Interleukin 15 protein complex and use thereof

Abstract

An interleukin 15 (IL-15) protein complex is provided. The IL-15 protein complex includes soluble fusion proteins (I) and (II), wherein the fusion protein (I) is an IL-15 polypeptide or a functional fragment thereof, and the soluble fusion protein (II) is an IL-15Rα polypeptide or a functional fragment thereof. The soluble fusion protein (I) has at least one amino acid residue mutated to a cysteine (Cys) residue, which pairs with a corresponding mutated Cys residue on the soluble fusion protein (II), or vice versa, to form one or more disulfide bonds. The IL-15 protein complex can be used for tumor therapy.

Claims

1. One or more nucleic acids encoding an IL-15 protein complex comprising a soluble fusion protein (I) and a soluble fusion protein (II), wherein: the soluble fusion protein (I) comprises an IL-15 polypeptide; and the soluble fusion protein (II) comprises an IL-15Rα polypeptide; wherein the soluble fusion protein (I) comprises an amino acid Cys substitution at a position corresponding to residue Q48, V49, L52 or E53 of the IL-15 polypeptide having the amino acid sequence of SEQ ID NO: 1, and the soluble fusion protein (II) comprises an amino acid Cys substitution at a position corresponding to residue A37, G38, S40 or L42 of the IL-15Rα polypeptide having the amino acid sequence of SEQ ID NO: 4, and a disulfide bond is formed by Cys residues of the soluble fusion protein (II) and the soluble fusion protein (I).

2. The one or more nucleic acids according to claim 1, wherein at least one of the soluble fusion protein (I) and soluble fusion protein (II) is covalently linked to an Fc fragment.

3. The one or more nucleic acids according to claim 1, wherein the soluble fusion protein (I) comprises the amino acid sequence of SEQ ID NO: 2.

4. The one or more nucleic acids according to claim 1, wherein the soluble fusion protein (I) comprises the amino acid Cys substitution at the position corresponding to residue L52 or E53 of the IL-15 polypeptide having the amino acid sequence of SEQ ID NO: 1; and the soluble fusion protein (II) comprises the amino acid Cys substitution at the position corresponding to residue A37, G38 or S40 of the IL-15Rα polypeptide having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

5. The one or more nucleic acids according to claim 4, wherein the soluble fusion protein (I) comprises the amino acid Cys substitution at the position corresponding to residue L52 of the IL-15 polypeptide; and the soluble fusion protein (II) comprises the amino acid Cys substitution at the position corresponding to residue S40 of the IL-15Rα polypeptide.

6. The one or more nucleic acids according to claim 1, wherein the soluble fusion protein (II) comprises the IL-15Rα polypeptide and an Fc fragment.

7. The one or more nucleic acids according to claim 6, wherein the IL-15Rα polypeptide is attached to the N-terminus of the Fc fragment.

8. The one or more nucleic acids according to claim 6, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO: 9.

9. The one or more nucleic acids according to claim 1, wherein the soluble fusion protein (II) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

10. The one or more nucleic acids according to claim 9, wherein the soluble fusion protein (II) comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

11. The one or more nucleic acids according to claim 1, wherein the IL-15 protein complex comprises the following combinations of the soluble fusion protein (I) and soluble fusion protein (II): TABLE-US-00012 No. soluble fusion protein (I) soluble fusion protein (II) 1 IL-15(L52C) IL-15Rα-ECD(S40C)-Fc (SEQ ID NO: 2) (SEQ ID NO: 5) 2 IL-15(L52C) Fc-IL-15Rα-ECD(S40C) (SEQ ID NO: 2) (SEQ ID NO: 6) 3 IL-15(L52C) IL-15Rα-sushi + (S40C)-Fc (SEQ ID NO: 2) (SEQ ID NO: 7) 4 IL-15(L52C) Fc-IL-15Rα-sushi + (S40C) (SEQ ID NO: 2) (SEQ ID NO: 8).

12. One or more isolated DNA vectors comprising the one or more nucleic acids according to claim 1.

13. An isolated host cell comprising the one or more isolated DNA vectors according to claim 12.

14. A method for preparing the IL-15 protein complex, the method comprising: culturing the host cell according to claim 13 under conditions sufficient for expression of the IL-15 protein complex; and expressing and purifying the IL-15 protein complex.

15. A pharmaceutical composition comprising the one or more nucleic acids according to claim 1, and a pharmaceutically acceptable excipient, diluent or carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a crystal complex structure of IL-15 and the receptor α, β, γ.

(2) FIG. 2 shows residues at the interface of IL-15 and IL-15Rα (receptor α).

(3) FIG. 3 shows relative positions of candidate mutant residues located on IL-15 and IL-15Rα.

(4) FIG. 4 is a model diagram of a disulfide bond formed between L52C on IL-15 and S40C on IL-15Rα.

(5) FIG. 5 shows Western blot analysis for detection of a His tag on the co-expressed molecule products 1-9 of the present invention.

(6) FIG. 6 shows Western blot analysis for detection of the Fc portion on co-expressed molecule products 1-18 of the present invention.

(7) FIG. 7 shows a structure diagram of protein complexes 1, 2, 3, 4 of the present invention.

(8) FIG. 8 shows the effect of the protein complex of the present invention on lung metastatic tumors in mice; “*” in the figure represents p<0.05, vs PBS.

(9) FIG. 9 shows the effect of the protein complex on the relative lung weight (lung weight/body weight) of mice.

(10) FIG. 10 shows the effect of the protein complex on the body weight of mice.

(11) FIG. 11 shows the half-life of protein complex 3 in rat.

DETAILED DESCRIPTION OF THE INVENTION

(12) Hereinafter, the present invention is further described with reference to examples. However, the scope of the present invention is not limited thereto.

(13) In the examples of the present invention, where specific conditions are not described, the experiments are generally conducted under conventional conditions, or under conditions proposed by the material or product manufacturers. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Ausubel et al, Greene Publishing Associates, Wiley Interscience, NY. Where the source of the reagents is not specifically given, the reagents are commercially available conventional reagents.

Example 1. Selection and Verification of the Mutants

(14) IL-15 is a promising cytokine for the treatment of cancer and viral diseases. It can be presented to IL-15 receptor β/γ located on the surface of T cells and NK cells by IL-15Rα (IL-15 receptor α), thereby stimulating the proliferation of the activated T cells. Therefore, increasing the binding capacity of IL-15 and IL-15 receptor α will significantly enhance the functions of a variety of lymphocytes, which is very important for immunotherapy.

(15) It can be seen that the three receptors bind to IL-15 in three different orientations, respectively, from the crystalline complex structure of IL-15 and the receptor α, β, γ (PDB ID: 4GS7) (FIG. 1). Therefore, when the residues present on the interface between IL-15 and receptor α are modified, the binding of IL-15 to receptors β and γ will not be affected.

(16) The present inventors selected the co-crystal structure (PDB ID: 2Z3Q) of IL-15 and receptor α as the initial structure (the crystal structure of Receptor α in this structure is slightly longer than that in 4GS7). The residues located on the interface of IL-15 and receptor α are summarized from the structure (Table 1). The cut-off value was set at 6 Å from the opposite molecule (FIG. 2).

(17) TABLE-US-00002 TABLE 1 Residues at the contacting interface of IL-15 and receptor α crystal complex Molecule Residues at the contacting interface IL-15 H20, I21, D22, A23, T24, L25, Y26, C42, L45, E46, Q48, V49, L52, E53, S54, G55, E87, C88, E89, E90, E93, K94 receptor α R24, R26, K34, R35, K36, A37, G38, T39, S40, S41, L42, E44, S60, I64, R65, D66, P67, A68, V70, H71, Q72

(18) Disulfide scanning was performed at the interface between IL-15 and receptor α by using Disulfide Scan in the Simulation software MOE. The basic principle of scanning is to look for a combination of residues located at IL-15 and IL-15 receptor α, respectively, within the disulfide bond length range, so as to obtain the following combination of residues (Table 2) and the relative positions of candidate mutant residues (FIG. 3).

(19) TABLE-US-00003 TABLE 2 Combination of the Mutated Residues at IL-15 and IL-15 Receptor α IL-15 Receptor α D stability (Kcal/mol) L45C A37C −4.1933 L45C G38C −3.4475 Q48C G38C −5.7596 V49C S40C −3.6957 L52C S40C −4.0172 E53C L42C −3.3652 E87C P67C −2.8417 C88C A37C −4.0382 E89C K34C −7.3065

(20) Selection for the mutation combination was performed according to the following principles. (1) Do not select the residues near the intramolecular disulfide bond, so as to avoid matching error, and then avoid mispairing of the original intramolecular disulfide bond. (2) Try to choose the residues which will not affect the three-dimensional structure of the protein following mutation. (3) Select the residues which will minimize the effect the energy on the whole structure following mutation.

(21) In order to meet the above requirement 1, from the crystal structure of complex, it can be seen that on the structure of IL-15, intramolecular disulfide bonds were formed between C35 and C85, and between C42 and C88, respectively. Therefore, it is possible to exclude the possibility of E87 and E89, upstream and downstream of C88, respectively, as the candidate residues on IL-15, and exclude the possibility of P67 and K34 on the corresponding receptor α as the candidate residues. In addition, it is necessary to exclude the possibility of a disulfide bond formed by the C88 residue at IL-15 with the A37 residue at the receptor α. On the structure of receptor α, C29 and C63 form a disulfide bond. No candidate residues were found near the pair.

(22) In order to meet the above requirement 2, the crystallization complex was analyzed. First, L45, Q48, V49, L52 and E53 were all located at the alpha helix on the IL-15 structure. In addition, L45, Q48 and V49 were all located in the middle of the alpha helix. If these residues are mutated to Cys, the torsion of the side chain caused by the formation of the disulfide bond may have an influence on the structure of the original alpha-helix, and then affect the whole protein structure. Therefore, L52 and E53 residues on the IL-15 were considered as preferred. Second, on the structure of IL-15 receptor alpha, L42 was located at the beta fold, A37, G38 and S40 were all located at the loop. Therefore, A37, G38 and S40 present on IL-15 receptor alpha were considered as preferred. In view of the two structures, L52 from IL-15 and S40 from the IL-15 receptor alpha were considered as preferred for mutation to Cys, and finally which led to the formation of a intermolecular disulfide bond.

(23) In order to meet the above requirement 3, alanine scanning was performed on all of the above residues by using Discovery Studio Computational Software. The results of energy change calculated in the mutations (Table 3) show that L52A present on IL-15 minimally affected the structural stability, and S40A present on IL-15 receptor α minimally affected the structural stability. Therefore, from the above results, L52 from IL-15 and S40 from the IL-15 receptor alpha can be considered as preferred candidates for mutation to Cys, and finally for the formation of the intermolecular disulfide bond (FIG. 4).

(24) TABLE-US-00004 TABLE 3 Alanine scanning results Mutation Energy Molecule Mutants (Kcal/mol) IL-15 LEU52 > ALA 0.65 IL-15 GLU89 > ALA 1.66 IL-15 GLN48 > ALA 1.67 IL-15 LEU45 > ALA 1.69 IL-15 GLU53 > ALA 2.12 IL-15 VAL49 > ALA 2.86 IL-15 Receptor α SER40 > ALA −0.81 IL-15 Receptor α ALA37 > ALA 0 IL-15 Receptor α GLY38 > ALA 1.31 IL-15 Receptor α LEU42 > ALA 2.65 IL-15 Receptor α LYS34 > ALA 2.98

(25) In summary, a total of 8 pairs of mutation residues were designed. Among these, L52 from IL-15 and S40 from the IL-15 receptor alpha are considered as preferred for mutation to Cys, and finally for the formation of the disulfide bond.

(26) Based on the above 8 pairs of the mutation residues, the molecules were designed for cell expression verification. There are two forms. One is IL-15-Fc fusion molecule with Cys mutation co-expressed with IL-15Rα with Cys mutation (combinations 10-18), and the other is IL-15-6His with Cys mutation co-expressed with IL-15Rα-Fc fusion molecule with Cys mutation (combinations 1-9). The cell supernatant obtained from the co-expression was subjected to Western blot analysis. The His labeled part of the co-expression product combinations 1-9 was detected with anti-mouse His (primary antibody, abcam, ab14923) and goat anti-mouse HRP (secondary antibody, Jackson, 115-035-062); and the Fc part of the co-expression product combinations 1-18 was detected with goat anti-human Fc-HRP (Jackson, 109-035-098). The specific co-expression combinations are shown in Table 4.

(27) TABLE-US-00005 TABLE 4 Co-expression combinations of different mutations Co- expression combination NO. Clone 1 IL-15-His IL-15Rα-linker-Fc 2 IL-15(L45C)-His IL-15Rα(A37C)-linker-Fc 3 IL-15(L45C)-His IL-15Rα(G38C)-linker-Fc 4 IL-15(Q48C)-His IL-15Rα(G38C)-linker-Fc 5 IL-15(V49C)-His IL-15Rα(S40C)-linker-Fc 6 IL-15(L52C)-His IL-15Rα(S40C)-linker-Fc 7 IL-15(E53C)-His IL-15Rα(L42C)-linker-Fc 8 IL-15(C88)-His IL-15Rα(A37C)-linker-Fc 9 IL-15(E89C)-His IL-15Rα(K34C)-linker-Fc 10 IL-15-linker-Fc IL-15Rα 11 IL-15(L45C)-linker-Fc IL-15Rα(A37C) 12 IL-15(L45C)-linker-Fc IL-15Rα(G38C) 13 IL-15(Q48C)-linker-Fc IL-15Rα(G38C) 14 IL-15(V49C)-linker-Fc IL-15Rα(S40C) 15 IL-15(L52C)-linker-Fc IL-15Rα(S40C) 16 IL-15(E53C)-linker-Fc IL-15Rα(L42C) 17 IL-15(C88)-linker-Fc IL-15Rα(A37C) 18 IL-15(E89C)-linker-Fc IL-15Rα(K34C)

(28) Western blot analysis showed that co-expression by pairing Fc-fused IL-15 and IL-15Rα was prone to result in mismatch, and to reduce the amount of correctly paired target product. However, following fusion with Fc, pairing Fc-fused IL-15Rα and IL-15 can result in a correctly pairing single molecule. Among them, the expression levels of co-expression combinations 5, 6 and 7 were highest, the product was highly homogeneous and the size of the bands was as expected (FIG. 5-6). The results were in good agreement with the prediction from simulation. Considering results from both computer simulation and the properties of products expressed by cells, the amino acid Cys mutation sites on IL15 were selected at L45, Q48, V49, L52, E53, C88 or E89, preferably at L52, E53 or E89, and more preferably at L52. The amino acid Cys mutation sites on IL-15Rα were selected at K34, L42, A37, G38 or S40, preferably at A37, G38 or S40, and more preferably at S40. The most preferable is the mutation L52C on IL-15 pairing with S40C on IL-15Rα. Furthermore, the stability of the IL-15 protein complex can be improved by selecting two or more pairs of disulfide bonds or introducing other non cysteine mutations between IL-15 and IL-15Rα.

Example 2. Construction of Related Vectors

(29) Materials:

(30) Eukaryotic expression vector pcDNA3.1 (+) (Life technologies, Cat. No. V790-20); IL-15 (DNA sequence 1), IL-15Rα ECD, IL15Rα-sushi+(73) and IgG1Fc DNA fragment were synthesized by a gene synthesis company (GENEWIZ, Inc., Suzhou);

(31) Primers were synthesized by a gene synthesis company (GENEWIZ, Inc., Suzhou).

(32) Procedure:

(33) 1. Fragment Ligation

(34) IL-15Rα-ECD-Fc fragment: Overlap PCR was used to form IL-15Rα-ECD-Fc fragment by joining three DNA fragments in the order of IL-15Rα-ECD, linker peptide and Fc (DNA sequence 2).

(35) Fc-IL-15Rα-ECD fragment: Overlap PCR was used to form Fc-IL-15Rα ECD fragment by joining three DNA fragments in the order of Fc, linker peptide and IL-15Rα-ECD (DNA sequence 3).

(36) IL-15Rα-sushi+-Fc fragment: Overlap PCR was used to form IL-15Rα-sushi+-Fc fragment by joining three DNA fragments in the order of IL-15Rα-sushi+, linker peptide and Fc (DNA sequence 4).

(37) Fc-IL-15Rα-sushi+fragment: Overlap PCR was used to form an Fc-IL-15Rα-sushi+ fragment by joining three DNA fragments in the order of Fc, linker peptide and IL-15Rα sushi+ (DNA sequence 5).

(38) Gene fragments containing a Cys mutation were obtained by point mutation, for example:

(39) IL-15 (L52C): on position 52, L was mutated to C (DNA sequence 6)

(40) IL15Rα-ECD (S40C)-Fc: on position 40, S was mutated to C (DNA sequence 7)

(41) Fc-IL-15Rα ECD (S40C) fragment: (DNA sequence 8)

(42) IL-15Rα-sushi+ (S40C)-Fc fragment: (DNA sequence 9)

(43) Fc-IL-15Rα-sushi+ (S40C) fragment: (DNA sequence 10).

(44) 2. Introducing Restriction Site and Signal Peptide Sequence:

(45) Restriction endonuclease KpnI site, Kozak sequence and the signal peptide sequence were introduced at the 5′-terminus of the gene fragment by PCR. The sequence between the KpnI site and the gene fragment is shown below:

(46) GGTACCTTGTGCCCGGGCGCCACCATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGTTCCCCGGCTCTCGGTGC (The underlined sequence is the KpnI restriction site, the italic sequence is the signal peptide); Termination codon TGA and NotI restriction enzyme site were introduced into the 3′-terminus of the three fragments, respectively.

(47) 3. Construction of Expression Vectors

(48) The above gene fragments were inserted into vector pcDNA3.1 (+) by KpnI and NotI restriction enzyme sites to construct the expression vectors, such as pcDNA3.1-IL-15, pcDNA3.1-IL-15Rα-ECD-Fc, pcDNA3.1-Fc-IL-15Rα-EC, pcDNA3.1-IL-15Rα-sushi.sup.+-Fc, pcDNA3.1-Fc-IL-15Rα-sushi.sup.+ and so on. The corresponding expression plasmids were obtained.

(49) 4. Site-Directed Mutations in Gene

(50) KOD kit (TOYOBO Cat. KOD-201) was used for site-directed mutation, with a 25 μL system comprising 2.5 μL 10×KOD buffer, 2.5 μL 2 mM dNTPs, 1 μL primer 1 (10 μM), 1 μL primer 2 (10 μM), 0.5 μL KOD plus, 1 μL 25 mM MgSO4 and 16 μL ddH2O. Synthesis procedure is as follows: 94° C. for 2 min, 94° C. for 30 sec, 55° C. for 30 sec, 68° C. for 11 min, for 25 amplification cycles, and PCR amplification was terminated following another 11 min at 68° C. PCR product was digested with 1 μL of DpnI (NEB Cat. R0176L) for 5 hours, and then transformed into DH5a competent cells. After that, a clone was picked up for sequencing to obtain desired plasmids pcDNA3.1-IL-15(L52C), pcDNA3.1-IL-15Rα-ECD(S40C)-Fc, pcDNA3.1-Fc-IL-15Rα-ECD(S40C), pcDNA3.1-IL-15Rα-sushi+(S40C)-Fc, pcDNA3.1-Fc-IL-15Rα-sushi+(S40C) and the other mutant genes. The protein complex 1 involved in the example of the present invention was obtained by expressing the expression vector containing DNA sequences 6 and 7. The protein complex 3 involved in the example of the present invention was obtained by expressing the expression vector containing DNA sequences 6 and 9. The protein complex 4 involved in the example of the present invention was obtained by expressing the expression vector containing DNA sequences 6 and 10. The protein complex 2 involved in the example of the present invention was obtained by expressing the expression vector containing DNA sequences 6 and 8.

(51) Constructing Nucleotide Sequence of Expression Plasmid

(52) The following sequences were used for vector construction, the single horizontal line represents a signal peptide DNA sequence, the dashed line represents a peptide linker DNA sequence, and the double horizontal line represents a mutated DNA sequence.

(53) TABLE-US-00006 IL-15 (DNA sequence 1, SEQ ID NO: 10): ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGG TTCCCCGGCTCTCGGTGCAACTGGGTGAATGTAATTAGTGATTTGAAA AAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATAT ACGGAAAGTGATGTTCACCCGAGTTGCAAAGTAACAGCAATGAAGTGC TTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGCGATGCAAGT ATTCATGATACAGTAGAAAATCTGATCATCTTAGCAAACAACAGTTTG TCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAA CTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATT GTCCAAATGTTCATCAACACTTCTTGA IL-15Rα-ECD-Fc (DNA sequence 2, SEQ ID NO: 11): ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGG TTCCCCGGCTCTCGGTGCATCACCTGCCCTCCACCTATGTCCGTGGAA CACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGC TACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACCTCCAGCCTG ACCGAGTGCGTGTTGAACAAGGCCACCAATGTCGCCCACTGGACAACC CCAAGTCTCAAATGCATTCGCGACCCTGCCCTGGTTCACCAACGCCCA GCGCCACCATCCACAGTAACCACTGCAGGCGTGACCCCACAGCCAGAG AGCCTCTCCCCTTCTGGCAAAGAGCCAGCAGCTTCATCTCCAAGCTCA AACAACACAGCGGCCACAACAGCAGCTATTGTCCCGGGCTCCCAGCTG ATGCCTTCAAAATCACCTTCCACAGGCACCACAGAGATCAGCAGTCAT GAGTCCTCCCACGGCACCCCATCTCAGACAACAGCCAAGAACTGGGAA CTCACAGCATCCGCCTCCCACCAGCCGCCAGGTGTGTATCCACAGGGC CCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCT GAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAG GATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTG GACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGAT GGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTAC AACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGAC TGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTG CCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGG GAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAG AATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGAC ATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAG ACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGC AAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGC TGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCT CTCAGCCTTTCTCCCGGCAAGTGA Fc-IL-15Rα-ECD (DNA sequence 3, SEQ ID NO: 12): ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGG TTCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACA TGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTT CTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCC GAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTG AAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACT AAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTG CTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGC AAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCT AAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCC AGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTG AAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGA CAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGAT GGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGG CAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCAC TGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGCTACATTTGTAAC TCTGGTTTCAAGCGTAAAGCCGGCACCTCCAGCCTGACCGAGTGCGTG TTGAACAAGGCCACCAATGTCGCCCACTGGACAACCCCAAGTCTCAAA TGCATTCGCGACCCTGCCCTGGTTCACCAACGCCCAGCGCCACCATCC ACAGTAACCACTGCAGGCGTGACCCCACAGCCAGAGAGCCTCTCCCCT TCTGGCAAAGAGCCAGCAGCTTCATCTCCAAGCTCAAACAACACAGCG GCCACAACAGCAGCTATTGTCCCGGGCTCCCAGCTGATGCCTTCAAAA TCACCTTCCACAGGCACCACAGAGATCAGCAGTCATGAGTCCTCCCAC GGCACCCCATCTCAGACAACAGCCAAGAACTGGGAACTCACAGCATCC GCCTCCCACCAGCCGCCAGGTGTGTATCCACAGGGCCACAGCGACACC ACTTGA IL-15Rα-sushi+ (73)-Fc (DNA sequence 4, SEQ ID NO: 13): ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGG TTCCCCGGCTCTCGGTGCATCACCTGCCCTCCACCTATGTCCGTGGAA CACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGC TACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACCTCCAGCCTG ACCGAGTGCGTGTTGAACAAGGCCACCAATGTCGCCCACTGGACAACC ACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCT TCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGC AGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGAT CCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAAT GCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTC GTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAG TATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAG ACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACA TTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACA TGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAG AGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTC GATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAG AGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAG GCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGC AAGTGAC Fc-IL-15Rα-sushi+ (73) (DNA sequence 5, SEQ ID NO: 14): ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGG TTCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACAC ATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTT TCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACC CGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGT GAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGAC TAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGT GCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTG CAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTC TAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCC CAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGT GAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGG ACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGA TGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTG GCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCA CTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGCTACATTTGTAA CTCTGGTTTCAAGCGTAAAGCCGGCACCTCCAGCCTGACCGAGTGCGT GTTGAACAAGGCCACCAATGTCGCCCACTGGACAACCCCAAGTCTCAA ATGCATTCGCGACCCTGCCCTGGTTCACCAACGCTGA IL-15(L52C) (DNA sequence 6, SEQ ID NO: 15): ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGG TTCCCCGGCTCTCGGTGCAACTGGGTGAATGTAATTAGTGATTTGAAA AAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATAT ACGGAAAGTGATGTTCACCCGAGTTGCAAAGTAACAGCAATGAAGTGC TTTCTCTTGGAGTTACAAGTTATTTCATGTGAGTCCGGCGATGCAAGT ATTCATGATACAGTAGAAAATCTGATCATCTTAGCAAACAACAGTTTG TCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAA CTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATT GTCCAAATGTTCATCAACACTTCTTGA IL-15Rα-ECD (S40C)-Fc (DNA sequence 7, SEQ ID NO: 16): ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGG TTCCCCGGCTCTCGGTGCATCACCTGCCCTCCACCTATGTCCGTGGAA CACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGC TACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACCTGCAGCCTG ACCGAGTGCGTGTTGAACAAGGCCACCAATGTCGCCCACTGGACAACC CCAAGTCTCAAATGCATTCGCGACCCTGCCCTGGTTCACCAACGCCCA GCGCCACCATCCACAGTAACCACTGCAGGCGTGACCCCACAGCCAGAG AGCCTCTCCCCTTCTGGCAAAGAGCCAGCAGCTTCATCTCCAAGCTCA AACAACACAGCGGCCACAACAGCAGCTATTGTCCCGGGCTCCCAGCTG ATGCCTTCAAAATCACCTTCCACAGGCACCACAGAGATCAGCAGTCAT GAGTCCTCCCACGGCACCCCATCTCAGACAACAGCCAAGAACTGGGAA CTCACAGCATCCGCCTCCCACCAGCCGCCAGGTGTGTATCCACAGGGC CCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCT GAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAG GATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTG GACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGAT GGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTAC AACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGAC TGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTG CCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGG GAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAG AATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGAC ATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAG ACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGC AAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGC TGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCT CTCAGCCTTTCTCCCGGCAAGTGA Fc-IL-15Rα-ECD (S40C) (DNA sequence 8, SEQ ID NO: 17): ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGG TTCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACA TGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTT CTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCC GAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTG AAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACT AAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTG CTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGC AAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCT AAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCC AGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTG AAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGA CAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGAT GGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGG CAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCAC TGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGCTACATTTGTAAC TCTGGTTTCAAGCGTAAAGCCGGCACCTGCAGCCTGACCGAGTGCGTG TTGAACAAGGCCACCAATGTCGCCCACTGGACAACCCCAAGTCTCAAA TGCATTCGCGACCCTGCCCTGGTTCACCAACGCCCAGCGCCACCATCC ACAGTAACCACTGCAGGCGTGACCCCACAGCCAGAGAGCCTCTCCCCT TCTGGCAAAGAGCCAGCAGCTTCATCTCCAAGCTCAAACAACACAGCG GCCACAACAGCAGCTATTGTCCCGGGCTCCCAGCTGATGCCTTCAAAA TCACCTTCCACAGGCACCACAGAGATCAGCAGTCATGAGTCCTCCCAC GGCACCCCATCTCAGACAACAGCCAAGAACTGGGAACTCACAGCATCC GCCTCCCACCAGCCGCCAGGTGTGTATCCACAGGGCCACAGCGACACC ACTTGA IL-15Rα-sushi+ (73) (S40C)-Fc (DNA sequence 9, SEQ ID NO: 18): ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGG TTCCCCGGCTCTCGGTGCATCACCTGCCCTCCACCTATGTCCGTGGAA CACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGC TACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACCTGCAGCCTG ACCGAGTGCGTGTTGAACAAGGCCACCAATGTCGCCCACTGGACAACC 0 ACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCT TCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGC AGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGAT CCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAAT GCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTC GTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAG TATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAG ACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACA TTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACA TGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAG AGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTC GATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAG AGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAG GCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGC AAGTGAC Fc-IL-15Rα-sushi+ (73)(S40C) (DNA sequence 10, SEQ ID NO: 19): ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGG TTCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACA TGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTT CTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCC GAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTG AAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACT AAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTG CTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGC AAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCT AAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCC AGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTG AAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGA CAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGAT GGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGG CAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCAC TGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGCTACATTTGTAAC TCTGGTTTCAAGCGTAAAGCCGGCACCTGCAGCCTGACCGAGTGCGTG TTGAACAAGGCCACCAATGTCGCCCACTGGACAACCCCAAGTCTCAAA TGCATTCGCGACCCTGCCCTGGTTCACCAACGCTGA Fc fragment: IgG1-Fc DNA (DNA sequence 11, SEQ ID NO: 20): GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCT CCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCC AAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTC GTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTG GATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAG TACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAG GACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCA CTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCA CGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACA AAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCA GACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTAC AAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTAC AGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTC AGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAA TCTCTCAGCCTTTCTCCCGGCAAG

Example 3. Characteristics of IL-15 Protein Complex

(54) The IL-15 protein complex provided in the present invention consists of a soluble fusion protein (I) and a soluble fusion protein (II), wherein the soluble fusion protein (I) comprises an IL-15 polypeptide covalently linked to a biologically active polypeptide or a functional fragment thereof. The soluble fusion protein (II) comprises an IL-15R alpha polypeptide covalently linked to a biologically active polypeptide or a functional fragment thereof, wherein the soluble fusion protein (I) or the soluble fusion protein (II) possesses Cys resulting from one or more amino acid mutation sites, and a disulfide bond is formed by the pairing of the corresponding Cys present in the soluble fusion protein (II) and the soluble fusion protein(I).

(55) In the present invention, a stable protein complex with obvious anti-tumor activity and prolonged in vivo half-life was constructed by a gene engineering method, and the complex molecule comprises an Fc fusion protein molecule of IL-15 or a derivative thereof and IL-15Rα or a derivative thereof.

(56) The fusion protein molecule has the following features:

(57) 1) The fusion protein comprises two major molecular moieties, one of which is a molecule having IL-15 biological activity and the other is an Fc fusion molecule having IL-15Rα or a functional fragment thereof;

(58) 2) The molecular moiety having IL-15 bioactivity has cysteine mutations at one or more amino acid sites on the basis of wild-type IL-15 or IL-15 functional mutants, and these cysteine mutation sites can be paired with the corresponding cysteine mutation sites on IL-15Rα or its functional fragment to form a disulfide bond;

(59) 3) The Fc fusion molecule moiety having an IL-15Rα or functional fragment has cysteine mutations at one or more amino acid sites on the basis of the entire extracellular domain fragment of IL-15Rα or an IL-15Rα functional fragment containing a shortened form of the sushi domain. These cysteines can be paired with the corresponding cysteine mutation sites on IL-15 or a functional mutant thereof to form a disulfide bond;

(60) 4) The fusion protein can be stably expressed by co-transfecting or constructing a single cell line with two plasmids, and a single molecule can be obtained by conventional separation methods.

(61) IL-15 used in the examples of the present invention refers to human interleukin 15 mature molecules (SEQ ID NO: 1) or variants thereof. The IL-15Rα ECD used in the examples of the present invention refers to human interleukin 15 receptor alpha extracellular domain fragment (SEQ ID NO: 3). The variant thereof is preferably a shortened version thereof, such as IL-15Rα-sushi+(SEQ ID NO: 4). The Fc fragment portion used in the examples of the present invention is an Fc fragment of human antibody IgG1, IgG2, IgG3, or IgG4, or a variant thereof, preferably an Fc fragment of human IgG1, more preferably SEQ ID NO: 9.

(62) In the present invention, IL-15Rα or a derivative thereof is fused to an Fc fragment or Fc variant through a linker peptide to form a soluble fusion protein (Π), in which the order of attachment of each protein component is not limited. The linker peptide can be a soft linker commonly used in the art, and preferably is (GGGGS) n, where n can be from 1 to 10, preferably from 1 to 5, and most preferably 2. In addition to binding IL-15 to IL-15R alpha, the soluble fusion protein (Π) and soluble fusion protein (I) can also be combined through a disulfide bond formed via pairing cysteine mutation sites. The stability of the molecule can be increased by the latter.

(63) Related protein sequences are as follows:

(64) TABLE-US-00007 IL-15 (SEQ ID NO: 1): (human Interleukin 15 amino acid sequence, and also the reference IL-15 sequence) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI SLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS IL-15(L52C) (SEQ ID NO: 2): (human Interleukin 15 amino acid sequence with a mutation L52C on position 52) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI SCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS IL-15Rα-ECD (SEQ ID NO: 3): (The amino acid sequence of the extracellular domain of human interleukin 15 receptor alpha) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPA ASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTA KNWELTASASHQPPGVYPQGHSDTT IL-15Rα-sushi+ (SEQ ID NO: 4): (The truncated form of the human interleukin 15 receptor fragment, containing 73 amino acids) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIRDPALVHQR IL-15Rα-ECD (S40C)-Fc (SEQ ID NO: 5): (fusion polypeptide of IL-15Rα extracellular domain fused to Fc, which contains an S40C mutation site) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKA TNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPA ASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTA KNWELTASASHQPPGVYPQGHSDTTGGGGSGGGGSEPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK Fc-IL-15Rα-ECD (S40C) (SEQ ID NO: 6): (fusion polypeptide of Fc fused to IL15Rα extracelullar region, which contains an S40C mutation) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSITCPPPMS VEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTT PSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNN TAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTAS ASHQPPGVYPQGHSDTT IL-15Rα-Sushi+(S40C)-Fc (SEQ ID NO: 7): (A truncated form of human interleukin 15 receptor α containing the sushi domain fused to the Fc via a linker, wherein sushi.sup.+ contains an S40C mutation and sushi.sup.+ is located at the N-terminus) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKA TNVAHWTTPSLKCIRDPALVHQRGGGGSGGGGSEPKSSDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK Fc-IL-15Rα-sushi+(S40C)(SEQ ID NO: 8): (A truncated form of human interleukin 15 receptor containing the sushi domain fused to the Fc via a linker, wherein sushi.sup.+ contains an S40C mutation and sushi.sup.+ is located at the C-terminus) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSITCPPPMS VEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTT PSLKCIRDPALVHQR Fc Fragment, IgG1-Fc (Protein)(SEQ ID NO: 9) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(65) In the experiments, the molecules to be tested were numbered as follows: protein complexes 1, 2, 3, and 4, wherein protein complex 1 was obtained by co-expression of SEQ ID NO: 2 and SEQ ID NO: 5; protein complex 2 was obtained by co-expression of SEQ ID NO: 2 and SEQ ID NO: 6; protein complex 3 was obtained by co-expression of SEQ ID NO: 2 and SEQ ID NO: 7; And protein complex 4 was obtained by co-expression of SEQ ID NO:2 and SEQ ID NO:8. A schematic diagram is shown in FIG. 7. The stability of the complex molecule was increased by increasing the formation of a disulfide bond by pairing the cysteine mutation sites.

(66) The list of protein complexes is as follows:

(67) TABLE-US-00008 NO. Sequence composition and description 1 Obtained by IL-15 (L52C) (SEQ ID NO: 2) and IL-15Rα-ECD (S40C)-Fc (SEQ ID NO: 5) co-expression 2 Obtained by IL-15(L52C) (SEQ ID NO: 2) and Fc-IL-15Rα-ECD(S40C) (SEQ ID NO: 6) co-expression 3 Obtained by IL-15(L52C) (SEQ ID NO: 2) and IL-15Rα-Sushi + (S40C)-Fc (SEQ ID NO: 7) co-expression 4 Obtained by IL-15(L52C)(SEQ ID NO: 2) and Fc-IL-15Rα-sushi + (S40C) (SEQ ID NO: 8) co-expression

Example 4. Obtaining the IL-15 Protein Complex

(68) 1. Protein Expression

(69) IL-15/IL-15Rα protein was transiently transfected and expressed by using FreeStyle 293 cells (GIBCO, Cat #R79007). FreeStyle 293 cells were suspension cultured in Freestyle 293 expression medium (GIBCO, Cat #12338018), and supplemented with Ultra Low IgG Fetal Bovine Serum (ultra low immunoglobulins FBS, GIBCO, Cat #16250078) at a final concentration of 1%. IL-15/IL-15Rα expression plasmids and transfection reagent PEI (Polysciences, Cat #239662) were prepared, and the two plasmids of IL-15 and IL-15Rα were co-transfected at a ratio ranging from 1:1 to 9:1, wherein the total amount of plasmids was 100 ug/100 ml cells, the ratio of plasmid to PEI was 1:2 by mass. Cell density on the day of transfection was 1×10.sup.6/ml. 1 L of FreeStyle 293 cells was prepared to be transfected. 50 ml of Opti-MEM (GIBCO, Cat #11058021) medium was mixed with the plasmid, kept still for 5 min and filtered. Another 50 ml of Opti-MEM medium was mixed with PEI, kept still for 5 min and filtered. The plasmid was mixed with PEI and kept still for 15 min. The mixture of plasmid and PEI was slowly added to the cells and cultured in a shaking incubator at 130 rpm at 37° C., 8% CO.sub.2. Five (5) days later, the supernatant was collected by centrifugation for protein purification.

(70) 2. Protein Purification

(71) Affinity Chromatography for IL-15 Fusion Protein:

(72) Supernatant was collected from cell culture after high speed centrifugation and subjected to affinity chromatography by using a Protein A column from GE. The equilibration buffer used in chromatography was 1×PBS (pH7.4). After cell supernatant was loaded and bound, it was washed with PBS until UV returned to baseline, and then the target protein was eluted with elution buffer (acidity, pH2.5-5). The pH was adjusted to neutral with Tris, and the target protein was stored.

(73) Ion Exchange Chromatography for IL-15 Fusion Protein:

(74) The pH of the product obtained during the affinity chromatography was adjusted to be 1-2 pH units lower or higher than pI. Then the sample was appropriately diluted to control the conductivity of the sample to less than 5 ms/cm. NaCl-gradient elution under corresponding pH conditions was performed by utilizing a suitable buffer corresponding to the pH, such as phosphate buffer, acetate buffer, and others, by utilizing conventional ion-exchange column chromatography methods in the art such as cation exchange or anion exchange. The target proteins corresponding to different absorption peaks were collected by using SDS-PAGE and stored.

(75) Size Exclusion Chromatography for IL-15 Fusion Protein:

(76) The product obtained during the ion exchange chromatography was concentrated by ultrafiltration and loaded for size exclusion chromatography, such as by using GE Superdex200 gel to remove possible polymer and other components, in order to obtain the desired product with high purity. Purity of the obtained protein was detected by SDS-PAGE and SEC-HPLC. Protein concentration was determined by UV spectrophotometry.

TEST EXAMPLES

Test Example 1. PBMC Proliferation Assay In Vitro

(77) Fresh PBMCs (human peripheral blood mononuclear cells, Shanghai Blood Center) were cultured in RPMI1640 medium (Thermo Fisher Chemical Products Co., Ltd (Beijing), Cat No. SH30809.01B) containing 10% FBS, centrifuged and resuspended to a cell density of 5×10.sup.5 cells/ml. 90 μl were added into each well of a 96-well plate. Samples were diluted at certain multiples to different concentrations with PBS. 10 μl were added into each well of a 96-well plate, and cultured in the incubator at 37° C., 5% CO.sub.2 for 48 hours. Thereafter, 50 μl were taken for detection of cell proliferation with CellTiter-Glo® Luminescent Cell Viability Assay kit.

(78) TABLE-US-00009 TABLE 5 Detection results of activity of protein complexes 1 and 3 of the present invention in PBMC proliferation assay in vitro EC50 relative activity Sample (ng/ml) of the cells IL-15 3.115 100 1 0.634 491 3 0.047 6627

(79) Table 5 shows the detection results of activity of protein complexes 1 and 3 of the present invention versus control IL-15 in a PBMC proliferation assay in vitro, which indicate that the protein complexes 1 and 3 of the present application significantly improved the proliferation activity of PBMC compared to control IL-15. In this experiment, the activity stimulated by protein complex 1 was increased by about 5 times, whereas the activity stimulated by protein complex 3 was improved by about 66 folds.

Test Example 2. Mo7e Cell Proliferation Assay In Vitro

(80) 1. Main Materials

(81) Mo7e (human megakaryocyte leukemia cell line) purchased from Peking Union Medical College;

(82) IL-15 purchased from Novoprotein, Cat No. C016, IL-15 analog was obtained from in-house preparation;

(83) Cell Counting Kit-8 (CCK-8) purchased from WST, Cat No. EX660;

(84) GM-CSF purchased from NOVOProtein, Cat No. CC79.

(85) 2. Procedures

(86) 1) Mo7e was cultured in modified RPMI-1640 medium (containing 2.05 mM L-glutamine, 10% FBS and 15 ng/ml GM-CSF) in the incubator at 37° C. (5% CO.sub.2);

(87) 2) Mo7e cells in good condition were centrifuged at room temperature, 150×g for 5 min. The supernatant was discarded;

(88) 3) The cell pellet was washed with GM-CSF-free medium twice and then counted;

(89) 4) Cell concentration was adjusted and plated in a 96-well plate with a cell number of 2×10.sup.4 per well and a volume of 90 μl (GM-CSF-free), kept in the cell incubator for culture;

(90) 5) IL-15 and its analog were 4-times diluted with PBS, 10 μl/well was added to the cell culture system after 2 hours of incubation of cells in 96-well plates. Each concentration was repeated in triplicate, blank wells (added with only PBS) were used as control;

(91) 6) Cell plates were cultured in the incubator for 3 days;

(92) 7) All test wells were added with 10 μl of CCK-8, and incubated in the incubator for 3 hours;

(93) 8) Absorbance at 450 nm (OD450) was detected.

(94) TABLE-US-00010 TABLE 6 Results of protein complexs 1-4 in Mo7e cell proliferation assay in vitro relative activity Sample EC50(nM)-Mo7e of the cells IL-15 15.5 100 1 0.42 3690 2 1.21 1281 3 0.07 22142 4 0.09 17222

(95) Table 6 shows the comparison of protein complexes 1-4 to control IL-15 in a Mo7e cell proliferation assay in vitro, which indicates that the protein complexes 1-4 significantly improved the proliferation activity compared to control IL-15, and that the proliferation activity stimulated by complexes 3 and 4 was significantly higher than that stimulated by protein complexes 1 and 2.

Test Example 3. Mouse Lung Metastasis Model

(96) 1. Animal Test Procedures

(97) Thirty-two (32) of C57BL/6 mice (SPF, Shanghai Super B&K Laboratory Animal Corp. Ltd.) were divided into 4 groups, each group having 8 mice. 1.5×10.sup.5 of B16F10 cells were intravenously injected into the mice via the tail-vein (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, TCM36). PBS, 2 μg of IL-15 or 5 μg or 15 μg of protein complex 3 was intraperitoneally injected into the mice on day 1. Weighing once every 2-3 days, one mouse from each group was killed on day 14, and the lung metastasis was observed. All mice were sacrificed on day 16. Lungs of all mice were removed and weighed, the black lung lumps observed and photographed, and then the lung was fixed in formaldehyde and the number of black lumps was counted.

(98) 2. Results

(99) Lungs of mice in the PBS group showed a large number of metastatic melanoma growing (73±43). Lungs of the IL-15 group showed a large number of melanoma lumps (65±29), about 90% of that in the PBS group. Lungs of protein complex 3-5 μg group showed a partial metastasis of melanoma lumps (30±16), about 41% of that in PBS group. Lungs of protein complex 3-15 μg groups showed a partial metastasis of melanoma lumps (24±13), about 33% of that in the PBS group.

(100) In the B16F10 mouse model, the efficacy of protein complex 3 was significantly superior to that of IL-15, as shown in FIG. 8.

(101) The relative lung weight in the PBS group was significantly higher than that in protein complex 3 group, as shown in FIG. 9.

(102) No significant decrease in body weight was observed in each group during the administration, suggesting that the administration dosage does not have significant toxicity, as shown in FIG. 10.

(103) In another B16F10 mouse model experiment with another dosing group, we observed significant anti-tumor activity when the dose of protein complex 3 was reduced to 0.5 μg/mouse, whereas no obvious abnormal symptoms was observed when the maximum tolerated dose was 30 μg/mouse.

(104) In summary, protein complex 3 can inhibit the metastasis of B16F10 cells in mice lungs, and has a dose-dependent effect and a good safety window.

Test Example 4. Mouse Subcutaneous Tumor Model

(105) 1. Animal Test Procedures

(106) 1.1 Mice were Adapted to the Laboratory Environment for 5 Days.

(107) 1.2 Tumor Cells Transplantation

(108) C57BL/6 mice (SPF, Shanghai Xi Puer Bei Kai Experimental Animal Co., Ltd.) were inoculated subcutaneously in the right rib with B16F10 cells (5×10.sup.6/mouse). Tumors grew for 7 days. When the volume of the tumor grew to 160±40 mm.sup.3, animals were randomly divided (d0) into 4 groups, each group of 7 mice.

(109) 1.3 Administration Dosage and Method

(110) Each group was intraperitoneally injected with test drug once on day 1 and day 5, totally twice, with PBS, or IL-15 (2 μg), or protein complex 3 (5 μg), or protein complex 3 (15 μg). Mice were measured for tumor volume and body weight every 2 days, and data was recorded.

(111) 1.4 Statistics

(112) Excel statistical software: mean value was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV/SQRT; P value between different groups was calculated as TTEST.
Tumor volume (V) was calculated as: V=½×L.sub.length×L.sub.short.sup.2
Relative volume (RTV)=V.sub.T/V.sub.0
Tumor Inhibition Rate (%)=(C.sub.RTV−T.sub.RTV)/C.sub.RTV (%)

(113) V.sub.0 and V.sub.T represent the tumor volume at the beginning of the experiment and at the end of the experiment, respectively. C.sub.RTV and T.sub.RTV represent blank control group (PBS) and relative tumor volume in the test group at the end of the experiment, respectively.

(114) 2. Results

(115) Since the transplanted B16F10 cell tumors grew very rapidly, the experiment was stopped on day 9. The growth inhibition effect of IL-15 protein on B16F10 tumor is shown in Table 1. After one-dose administration on days 1 and 5, respectively, IL-15 did not inhibit the growth of transplanted B16F10 cell tumors on day 9. However, the inhibitory rate in the protein complex 3-5 μg group and 3-15 μg group was 30% and 73%, respectively, wherein the protein complex 3-15 μg group significantly inhibited the tumor growth.

(116) In conclusion, the protein complex 3 has an effect on inhibiting the growth of B16F10 xenografts in this study, and has obvious dose-dependent effects, see Table 7.

(117) TABLE-US-00011 TABLE 7 Therapeutic effects of administered proteins on B16F10 xenografts in mice Mean tumor Mean tumor Relative tumor volume (mm3) volume (mm3) volume % Inhibition p Group Administration Pathway D1 SEM D9 SEM D28 SEM Rate D28 (vs blank) PBS d1/5 i.p. 162.39 16.32 2703.46 393.15 16.32 2.18 IL-15-2 μg d1/5 i.p. 161.74 16.64 3219.01 644.69 18.32 2.99 −12%  0.60 3-5 μg  d1/5 i.p. 168.26 19.22 1892.20 315.12 11.47 1.58   30%** 0.10 3-15 μg d1/5 i.p. 168.30 19.17 824.38 170.63 4.48 0.61   73%** 0.00 **p < 0.01, vs PBS

Test Example 5. Determination of the Metabolic Half-Life of the Protein Complexes

(118) 1. Animal Test Procedures

(119) The SD rats (n=2, provided by Sipur-Bikai Experimental Animal Co., Ltd.) were administered via intraperitoneal injection with a dose of 188 μg/kg in a volume of 5 ml after fasting overnight. 0.2 ml blood samples were taken through the rat retro-orbital plexus before and after administration and at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 11 h, 24 h, 48 h, 72 h and 96 h. The blood samples were collected in a tube and kept in the tube for 30 min at 4° C., then centrifuged at 3500 rpm for 10 min and the serum was isolated. Stored at −80° C. The rats were fed 2 h after administration.

(120) 2. Results

(121) Protein complex 3 in rat serum was captured by an ELISA plate coated with anti-IL-15 antibody. Anti-human IgG Fc antibody was used to detect the concentration curve, and the measured half-life in vivo of protein complex 3 in rat was about 13.7 h (FIG. 10). It is reported that the half-life in vivo of IL-15 is less than 1 h (J Immunol 2006; 177:6072-6080), which indicates that the protein complex 3 has a significantly prolonged half-life in vivo.