CN111518221B

CN111518221B - Recombinant IL-15 fusion protein and application thereof

Info

Publication number: CN111518221B
Application number: CN202010630346.5A
Authority: CN
Inventors: 张鹏; 马琳
Original assignee: Suzhou Pharmaceutical Technology Co ltd
Current assignee: Suzhou Pharmaceutical Technology Co ltd
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2020-11-27
Anticipated expiration: 2040-07-03
Also published as: CN111518221A

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a recombinant IL-15 fusion protein and application thereof. The recombinant IL-15 fusion protein provided by the invention comprises interleukin-15, a Sushi structural domain of interleukin-15 receptor alpha and an anti-human serum albumin nano antibody; the interleukin-15, the Sushi structural domain of the interleukin-15 receptor alpha and the anti-human serum albumin nano antibody are directly connected or indirectly connected through a connecting peptide. The recombinant IL-15 fusion protein can be combined with human serum albumin, and can effectively activate T cells at low dose concentration, so that an immune system can play a role, and the aim of preventing and treating tumors is fulfilled.

Description

Recombinant IL-15 fusion protein and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a recombinant IL-15 fusion protein and application thereof.

Background

Cytokines, such as lymphokines, chemokines, and growth factors, are produced by a variety of cell types, including lymphocytes, monocytes, and have a wide variety of roles in human physiology and disease control. These cytokines have been clinically proven to be effective in treating various diseases. For example, Interleukins (ILs) are a class of cytokines produced by and acting on a variety of cells that play an important role in transmitting information, activating and modulating immune cells, mediating T, B cell activation, proliferation and differentiation, and in inflammatory responses.

Among them, interleukin-15 (IL-15) belongs to a member of four small alpha helical bundle cytokine families. IL-15 needs to exert biological activity by binding to its receptor. The IL-15 receptor consists of three receptor subunits: IL-15 receptor alpha (IL-15R alpha), IL-2 receptor beta (IL-2R beta, also known as IL-15R beta or CD 122), and yc (also known as CD 132). IL-15 Ra contains a Sushi domain, can bind to IL-15, and is necessary for the bound IL-15 to exert biological functions. IL-15 has a wide range of immunomodulatory activities and can be involved in the regulation of survival, proliferation and function of a variety of immune cells, including NK cells, memory CD8+ T cells, NKT cells, and the like.

Native IL-15 must often be administered at high doses during the course of treatment, limiting its utility due to toxicity. IL-15 has a half-life of roughly 1 hour after administration and must be administered daily for treatment. The development of fusion proteins containing human antibody Fc fragments by genetic engineering is a common way to increase the half-life of proteins. Patent CN108341884A discloses an immune factor, which comprises polypeptide of amino acid sequence of interleukin 15 or its derivatives, polypeptide of amino acid sequence of sushi domain of IL-15R α or its derivatives, antibody directly or indirectly covalently linked to the conjugate, or fragment of the antibody, and can prolong half-life of IL-15 and enhance immune effect. In patent CN108463239A, IL-15 RalphaSushi-Fc fused with a fragment of the IL-15 receptor using a mutant IL-15N72D of IL-15 forms a tetrameric fusion protein containing a human Fc fragment under co-expression for extending the half-life of IL-15, and the superagonist mutant of IL-15 (IL-15N 72D) increases IL-2 Rbeta binding capacity (4 to 5 fold higher than native IL-15) and has been identified for therapeutic use. In patent CN106459219B, IL-15 containing human Fc fragment and its receptor IL-15R alpha Sushi fusion protein are obtained by mutating and modifying the way of additionally introducing disulfide bond. The IL-15 protein is modified by CN102145178A, CN108472324A and Nektar Therapeutics company by using a PEG technology, so that the removal rate of the modified protein in vivo is reduced, and the purpose of long-acting use is achieved. However, pegylation requires a high process, and pegylation has problems that pegylation cannot be performed on a target protein at a fixed point, and the activity of the protein after pegylation is reduced. Therefore, there is still a need to develop new IL-15 fusion proteins using new fusion approaches.

Disclosure of Invention

Aiming at the defects in the prior art, the technical problem to be solved by the invention is to construct a novel recombinant IL-15 fusion protein on the basis of an anti-human serum albumin nano antibody, wherein the recombinant IL-15 fusion protein can be combined with human serum albumin with high affinity, shows excellent T cell activation effect and can be used for preventing and treating tumors.

The terms:

1. interleukin-15 (interleukin-15, IL-15): refers to cytokines having a structure similar to IL-2 (GRABSTEIN et al, Science, vol.264 (5161), p: 965-968, 1994). This cytokine is also known as IL-15, IL15 or MGC 9721. This cytokine and IL-2 share many biological activities and are found to bind to the same erythropoietin receptor subunit. Thus, IL-15 and IL-2 may compete for the same receptor, thereby down-regulating each other's activity. IL-15 has been shown to regulate the activation and proliferation of T cells and natural killer cells, and it has been demonstrated that the number of CD8+ memory cells is controlled by the balance between IL-15, a cytokine, and IL-2. In the present application, the English symbol "IL-15" is used.

2. Sushi domain of interleukin-15 receptor alpha: refers to a domain that begins at the first cysteine residue (C1) after the signal peptide of interleukin-15 receptor alpha (IL-15 Ra) and ends at the fourth cysteine residue (C4) after the signal peptide. The Sushi domain, which corresponds to a portion of the extracellular region of IL-15R α, is necessary for its binding to IL-15 (WEI et al, J.Immunol., vol.167 (1), p: 277, 282, 2001). In this application, the English notation "IL-15R α Sushi" is used.

3. Anti-human serum albumin nanobody: the nano antibody is a heavy chain antibody derived from camelids or nurse sharks. In 1993 scientists first discovered that in alpaca peripheral blood there was a naturally light chain deleted antibody which contained only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. The VHH structure cloned and expressed by the gene has the structural stability equivalent to that of the original heavy chain antibody and the binding activity with antigen. The anti-human serum albumin nano antibody is a nano antibody capable of being combined with human serum albumin, and the nano antibody can prolong the serum half-life of protein after being fused with other protein such as IL-15. The anti-human serum albumin nanobody is denoted by the English symbol "anti-HSA VHH" in the present application.

4. Amino acid sequence: the type and arrangement of amino acid residues constituting a protein or polypeptide are usually indicated by three-letter method, which is conventionally used in the art, or one-letter method, which is conventionally used in the art, and those skilled in the art should be able to convert the three-letter amino acid sequence into the one-letter amino acid sequence. It should be clear that a person skilled in the art can understand and convert it no matter what way the application is presented. For example: alanine, a single letter is a, and a three letter is Ala; arginine, wherein one letter is R, and the three letters are Arg; aspartic acid, wherein the single letter is D, and the three letters are Asp; cysteine, with the single letter being C and the three letters being Cys; glutamine, one letter is Q, and the three letters are Gln; glutamic acid, wherein one letter is E, and the three letters are Glu; histidine, H in single letter and His in three letters; isoleucine, I in single letter and Ile in three letters; glycine, G is a single letter, and Gly is a three letter; asparagine, with N as the single letter and Asn as the three letters; leucine with the single letter of L and the three letters of Leu; lysine, wherein one letter is K, and the three letters are Lys; methionine, with a single letter of M and a three letter of Met; phenylalanine, with one letter F and three letters Phe; proline, P in one letter and Pro in three letters; serine, wherein the single letter is S, and the three letters are Ser; threonine, wherein one letter is T, and the three letters are Thr; tryptophan with W as a single letter and Trp as a three-letter; tyrosine, with one letter being Y and the three letters being Tyr; valine, V in single letter and Val in three letters.

5. English notation "IL-15/IL-15R α Sushi/anti-HSA VHH": according to the explanations of the above 1-3, the symbol in the present application represents a recombinant IL-15 fusion protein comprising, in order, interleukin-15, the Sushi domain of interleukin-15 receptor alpha, and the anti-human serum albumin nanobody; wherein the symbol "/" denotes a connection; it should be noted that this "linkage" may be a direct linkage or an indirect linkage via a linking peptide.

6. English notation "IL-15R α Sushi/IL-15/anti-HSA VHH": according to the explanations of the above 1-3, the symbol in the present application represents a recombinant IL-15 fusion protein comprising, in order, the Sushi domain of interleukin-15 receptor alpha, interleukin-15, and anti-human serum albumin nanobody; wherein the symbol "/" denotes a connection; it should be noted that this "linkage" may be a direct linkage or an indirect linkage via a linking peptide.

7. English symbol "anti-HSA VHH/IL-15/IL-15R α Sushi": according to the explanations of the above 1-3, the symbol in the present application represents a recombinant IL-15 fusion protein comprising successively an anti-human serum albumin nanobody, interleukin-15, and the Sushi domain of interleukin-15 receptor alpha; wherein the symbol "/" denotes a connection; it should be noted that this "linkage" may be a direct linkage or an indirect linkage via a linking peptide.

8. English notation "anti-HSA VHH/IL-15R α Sushi/IL-15": according to the explanations of the above 1-3, the symbol in the present application represents a recombinant IL-15 fusion protein comprising, in order, an anti-human serum albumin nanobody, a Sushi domain of interleukin-15 receptor alpha, and interleukin-15; wherein the symbol "/" denotes a connection; it should be noted that this "linkage" may be a direct linkage or an indirect linkage via a linking peptide.

9. Human Serum Albumin (Human Serum Albumin, HSA): is a protein in human plasma, and human serum albumin can transport fatty acids, cholesterol, amino acids, steroid hormones, metal ions, and many therapeutic molecules, etc. in body fluids: while maintaining normal osmotic pressure of the blood. In clinic, human serum albumin can be used for treating shock and burn, supplementing blood loss caused by operation, accident or hemorrhage, and can also be used as blood plasma bulking agent. In the present application, human serum albumin is denoted by the english symbol "HSA", and its amino acid sequence is SEQ ID NO: 8, or a sequence shown in figure 8.

In order to solve the technical problems, the invention provides the following technical scheme:

in one aspect, the invention provides a recombinant IL-15 fusion protein.

Specifically, the recombinant IL-15 fusion protein comprises interleukin-15, a Sushi structural domain of interleukin-15 receptor alpha and an anti-human serum albumin nano antibody;

the interleukin-15, the Sushi structural domain of the interleukin-15 receptor alpha and the anti-human serum albumin nano antibody are directly connected or indirectly connected through a connecting peptide;

the general formula of the amino acid sequence of the anti-human serum albumin nano antibody is as follows:

EVQLVESGGGLVQPGNSLRLSCAASGFTFSSX1X2X3X4 WVRQACPGKGLEWVSSISGSGSGSX 5X6X7X8 ADSVKGRIDNISDNAKTLYLQMNNSLREDTAVYYCX 9X10X11X12X13X 15X16WGQGTLVTVSS, wherein: x1 is any one of amino acids H, S, F, Y, N, X2 is any one of amino acids G, A, W, Y, X3 is any one of amino acids W, I, M, X4 is any one of amino acids H, S, G, X5 is any one of amino acids S, G, N, D, X6 is any one of amino acids K, T, I, X7 is any one of amino acids D, L, Y, N, X8 is any one of amino acids Y, F, X9 is any one of amino acids A, T, V, X10 is any one of amino acids A, R, T, I, X11 is any one of amino acids E, G, D, X12 is any one of amino acids Y, D, G, R, X13 is any one of amino acids S, G, Y, L, X14 is any one of amino acids Y, S, G, X15 is amino acid G, amino acid B, s, D, N, A, and X16 is any one of amino acids K, S, Y, R, I.

Specifically, the recombinant IL-15 fusion protein comprises the following components from the N end to the C end in sequence:

interleukin-15, the Sushi domain of interleukin-15 receptor alpha, and anti-human serum albumin nanobody;

or, the Sushi domain of interleukin-15 receptor alpha, interleukin-15 and anti-human serum albumin nanobody;

or, anti-human serum albumin nanobody, interleukin-15 and the Sushi domain of interleukin-15 receptor alpha;

or anti-human serum albumin nano-antibody, the Sushi structural domain of interleukin-15 receptor alpha and interleukin-15.

Specifically, the amino acid sequence of the interleukin-15 is SEQ ID NO: 1, or a fragment thereof.

Specifically, the amino acid sequence of the Sushi structural domain of the interleukin-15 receptor alpha is SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

Specifically, the interleukin-15 and the Sushi structural domain of the interleukin-15 receptor alpha are connected through a connecting peptide; the connecting peptide is (G)_xS_y）_nWherein: g is glycine, S is serine, x is 1, 2, 3 or 4, y is 1, 2, 3 or 4, and n is 1, 2, 3, 4, 5 or 6.

Preferably, the amino acid sequence of the connecting peptide is SEQ ID NO: 3, and (b) is the sequence shown in the specification.

Preferably, the amino acid sequence of the anti-human serum albumin nanobody is SEQ ID NO: 4, or a sequence shown in the figure.

Preferably, the amino acid sequence of the recombinant IL-15 fusion protein is shown in SEQ ID NO: 5, respectively.

In another aspect, the present invention provides a biological molecule, vector or host cell comprising a nucleic acid molecule encoding a recombinant IL-15 fusion protein as described above.

In still another aspect, the present invention provides the use of the above recombinant IL-15 fusion protein and/or biological molecule, vector or host cell in the preparation of a tumor-associated medicament.

Specifically, the tumor includes, but is not limited to, any one or more of melanoma, lung cancer, head and neck cancer, colorectal cancer, non-small cell lung cancer, squamous cell cancer of the head and neck, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colon cancer, renal cancer, gastric cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, glioma, lymphoma, leukemia, myeloma, sarcoma, or virus-related tumor.

In another aspect, the invention also provides a pharmaceutical composition.

In particular, the pharmaceutical composition comprises the recombinant IL-15 fusion protein and/or a biological molecule, vector or host cell.

Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

More specifically, the pharmaceutically acceptable carriers include, but are not limited to: diluents, excipients, fillers, wetting agents, disintegrants, flavoring agents and binders.

Compared with the prior art, the invention has the following beneficial effects:

the recombinant IL-15 fusion protein provided by the invention can be combined with human serum albumin, and can effectively activate T cells at a low dose concentration, so that an immune system can play a role, and the aim of preventing and treating tumors is fulfilled.

Drawings

FIG. 1 shows the SDS-PAGE results of recombinant IL-15 fusion proteins. Wherein, the loading information represented by different lanes is as follows: lane A is the expression supernatant, lane B is the purification flow-through, lane C is the eluate (reduced), lane D is the eluate (non-reduced), and lane E is the protein standard.

FIG. 2 shows the result of CTLL2 cell proliferation induced by recombinant IL-15 fusion protein.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

Example 1 Gene Synthesis and vector construction of recombinant IL-15 fusion proteins

Assembling amino acid sequences of IL-15 (SEQ ID NO: 1), IL-15R alpha Sushi (SEQ ID NO: 2) and anti-human serum albumin nano antibody according to the sequence of interleukin-15, the Sushi domain of interleukin-15 receptor alpha and anti-human serum albumin nano antibody, indirectly connecting IL-15 and IL-15R alpha Sushi through flexible connecting peptide (SEQ ID NO: 3) to form a recombinant IL-15 fusion protein sequence (SEQ ID NO: 5), performing codon optimization according to human host cells and conventionally synthesizing genes, and then sequentially adding an EcoRI enzyme cutting site and 5'UTR (SEQ ID NO: 6) at the 5' end; 3'UTR ([ TGATGA ]) and HindIII cleavage sites were added to the 3' end in this order, and the gene was cloned into vector pTT5 (SEQ ID NO: 7) by 5 'EcoRI and 3' HindIII. Selecting clone for sequencing, selecting correctly sequenced thallus for conservation and enlarged culture, and using the enlarged thallus for extraction of plasmid.

EXAMPLE 2 expression and purification of recombinant IL-15 fusion proteins

Cell transfection and protein isolation and purification of the extracted plasmid were performed as follows:

1. the cell density is determined, the activity should be more than 95%, the 293 cell density is adjusted to 3X 10 by using the pre-heated 293 medium⁶cells/mL, gently shaken and aliquoted (transfection system 90%), the volume of cells in the flask did not exceed 1/3, which is the specification for the flask, and placed on a shaker until ready for use.

2. The volume of the transfection buffer opti-MEM was calculated from the volume of transfected cells and was 1/10 for the transfection system; calculating the amount of PEI as a transfection reagent, wherein the proportion of PEI is 3 mu L/mL of transfected cells; the total amount of transfected DNA was calculated in a ratio of 1. mu.g/mL of transfected cells.

The specific transfection procedure was as follows:

adding 10% of transfection buffer opti-MEM into 1 50mL centrifuge tube, adding plasmid, mixing, filtering, standing for 5min, adding PEI into the DNA suspension, mixing gently (mixing gently by inversion for 2-3 times), and standing for 15-20 min. Then, the compound is gently added into the subpackaged cells, and the shake flask is gently shaken while the compound is added; the transfected cells were cultured in a shaker at 37 ℃.

Feeding on day 1: after 20h, the corresponding amount of enhancer was added at 1:1000 transfected cell volume and the feed medium at 2% transfected cell volume.

Determining the expression amount of the transfected cells on day 4: taking 200 mu L of sample to be tested, detecting the protein expression quantity by using a label-free analyzer, using 293 culture medium as a negative control, and using a solution with known protein concentration as a positive control during the analysis of the analyzer.

Collecting samples on 5-6 days: detecting the cell viability (65-75% or more preferably), centrifuging the sample at 3000-.

The SDS-PAGE detection result of the protein purified by the expression of the obtained molecules is shown in figure 1.

Example 3 binding assay of recombinant IL-15 fusion protein to HSA

By using Gator^TMThe non-labeled bioanalyzer was used to detect the affinity between the recombinant IL-15 fusion Protein and HSA Protein (purchased from ACROBIOSystems, cat # HSA-H5220), and Protein A biosensor was used to capture the antibody sample, and then the kinetic detection of the binding and dissociation of the captured recombinant IL-15 fusion Protein and HSA Protein was carried out. Kinetics were performed using 1:1 the fitting analysis is performed in conjunction with the model. The brief steps are as follows: protein loading for 200 s; binding for 180 s; dissociation for 300 s; and regenerating for 30 s.

Using Gator^TMThe affinity data measured by the instrument are shown in table 1 below.

TABLE 1 affinity assay results for recombinant IL-15 fusion proteins and HSA proteins

Antigens

Concentration of

Fusion proteins

Response value

Affinity (M)

Dissociation constant (1/s)

Binding constant (1/Ms)

HSA

10 μg/ml

83 nM

0.473

2.58E-08

6.83E-03

2.65E+05

The detection result shows that the expressed recombinant IL-15 fusion protein can be combined with HSA protein with higher affinity, which ensures that the expressed recombinant IL-15 fusion protein can exist in vivo for a longer time, thereby effectively prolonging the half-life of the medicament.

Example 4 Activity assay of recombinant IL-15 fusion proteins

CTLL2 cells are mouse T lymphocytes, CTLL2 cells express receptors for test proteins, which, when bound to CTLL2, promote proliferation of the cells. CellTiter-Glo was used in this example^®The reagent detects the proliferation effect of the expressed recombinant IL-15 fusion protein on CTLL2 cells. CellTiter-Glo^®The reagent quantifies the number of cells by detecting the content of ATP in the living cells, and the more cells, the stronger the detected luminescent signal.

The method comprises the following specific steps:

1. CTLL2 cells were cultured with RPMI 1640 medium (ATCC Modification, Gibco # A1049101) +10% FBS +2mM L-Glutamine +1mM sodium pyruvate +10ng/mL hIL-2, the cells which had been expanded were collected, centrifuged at 1000rpm for 5 minutes after being blown out uniformly, washed 3 times with RPMI 1640 medium (ATCC Modification, Gibco # A1049101) +10% FBS +2mM L-Glutamine +1mM sodium pyruvate, then resuspended and counted in this medium and the cell concentration was adjusted to 2E5/mL, and 50. mu.L of the cells were plated in a 96-well plate.

2. The recombinant IL-15 fusion protein was diluted in a 4-fold concentration gradient from 10nM, 50. mu.L per well, in cells at 37 ℃ with 5% CO₂Incubate for 48 hours.

3. After 48 hours, the plates were removed from the incubator and allowed to equilibrate at room temperature for 10 minutes, 100. mu.L CelTiter-Glo were added to each well^®The cells were lysed thoroughly by shaking on a microplate mixer at 500rpm for 10 minutes.

4. Luminescence signal values were read on a microplate reader and the results were analyzed using GraphPad Prism 5.

The analysis result shows (as figure 2), the expressed recombinant IL-15 fusion protein can induce CTLL2 cell proliferation, and has obvious dose dependence, EC₅₀About 1.6nM, which indicates that the expressed recombinant IL-15 fusion protein can effectively activate T cells at very low dose levels, and thus kill tumor cells.

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

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Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

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ctgccccttc ttcgcggggc agtgcatgta atcccttcag ttggttggta caacttgcca 1500

actgaaccct aaacgggtag catatgcttc ccgggtagta gtatatacta tccagactaa 1560

ccctaattca atagcatatg ttacccaacg ggaagcatat gctatcgaat tagggttagt 1620

aaaagggtcc taaggaacag cgatgtaggt gggcgggcca agataggggc gcgattgctg 1680

cgatctggag gacaaattac acacacttgc gcctgagcgc caagcacagg gttgttggtc 1740

ctcatattca cgaggtcgct gagagcacgg tgggctaatg ttgccatggg tagcatatac 1800

tacccaaata tctggatagc atatgctatc ctaatctata tctgggtagc ataggctatc 1860

ctaatctata tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc 1920

ctaatttata tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc 1980

ctaatctata tctgggtagt atatgctatc ctaatctgta tccgggtagc atatgctatc 2040

ctaatagaga ttagggtagt atatgctatc ctaatttata tctgggtagc atatactacc 2100

caaatatctg gatagcatat gctatcctaa tctatatctg ggtagcatat gctatcctaa 2160

tctatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa 2220

tctatatctg ggtagtatat gctatcctaa tttatatctg ggtagcatag gctatcctaa 2280

tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa 2340

tctgtatccg ggtagcatat gctatcctca tgataagctg tcaaacatga gaattaattc 2400

ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 2460

ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 2520

atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 2580

tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 2640

cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 2700

agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 2760

taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 2820

tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg 2880

catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 2940

ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 3000

ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 3060

catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 3120

aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt 3180

aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 3240

taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 3300

atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 3360

gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 3420

tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 3480

ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 3540

gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 3600

agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 3660

aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 3720

agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 3780

tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 3840

atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 3900

taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 3960

gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 4020

gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 4080

aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 4140

tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 4200

gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 4260

cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 4320

ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 4380

cgagtcagtg agcgaggaag c 4401

<210> 8

<211> 585

<212> PRT

<213> Artificial Sequence

<400> 8

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln

20 25 30

Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

100 105 110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His

115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala

165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser

180 185 190

Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu

195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

210 215 220

Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

275 280 285

Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

290 295 300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

325 330 335

Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr

340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

355 360 365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

370 375 380

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

435 440 445

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

450 455 460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

465 470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr

485 490 495

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

500 505 510

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

515 520 525

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

530 535 540

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

545 550 555 560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

565 570 575

Ala Ala Ser Gln Ala Ala Leu Gly Leu

580 585

Claims

1. A recombinant IL-15 fusion protein, wherein the amino acid sequence of the recombinant IL-15 fusion protein is SEQ ID NO: 5, and (c) a sequence shown in (5).

2. A vector comprising a nucleic acid molecule encoding the recombinant IL-15 fusion protein of claim 1.

3. A host cell comprising a nucleic acid molecule encoding the recombinant IL-15 fusion protein of claim 1.

4. Use of a recombinant IL-15 fusion protein according to claim 1, a vector according to claim 2 and/or a host cell according to claim 3 for the preparation of a tumor-associated medicament.

5. The use of claim 4, wherein the tumor is any one or more of melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, renal cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, glioma, lymphoma, leukemia, myeloma, sarcoma.

6. The use according to claim 5, wherein the tumor is any one or more of non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, gastric cancer.

7. The use according to claim 6, wherein the colorectal cancer is colon cancer.

8. The use of claim 4, wherein the neoplasm is a virus-associated neoplasm.

9. A pharmaceutical composition comprising the recombinant IL-15 fusion protein of claim 1, the vector of claim 2, and/or the host cell of claim 3.