGGTGGTGTTCG Analog IL-15-GS-IVEHRKKK 170 9 IL-15-ggtagcattgtagaacaccgtaagaaaaag 171 Forward primer for Analog 9: 47 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 9: 48 ctttttcttacggtgttctacaatgctaccGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 9: 49 gtggtggtggtggtgctcgagTTACTTTTTCTTACGGTGTTCTACAATGC Analog IL-15-GS-VPKTGRRR 172 10 IL-15-ggtagcgtaccaaaaactggtcgtcgccgt 173 Forward primer for Analog 10: 50 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 10: 51 acggcgacgaccagtttttggtacgctaccGCTGGTGTTAATAAACATTTGAA CG Reverse primer 2 for Analog 10: 52 gtggtggtggtggtgctcgagTTAACGGCGACGACCAGTTTTT Analog IL-15-GS-LVASGKK 174 11 IL-15-ggtagcctggttgctagcggtaaaaag 175 Forward primer for Analog 11: 53 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 11: 54 ctttttaccgctagcaaccaggctaccGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 11: 55 gtggtggtggtggtgctcgagTTACTTTTTACCGCTAGCAACCAGG Analog IL-15-GS-HRKSGHHH 176 12 IL-15-ggtagccatcgtaaatctggtcaccatcat 177 Forward primer for Analog 12: 56 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 12: 57 atgatggtgaccagatttacgatggctaccGCTGGTGTTAATAAACATTTGAA CG Reverse primer 2 for Analog 12: 58 gtggtggtggtggtgctcgagTTAATGATGGTGACCAGATTTACGATG Analog IL-15-GS-LPKTGRHK 178 13 IL-15-ggtagccttccaaaaactggtcgtcacaag 179 Forward primer for Analog 13: 59 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 13: 60 cttgtgacgaccagtttttggaaggctaccGCTGGTGTTAATAAACATTTGAA CG Reverse primer 2 for Analog 13: 61 gtggtggtggtggtgctcgagTTACTTGTGACGACCAGTTTTTGGA Analog IL-15-KKKTGRRH 180 14 IL-15-aaaaagaagactggtcgtcgccat 181 Forward primer for Analog 14: 62 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 14: 63 atggcgacgaccagtcttctttttGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 14: 64 gtggtggtggtggtgctcgagTTAATGGCGACGACCAGTCTTCTT Analog IL-15-LPRSGRHK 182 15 IL-15-cttccacgttctggtcgtcataag 183 Forward primer for Analog 15: 65 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 15: 66 cttatgacgaccagaacgtggaagGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 15: 67 gtggtggtggtggtgctcgagTTACTTATGACGACCAGAACGTGGA Analog IL-15-LVETHHHH 184 16 IL-15-ctggttgaaactcaccatcatcac 185 Forward primer for Analog 16: 68 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 16: 69 gtgatgatggtgagtttcaaccagGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 16: 70 gtggtggtggtggtgctcgagTTAGTGATGATGGTGAGTTTCAACCAG Analog IL-15-VRPETHHH 186 17 IL-15-gttcgtccagaaactcaccatcat 187 Forward primer for Analog 17: 71 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 17: 72 atgatggtgagtttctggacgaacGCT GGT GTTAATAAACATTTGAACG Reverse primer 2 for Analog 17: 73 gtggtggtggtggtgctcgagTTAATGATGGTGAGTTTCTGGACGAA Analog IL-15-KKK 188 18 IL-15-aaaaaaaag 189 Forward primer for Analog 18: 74 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 18: 75 cttttttttGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 18: 76 gtggtggtggtggtgctcgagTTACTTTTTTTTGCTGGTGTTAATAAACA Analog IL-15-RHHHH 190 19 IL-15-cgtcaccatcatcat 191 Forward primer for Analog 19: 77 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 19: 78 atgatgatggtgacgGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 19: 79 gtggtggtggtggtgctcgagTTAATGATGATGGTGACGGCTGG Analog IL-15-KRETHHHH 192 20 IL-15-aagcgtgaaactcaccatcatcat 193 Forward primer for Analog 20: 80 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 20: 81 atgatgatggtgagtttcacgcttGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 20: 82 gtggtggtggtggtgctcgagTTAATGATGATGGTGAGTTTCACGCT Analog IL-15-GS-LPETG-GSGGSHHHHHH 194 21 IL-15-ggttctctgccggaaaccggtggttctggtggttctcaccaccaccaccaccac 195 Forward primer for Analog 21: 83 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 21: 84 accaccagaaccaccggtttccggcagagaaccGCTGGTGTTAATAAACATT Reverse primer 2 for Analog 21: 85 gtggtggtggtggtgctcgagTTAGTGGTGGTGGTGGTGGTGAGAACC ACCAGAACC Analog IL-15-HLETGKKK 196 22 IL-15-caccttgaaactggtaaaaagaag 197 Forward primer for Analog 22: 86 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 22: 87 cttctttttaccagtttcaaggtgGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 22: 88 gtggtggtggtggtgctcgagTTACTTCTTTTTACCAGTTTCAAGGTGG Analog IL-15-HVESGRRR 198 23 IL-15-catgttgaatctggtcgtcgccgt 199 Forward primer for Analog 23: 89 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 23: 90 acggcgacgaccagattcaacatgGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 23: 91 gtggtggtggtggtgctcgagTTAACGGCGACGACCAGATTCA Analog IL-15-RRHTGKKK 200 24 IL-15-cgtcgtcatactggtaaaaagaag 201 Forward primer for Analog 24: 92 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 24: 93 cttctttttaccagtatgacgacgGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 24: 94 gtggtggtggtggtgctcgagTTACTTCTTTTTACCAGTATGACGACGG Analog IL-15-HVKTGHHH 202 25 IL-15-catgttaagactggtcaccatcat 203 Forward primer for Analog 25: 95 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 25: 96 atgatggtgaccagtcttaacatgGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 25: 97 gtggtggtggtggtgctcgagTTAATGATGGTGACCAGTCTTAACATGG Analog IL-15-HVKSGRHH 204 26 IL-15-catgttaagtctggtcgtcatcat 205 Forward primer for Analog 26: 98 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 26: 99 atgatgacgaccagacttaacatgGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 26: 100 gtggtggtggtggtgctcgagTTAATGATGACGACCAGACTTAACATGG Analog IL-15-HVKSSHRH 206 27 IL-15-catgttaagtctagccatcgtcac 207 Forward primer for Analog 27: 101 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 27: 102 gtgacgatggctagacttaacatgGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 27: 103 gtggtggtggtggtgctcgagTTAGTGACGATGGCTAGACTTAACATGG Analog IL-15-(GS).sub.5-LVKSGHHH 208 28 IL-15-ggtagcggtagcggtagcggtagcggtagcctggtaaagtctggtcaccatcat 209 Forward primer for Analog 28: 104 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 28: 105 gctaccgctaccgctaccgctaccgctaccGCTGGTGTTAATAAACATTTGAA CG Reverse primer 2 for Analog 28: 106 atgatggtgaccagactttaccagGCTACCGCTACCGCTACCG Analog 28 Reverse primer for 3: 107 gtggtggtggtggtgctcgagTTAATGATGGTGACCAGACTTTACCAG Analog IL-15-RPKSGHHK 210 29 IL-15-cgtccaaagagcggtcaccataag 211 Forward primer for Analog 29: 108 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 29: 109 cttatggtgaccgctctttggacgGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 29: 110 gtggtggtggtggtgctcgagTTACTTATGGTGACCGCTCTTTGGA Analog IL-15-KKC 212 30 IL-15-aaaaagtgt 213 Forward primer for Analog 30: ill taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG ReverseprimerforAnalog30: 112 gtggtggtggtggtgctcgagTTAacactttttGCTGGTGTTAATAAACATTTG AACG Analog IL-15-LHKAGKHH 214 31 IL-15-cttcacaaggctggtaaacaccat 215 Forward primer for Analog 31: 113 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer 1 for Analog 31: 114 atggtgtttaccagccttgtgaagGCTGGTGTTAATAAACATTTGAACG Reverse primer 2 for Analog 31: 115 gtggtggtggtggtgctcgagTTAATGGTGTTTACCAGCCTTGTGAA Analog IL-15-K 216 32 IL-15-aaa 217 Forward primer for Analog 32: 116 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer for Analog 32: 117 gtggtggtggtggtgctcgagTTAtttGCTGGTGTTAATAAACATTTGAAC G Analog IL-15-KK 218 33 IL-15-aaaaag 219 Forward primer for Analog 33: 118 taagaaggagatatacatatgAACTGGGTGAACGTTATCAGCG Reverse primer for Analog 33: 119 gtggtggtggtggtgctcgagTTActttttGCTGGTGTTAATAAACATTTGAA CG
The analogs 34 to 43 were formed by introducing mutations into the wild-type IL-15 to form IL-15 mutants, and then adding the sequence GSLPETGGGSGGSHHHHHH (SEQ ID NO: 220) to the C-terminals based on the IL-15 mutants. The analogs 34- to 43- were the mutants of IL-15 formed just after the mutations were introduced, without further adding the sequence GSLPETGGGSGGSHHHHHH (SEQ ID NO: 220) to the C-terminals. The nucleotide sequences of the mutated analogs 34 to 43 and analogs 38- to 43- without adding the sequence GSLPETGGGSGGSHHHHHH (SEQ ID NO: 220) to the C-terminals were synthesized by the Genewiz, Inc., and cloned into the pET41a vectors.
TABLE-US-00005 TABLE 4 Analogs 34-43 SEQ Identity SEQ ID NO: ID with IL- (Amino Acid Name Sequence of Protein NO: 15 Sequence) IL-15 MNWVNVISDLKKIEDLIQSMHIDATLYTESDVH 1 2 PSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIILANNSLSSNGNVTESGCKECEELEEKNIKEF LQSFVHIVQMFINTS Analog MNWVNVISDLKKIEDLIQSMHIDATLYTESDVH 3 99% 13 34 PSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIILANNSLSSNANVTESGCKECEELEEKNIKEF LQSFVHIVQMFINTSGSLPETGGSGGSHHHHHH Analog MANWVNVISDLKKIEDLIQSMHIDATLYTESDV 4 97% 14 35 HPSCKVTAMKCFLLELQVISLESGDASIHDTVE NLIILANNSLSSNANVTESGCKECEELEEKNIKE FLQSFVHIVQMFINTSGSLPETGGSGGSHHHHH H Analog MNWVNVISDLKKIEDLIQSMHIRGDLYTESDV 5 96% 15 36 HPSCKVTAMKCFLLELQVISLESGDASIHDTVE NLIILANNSLSSNANVTESGCKECEELEEKNIKE FLQSFVHIVQMFINTSGSLPETGGSGGSHHHHH H Analog MSNWVNVISDLKKIEDLIQSVHIRGDLYTESDV 6 93% 16 37 HPSCKVTAMKCFLLELQVISLESGDGSIHDTVE NLIILAQQSLSSNANVTESGCKECEELEEKNIKE FLQSFVHIVQMFINTSGSLPETGGSGGSHHHHH H Analog MSNWVNVISDLKKIEDLIQSVHIRGDLYTESDV 7 90% 17 38 HPSCKVTAMKCFLLELQVISLESGDGSIHDTVE NLIILAQQSLSSNANVTESGCKECEELSEKNIKE FLQSFVHIVQVFINTSGSLPETGGSGGSHHHHH H Analog MSNWVNVISDLRKIRGDIQSVHIDATLYTESDV 8 90% 18 39 HPSCRVTAMKCFLLELQVISLESGDASIHDTVE NLIILANQSLSSNANVTESGCKECEELEEKNIKE FLQSFVHIVQLFIQTSGSLPETGGSGGSHHHHH H Analog MSNWVNVISDLRKIRGDLNAVHVDATLYTESD 9 85% 19 40 VHPSCRVTAMKCFLLELQVISLESGDASIHDTV ENLIILANQSLSSNANVTESGCKECEELEEKNIK EFLQSFVHIVQLFIQTSGSLPETGGSGGSHHHH HH Analog MSNWVNVISDLRKIRGDIQSVHIDATLYTESDV 10 85% 20 41 HPSCRVTAMKCFLLELQVISLESGDASIHDTVE NLIILANQSLAAQAQLTESGCKECEELEEKNIKE FLQSFVHIVQLFIQTSGSLPETGGSGGSHHHHH H Analog MSNWVNVISDLRKIRGDLNAVHVDATLYTESD 11 80% 21 42 VHPSCRVTAMKCFLLELQVISLESGDASIHDTV ENLIILANQSLAAQAQLTESGCKECEELEEKNIK EFLQSFVHIVQLFIQTSGSLPETGGSGGSHHHH HH Analog MSNWVNVISDLRKIRGDIQSVHIDATLYTESDV 12 80% 22 43 HPSCRVTAMKCFLLELQLISLDSGDASIHETVE QLILLANQSLAAQAQLTESGCKECEELEEKNIK EFLQSFVHIVQLFIQTSGSLPETGGSGGSHHHH HH
[0094] (2) PCR Amplification
[0095] The PCR amplification was classified into three cases as follows.
[0096] In the first case, only one reverse primer was required, and a total of 1 run of PCR was performed.
[0097] Sample loading system: 50 μL (1 tube)
[0098] Primers: forward primer and reverse primer
[0099] Template: IL-15
[0100] Annealing temperature: 61° C.
[0101] Extension time: 30 s
[0102] In the second case, two reverse primers were required, and a total of 2 runs of PCR were performed.
[0103] 1.sup.st Run of PCR
[0104] Sample loading system: 20 μL (1 tube)
[0105] Primers: forward primer and reverse primer 1
[0106] Template: IL-15
[0107] Annealing temperature: 61° C.
[0108] Extension time: 30 s
[0109] 2.sup.nd run of PCR
[0110] Sample loading system: 50 μL (1 tube)
[0111] Primers: forward primer and reverse primer 2
[0112] Template: PCR product of 1.sup.st run
[0113] Annealing temperature: 61° C.
[0114] Extension time: 30 s
[0115] In the third case, three reverse primers were required, and a total of 3 runs of PCR were performed.
[0116] 1.sup.st run of PCR
[0117] Sample loading system: 20 μL (1 tube)
[0118] Primers: forward primer and reverse primer 1
[0119] Template: IL-15
[0120] Annealing temperature: 61° C.
[0121] Extension time: 30 s
[0122] 2.sup.nd run of PCR
[0123] Sample loading system: 20 μL (1 tube)
[0124] Primers: forward primer and reverse primer 2
[0125] Template: PCR product of 1.sup.st run
[0126] Annealing temperature: 61° C.
[0127] Extension time: 30 s
[0128] 3.sup.rd run of PCR
[0129] Sample loading system: 50 μL (1 tube)
[0130] Primers: forward primer and reverse primer 3
[0131] Template: PCR product of 2.sup.nd run
[0132] Annealing temperature: 61° C.
[0133] Extension time: 30 s
[0134] Sample Loading System for PCR: 20 μL
TABLE-US-00006 5 × TransStart ® FastPfu Fly buffer 4 μL Forward primer (5 μM) 1.2 μL Reverse primer (5 μM) 1.2 μL dNTPs (2.5 mM) 1.6 μL Template 0.5 μL Pfu DNA polymerase 0.4 μL ddH.sub.2O making up to 20 μL
[0135] Sample Loading System for PCR: 50 μL
TABLE-US-00007 5 × TransStart ® FastPfu Fly buffer 10 μL Forward primer (5 μM) 3 μL Reverse primer (5 μM) 3 μL dNTPs (2.5 mM) 4 μL Template 0.5 μL Pfu DNA polymerase 1 μL ddH.sub.2O making up to 50 μL
[0136] Amplification Procedure for PCR:
TABLE-US-00008 98° C. 5 min 98° C. 20 s {close oversize brace} 61° C. 20 s 72° C. 30 s 72° C. 10 min
[0137] (3) Enzyme Digestion of Vector
[0138] Reaction system: 30 μL, incubating at 37° C. overnight after mixing
TABLE-US-00009 pET41a vector 5 μg NdeI 1 μL XhoI 1 μL 10 × H buffer 3 μL ddH.sub.2O making up to 30 μL
[0139] (4) PCR Products and Digested Vectors in the Gels were Recovered.
[0140] (5) Loading of Recombinants
[0141] Reaction system: 20 μL
TABLE-US-00010 Digested pET41a vector 13.5 μL PCR product 0.5 μL 5 × CE II buffer 4 μL Exnase II 2 μL
[0142] The molar ratio of vector to fragment was 2:1. After reacting at 37° C. for 30 min, it was immediately cooled on ice and transformed into DH5α.
[0143] (6) Sterility Test
[0144] 0.5 mL of Amp-resistant LB media was added to a 1.5 mL centrifuge tube and well-grown single colonies were inoculated thereto. A total of 5 tubes were inoculated and they were incubated in a shaker under shaking at 37° C. After incubation for 3 h, 1 μL of the culture was taken as a template for PCR of the bacterial suspension.
[0145] Reaction system for PCR: 10 μL
TABLE-US-00011 10 × Taq DNA polymerase buffer 1 μL T7-P (5 μM) 1 μL T7-T (5 μM) 1 μL dNTPs (2.5 mM) 0.8 μL Template 1 μL Taq DNA polymerase 0.1 μL ddH.sub.2O 5.1 μL
[0146] Amplification Procedure for PCR:
TABLE-US-00012 94° C. 5 min 94° C. 30 s {close oversize brace} 55° C. 30 s 72° C. 1 min 72° C. 5 min
[0147] After PCR was completed, it was detected by agarose gel electrophoresis. Three positive clones were selected for sequencing (Sangon Biotech).
[0148] (7) Preservation of Positive Bacterial Suspension and Extraction of Plasmids
[0149] The positive clones with correct sequencing were inoculated into 5 mL of Amp-resistant LB liquid media with an inoculation volume of 10 μL. 500 μL of bacterial suspension was added into a 1.5 mL centrifuge tube. 500 μL of 40% glycerol was added for storage. It was labeled with the name, host bacteria and date, capped and stored in a refrigerator at −80° C.
[0150] The remaining bacterial suspension was collected by centrifugation for plasmid extraction.
[0151] 1.2 Protein Expression of Wild-Type IL-15 and IL-15 Analogs
[0152] (1) Transformation of BL21 (DE3)
[0153] a) 2 μL of plasmid was added to 100 μL of BL21 (DE3) competent cells, and it was mixed immediately and placed on ice for 30 min.
[0154] b) It was heat-shocked at 42° C. for 90 s, followed by a rapid ice bath for 2 min.
[0155] c) 500 μL of LB media was added, then it was incubated at 37° C. under shaking (≤200 rpm) for 60 minutes.
[0156] d) It was centrifuged at 6000 rpm for 1 min. Most of the supernatant was discarded, and about 100 to 150 μL of the supernatant was retained. After the pellet was resuspended, they were spread on LB plates containing Amp and cultured at 37° C. overnight.
[0157] (2) Small-Scale Expression
[0158] a) Bacteria preservation: One single clone was picked into 1 mL of Amp-resistant LB media. It was incubated at 37° C. under shaking at 220 rpm for about 5 h. 1 mL of 40% glycerol was added. It was divided into 2 tubes and cryopreserved at −80° C.
[0159] b) 2.5 mL of LB liquid media containing Amp was added to the tube in the previous step. The culture was incubated at 37° C. under shaking at 220 rpm overnight.
[0160] c) The bacterial suspension incubated overnight was inoculated into 20 mL of LB media containing Amp at a ratio of 1:50. It was incubated at 37° C. under shaking at 220 rpm to reach OD600=0.6 (about 3 h). IPTG was added at a final concentration of 0.5 mM. It was incubated at 37° C. under shaking at 220 rpm for 3 h.
[0161] (3) Expression Level Determination by SDS-PAGE
[0162] a) The cultural suspension was determined for OD600. 10 OD bacterial suspension was taken, and centrifuged at 10000 rpm for 2 min. The supernatant was removed.
[0163] b) The pellet was resuspended with 1 mL of lysis buffer (10 mM Tris-HCl, pH 8.0) placed on ice and lysed by ultrasonication. Ultrasonic conditions: 130 W, 4 min, on 3 s, off 3 s.
[0164] c) After ultrasonication, 80 μL was taken and labeled as “T (total)”. The remaining liquid was centrifuged at 12000 rpm for 10 min to obtain “S (supernatant)” and “P (pellet)”. 20 μL of 5×Reducing Loading Buffer was added to 80 μL of T, P and S, respectively. Then they were heated at 95° C. for 5 min and 12.5 μL (0.1 OD) of each sample was taken for SDS-PAGE electrophoresis.
[0165] The electrophoretogram of wild-type IL-15 was shown in
[0166] (4) Expression Level Determination by HPLC
[0167] a) After expression, 10 OD cells were collected and resuspended with 1 mL of 10 mM Tris-HCl buffer at pH 8.0.
[0168] b) Sonication was performed under the same conditions as those for running gel electrophoresis as described in the above step (3).
[0169] c) After sonication, it was centrifuged at 12000 rpm for 10 min. The supernatant was discarded.
[0170] d) The pellet was added with 1 mL of freshly prepared 8 M Urea/10 mM Tris-HCl (pH 8.0, 10 mM DTT) and dissolved by shaking at room temperature for about 1 h.
[0171] e) It was filtered with a 0.2 μm filter and loaded for HPLC analysis. The analysis was performed using a C4 analytical column with 0.1% TFA in deionized water as mobile phase A and 0.1% TFA in acetonitrile as mobile phase B, followed by a 15-minute gradient from 20% B to 60% B.
[0172] As shown by the HPLC quantitative results in
Example 2: In Vitro Cell Activity Assay of IL-15 Analogs
[0173] 2.1 Preparation of IL-15 Analogs
[0174] The IL-15 analogs 11, 18, 21 and 28 expressed by the inclusion bodies were dissolved in 8 M urea solution, and purified by ion exchange and reversed-phase chromatography (for details, see: Yunier Rodriguez-Alvarez et al, Preparative Biochemistry and Biotechnology, 47: 9, 889-900), to obtain relatively pure proteins. The SDS-PAGE electrophoretograms were shown in
[0175] 2.2 CTLL-2 Cell Proliferation Assay
[0176] The CTLL-2 cell proliferation assay is commonly used to detect the activity of immune cells stimulated by interleukin at the cellular level. Therefore, the biological activity of IL-15 analogs was determined herein by the proliferative effect of the wild-type IL-15 and IL-15 analogs on CTLL-2 cells.
[0177] 1) Preparation of CTLL-2 Cells: The Cells were Resuspended in Media Containing FBS and Rat-T-Stim.
[0178] 2) Loading: the cells were seeded in a 96-well culture plate at 0.1 mL per well. At the same time, the proteins samples of IL-15 analogs 11, 18, 21 and 28 to be tested (i.e., the proteins prepared in step 2.1) were diluted by multiples, respectively. 0.1 mL was added to each well, and 3 replicate wells were set for each dilution concentration. The control well for culture media was set (100 μL cells+100 μL culture media). The plate was incubated at 37° C. with 5% CO.sub.2 for 72 hours.
[0179] 3) MTS addition: 20 μL of CellTiter96® AQueous One Solution Reagent was added to each well, and the plate was incubated at 37° C. with 5% CO.sub.2 for 2 to 4 hours.
[0180] 4) Detection: the absorbance value (A) was measured at a wavelength of 490 nm with a microplate reader and the EC50 value was calculated.
[0181] The results were shown in Table 5. There was no significant difference in the cellular activity of the IL-15 analogs and wild-type IL-15.
TABLE-US-00013 TABLE 5 CTLL-2 cell viability assay EC50 (ng/mL) IL-15 0.05445 Analog 11 0.05252 Analog 18 0.05609 Analog 21 0.05235 Analog 28 0.05085
Example 3: Preparation of Conjugates of IL-15 Analogs (Enzymatic Reaction Method)
[0182] The purified IL-15 Analog 21 (IL-15-GS-LPETG-GSGGSHHHHHH) was used in this example. The fatty acid chain (816366) with GGG at the N-terminal was linked to IL-15-GS-LPETG-GSGGSHHHHHH by a ligation reaction catalyzed by the transpeptidase Sortase A. The reaction was carried out in a Sortase A: IL-15: fatty acid chain ratio of 1:6:30, wherein the reaction buffer was 50 mM Tris-HCl (1 mM CaCl, 150 mM NaCl, pH 8.0). After reacting at room temperature for 3 hours, purification was carried out. Reversed-phase chromatography C8 (Sepax Technologies, Inc.) was used for purification to separate the unconjugated IL-15 Analog 21 and the unreacted fatty acid chains from the conjugated products. The purity of the final product was determined by UPLC (
Example 4: Preparation of Conjugates of IL-15 Analog (Coupling the N-Terminal Amino Group of the IL-15 Analog to a Fatty Acid Chain)
[0183] The purified IL-15 Analog 21 was coupled to a fatty acid chain with a succinimidyl ester (820044) through the amino group at N-terminal thereof at neutral pH. The reaction was carried out in an IL-15: fatty acid chain ratio of 1:1, wherein the reaction buffer was PBS at pH 7.2. After reacting at room temperature for 1 hour, purification was carried out. Reversed-phase chromatography C8 (Sepax Technologies, Inc.) was used for purification to separate the unconjugated IL-15 and the unreacted fatty acid chains from the conjugated products. The final product was identified by LC-MS. As shown in
Example 5: Binding Assay of the IL-15 Analog 21 and Conjugates of IL-15 Analog 21 to the IL15Rα Receptor
[0184] The conjugates of IL-15 analogs used in this example were prepared in Example 3.
[0185] In this example, biolayer interferomeory (BLI) was used to determine the affinity between the target protein and the receptor. For procedures, see Patricia Estep et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning, MAbs 2013, 5(2): 270-278. The receptor protein IL15Rα-His used in the experiment was produced by Leto Laboratories Co. Ltd. The formulation of buffer was: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20. The receptor IL15Rα-His was pre-immobilized on a HISIK sensor (Pall Fortebio, Catalog #18-5120), followed by an established process comprising the steps of setting baseline, loading, baseline, association and dissociation. Data acquisition and analysis were carried out using the software Data acquisition 11.0 and Data analysis 11.0 installed with Octet RED96, respectively.
[0186] The results for affinity assay of the IL-15 analog and the conjugated products of IL-15 analogs and fatty acid chains to the IL15Rα receptors are shown in Table 6. As compared to the IL-15 analog before conjugation, the affinity of the conjugated products of IL-15 analog and fatty acid chains to the IL15Rα receptor did not change significantly.
TABLE-US-00014 TABLE 6 Results for affinity assay Name of Protein Affinity to IL15Rα (M) IL-15Analog 21 1.18E−09 IL-15Analog 21-816366 6.6E−09.sup. IL-15Analog 21-823479 4E−09 IL-15Analog 21-823480 8E−10
Example 6: Half-Life of IL-15 Analog 21 and Conjugates of IL-15 Analog 21 in Mice
[0187] The conjugates of IL-15 analogs used in this example were prepared in Example 3.
[0188] Eight C57BL/6 mice were divided into 2 groups with 4 mice in each group. Blood was collected from two mice at each time point, and blood was collected cyclically. For one group, the mice were injected with IL-15 at 0.5 mg/kg via tail vein. Blood samples were collected immediately, and then at 10 min, 30 min, 1.5 h and 4 h after administration. For the other group, the mice were injected with long-acting IL-15 at 0.5 mg/kg via tail vein. Blood samples were collected immediately, and then at 1 h, 2 h and 4 h after administration. At each time point, 50 to 100 μL of blood was collected from eye orbit. Then serum was collected for ELISA assay of IL-15.
[0189] The calculation formula of half-life is: t.sub.1/2=0.693/k, k=(lnc.sub.0−lnc)/t. Upon calculation, the half-life of IL-15 was about 5 min, and the half-life of long-acting IL-15 could reach 1.5 h.
Example 7: In Vivo Tumor Inhibition Assay for IL-15 Analog 21 and Conjugates of IL-15 Analog in Mice
[0190] The conjugates of IL-15 analogs used in this example were prepared in Example 3.
[0191] Fifteen C57BL/6 mice aged 6 to 8 weeks were randomly divided into 3 groups, namely the reagent control group (PBS), the IL-15 group and the long-acting IL-15 group, with 5 mice in each group. On Day 1, the mice were subcutaneously inoculated with B16-F10 cells on the back of the neck, with 2×10.sup.5/100 μL/mouse. On Days 4 to 8, the PBS group and the IL-15 group were administered intravenously (i.v.) for 5 consecutive days, respectively, at a dose of 20 μg/100 μL/mouse (IL-15 group) or 100 μL/mouse (PBS group). The IL-15 analog conjugate group was administered twice in the same way on Day 4 and Day 7, with the same dosage of 20 μg/100 μL/mouse each time. Tumor sizes were observed from Day 10 and continued for 6 days.
[0192] The results are shown in