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CN113943374B - Interleukin 15 and polypeptide complex of receptor thereof - Google Patents

Interleukin 15 and polypeptide complex of receptor thereof Download PDF

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Publication number
CN113943374B
CN113943374B CN202111123534.XA CN202111123534A CN113943374B CN 113943374 B CN113943374 B CN 113943374B CN 202111123534 A CN202111123534 A CN 202111123534A CN 113943374 B CN113943374 B CN 113943374B
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CN113943374A (en
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芦迪
霍永庭
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Guangdong Fapon Biopharma Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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Abstract

The invention relates to the field of biological medicine, in particular to a polypeptide complex containing interleukin 15 and a receptor thereof. The IL15 and the IL15R alpha have a non-natural inter-chain bond, the non-natural inter-chain bond is formed between a first mutation residue of the IL15 and a second mutation residue of the IL15R alpha, the first mutation residue of the IL15 is E at the 90 th position and is mutated into C, and the second mutation residue of the IL15R alpha is P at the 67 th position and is mutated into C.

Description

Interleukin 15 and polypeptide complex of receptor thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a polypeptide complex containing interleukin 15 and a receptor thereof.
Background
Cytokines play an important role in human body immunoregulation, and are also involved in the immunoregulation of tumors, and are closely related to the occurrence and development of tumors. In immunotherapy, cytokines can directly act on immune effector cells in the tumor microenvironment, enhancing tumor suppression effects. Through clinical studies and animal experiments, many cytokines have been demonstrated to have significant anti-tumor activity, and many cytokines have been FDA approved for sale.
Interleukin 15 (IL-15) is a cytokine found by Grabstein et al in 1994 to be about 12-14kD and can play a role in the normal immune response of the body, such as promoting proliferation of T cells, B cells, natural Killer (NK) cells.
IL-15 belongs to a member of the four Small alpha helix bundle cytokine families (Small four alpha-helix bundle familyof cytokines). 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-15 Rα), IL-2 receptor beta (IL-2 Rβ, also known as IL-15Rβ or CD 122), and yc (also known as CD 132). IL-15Rα contains a Sushi domain, binds to IL-15, and is required for the bound IL-15 to perform biological functions.
In recent years, IL15 and IL15 receptors have been increasingly used for constructing fusion proteins, and in order to improve the stability of the fusion proteins, disulfide bonds have been introduced between interleukin 15 and its receptor.
Disclosure of Invention
The invention provides an IL15/IL15Rα polypeptide complex, wherein an unnatural inter-chain bond is formed between IL15 and IL15Rα, the unnatural inter-chain bond is formed between a first mutation residue of IL15 and a second mutation residue of IL15Rα, the first mutation residue of IL15 is E mutation at position 90 into C, and the second mutation residue of IL15Rα is P mutation at position 67.
In some embodiments, the full length of the extracellular region of IL15 ra or the IL15 ra sushi domain.
In some embodiments, the IL15Rα has 73 to 175 amino acids.
In some embodiments, the IL15 has the amino acid sequence set forth in SEQ ID No. 9.
In some embodiments, the IL15Rα has an amino acid sequence set forth in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, or SEQ ID NO. 14.
The invention also relates to vectors containing the nucleic acids as described above.
The invention also relates to a host cell containing a nucleic acid as described above or a vector as described above.
The invention also relates to a method for preparing the IL15/IL15Rα polypeptide complex, which comprises the following steps:
Transforming a host cell with a vector as described above;
Culturing the transformed host cell; and
Collecting the IL15/IL15Rα polypeptide complex expressed in the host cell.
The invention also relates to pharmaceutical compositions comprising an IL15/IL15 ra polypeptide complex as described above and a pharmaceutically acceptable carrier, excipient, or stabilizer.
The invention also relates to the use of an IL15/IL15Rα polypeptide complex as described above for the preparation of a medicament for the treatment of a disease.
Drawings
FIG. 1 is a schematic diagram of the interaction of IL15/IL15Rα and IL2/15Rβ/γC complexes;
FIG. 2 is a schematic diagram showing the molecular disulfide mutation pair of IL15 (ligand) and IL15Rα (receptor) in the embodiment of the invention;
FIG. 3 is a schematic diagram of an exemplary IL15/IL15Rα complex structure;
FIG. 4 is a schematic diagram of an exemplary IL15/IL15Rα complex specific application scenario;
FIG. 5 shows the results of gel electrophoresis detection of disulfide-engineered IL15/IL15Rα complexes;
FIG. 6 is a graph showing the results of the detection of binding force of disulfide bond engineered and non-engineered IL15/IL15Rα complexes to the targeting region (@ TIGIT);
FIG. 7 is a graph showing the results of the detection of binding force of disulfide bond engineered and non-engineered IL15/IL15Rα complexes to the targeting region (@ PD-L1).
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment. All documents cited in this disclosure, including publications, patents, and patent applications, are incorporated by reference in their entirety.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Embodiments of the present invention will be described in detail below with reference to examples.
EXAMPLE 1 disulfide bond engineering of IL15/IL15Rα complexes
Disulfide engineering between IL15, IL15 ra, helps to form covalent disulfide linkages between non-covalently linked IL15 and IL15 ra. According to the sequence information of IL-15 (accession number: NP-000576), IL-15Rα (accession number: NP-002180) and Chinese patent CN110214148A in the public database UniProt, the amino acid sequence of the mature form of wild-type human IL15 is shown as SEQ ID NO.15, and the amino acid sequence of the whole extracellular region of IL15Rα is shown as SEQ ID NO. 16;
Amino acid sequence of mature form of wild-type human IL15 (SEQ ID No. 15):
wild-type IL15R alpha extracellular region full-length amino acid sequence (SEQ ID NO. 16):
The mutation sites are shown in the following table:
Combination of two or more kinds of materials IL15 IL15Rα
1 E87C D96+C97
2 E90C P67C
3 E93C R35C
Exemplary selection of a use scenario for the IL15/IL15Rα complex: the heavy chain variable region (VH) variable region of the target antigen is connected to IL15Rα through Linker, and then connected with Fc of the antibody through finger; the light chain variable region (VL) of the targeting same antigen is connected to IL15 through a Linker to prepare the targeting IL15/IL15Rα complex.
The above complex can be applied to molecules targeting the same antigen or target, such as the structure of fig. 4B or fig. 4C (the scfv targets the same antigen or target), and can also be used to target 2 or more antigen or target molecules, such as fig. 4A or fig. 4C (the scfv targets different antigens or targets), and the application scenario of the complex is not limited to the above examples.
Example 2 preparation of a Complex sample
Transient protein expression:
the plasmid containing the target gene is introduced into host cell Expi293 after forming a cationic complex with transfection reagent PEI, and the exogenous gene on the plasmid is transcribed and translated in the cell during the period of the plasmid in the cell, thereby obtaining the target protein.
The Expi293 was incubated at 37℃with 8% carbon dioxide at 130rpm and 2E6 cells were inoculated into 1L shake flasks by cell counting prior to transfection, the culture system being approximately 300ml. Preparing transfection complex for transfection: firstly, 750 mug of target plasmid is added into a 50ml centrifuge tube containing 15mlOpti-MEM reagent, and the mixture is gently mixed and marked as a tube A; 1.5mg of the transfection reagent PEI was added to a 50ml centrifuge tube containing 15mlOpti-MEM reagent, gently mixed, incubated at room temperature for 5min, and labeled as tube B; and (3) dropwise adding the PEI diluent of the B tube into the DNA diluent of the A tube, slightly mixing, incubating for 15min at room temperature, adding the PEI-target plasmid complex into the Expi293 cells after incubation, and placing the mixture in a shaking table at 37 ℃ for continuous culture. Until D7-D10, the sample is collected.
Purification of complex samples:
The transient cell expression liquid is centrifuged at 9000rpm/20min, and the supernatant is collected and sterilized and filtered by a 0.22 mu m filter membrane. ProA affinity chromatography is adopted for purification. The procedure is as follows, using AKTAAVANT.sup.150 chromatography apparatus, the column (e.g. MabSelectSuRe LX, GE) is equilibrated with at least 5CV equilibration buffer (10 mM PBS), and the sample is loaded onto the column, allowing the target protein to adsorb onto the column while other impurities penetrate the column. After loading was completed, the column was again rinsed with at least 5CV of equilibration buffer (10 mM PBS), followed by elution of the target protein with elution buffer (20 mM naac, ph=3.4), and the collection tube was pre-loaded with neutralization buffer (1 m tris, ph 8.0) at a volume of 10% of the elution volume depending on the estimated amount of eluted sample.
EXAMPLE 3 disulfide engineering IL15/IL15Rα Complex gel electrophoresis detection
SDS-PAGE electrophoresis detection is carried out on the disulfide bond modified IL15/IL15Rα complex, the detection result is shown in figure 5, and a band exists between the molecular weight of 25KD and 35KD, which indicates that a free light chain exists; complex 1 and complex 2 (disulfide bond modification position IL15 (E90C)/IL 15Rα (P67C)) were free of bands between molecular weights 25KD and 35KD, indicating successful disulfide bond modification, complex 3 (disulfide bond modification position IL15 (E87C)/IL 15Rα (D96+C97)), and complex 4 (disulfide bond modification position IL15 (E93C)/IL 15Rα (R35C)) were free of bands between molecular weights 25KD and 35KD, indicating failure of disulfide bond modification.
Example 4 targeting moiety affinity detection
TIGIT end binding Activity assay
The binding activity of the diabody molecule (TIGIT end) to CHO-TIGIT cells was detected by FCM assay. Preparing 3% BSA buffer: weighing 4.5gBSA to 150mL of 1XPBS, uniformly mixing, and placing on ice for later use; antibody dilution: the test antibody and the positive control are diluted with 3% BSA to an initial concentration of 800nM, the subtype control is diluted to an initial concentration of 20 mug/mL, the volume is 300 mug, and the total of 10 points are 3 times of gradient dilution (100+200); binding activity assay: cell count and plating: after counting the R0254-3 cells, the cells were separated into 96-well V-shaped plates at 100. Mu.L, 2E+05/well; firstly adding 50 mu L of antibodies with different concentrations into cells, incubating for 0.5h at 2-8 ℃, then adding 50 mu L of ligand, and incubating for 0.5h at 2-8 ℃; after centrifugation at 350Xg for 5min, the supernatant was removed and 200. Mu.L/well of 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed, fluorescent antibodies PE Goat anti-human IgG Fc and PE Goat anti-mouse IgG Fc (1:500Xdilution) were formulated in 3% BSA, added to the corresponding 96-well plates at 100. Mu.L/well and incubated at 2-8℃for 30min; centrifugation at 350g for 5min, removal of supernatant, washing of cells with 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed and 1XPBS was added at 100. Mu.L/well to resuspend cells; the results of the on-press detection according to CytoFLEX flow cytometer standard protocol are shown in figure 6, which compares the affinity of the molecules without disulfide modification (the sequence is identical to complex 1 except for disulfide modification), indicating that disulfide modification does not affect the affinity of the targeting region.
PD-L1 end binding Activity assay
The binding activity of the diabody molecule (PD-L1 end) to CHO-PD-L1 cells was examined by FCM assay. Preparing 3% BSA buffer: weighing 4.5gBSA to 150mL of 1XPBS, uniformly mixing, and placing on ice for later use; antibody dilution: the test antibody and the positive control are diluted with 3% BSA to an initial concentration of 800nM, the subtype control is diluted to an initial concentration of 20 mug/mL, the volume is 300 mug, and the total of 10 points are 3 times of gradient dilution (100+200); binding activity assay: cell count and plating: after counting the R0254-3 cells, the cells were separated into 96-well V-shaped plates at 100. Mu.L, 2E+05/well; firstly adding 50 mu L of antibodies with different concentrations into cells, incubating for 0.5h at 2-8 ℃, then adding 50 mu L of ligand, and incubating for 0.5h at 2-8 ℃; after centrifugation at 350Xg for 5min, the supernatant was removed and 200. Mu.L/well of 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed, fluorescent antibodies PE Goat anti-human IgG Fc and PE Goat anti-mouse IgG Fc (1:500Xdilution) were formulated in 3% BSA, added to the corresponding 96-well plates at 100. Mu.L/well and incubated at 2-8℃for 30min; centrifugation at 350g for 5min, removal of supernatant, washing of cells with 3% BSA; after centrifugation at 350Xg for 5min, the supernatant was removed and 1XPBS was added at 100. Mu.L/well to resuspend cells; and (3) performing on-machine detection according to CytoFLEX standard operation procedures of the flow cytometer. The results of the assay are shown in FIG. 7, which shows that the disulfide modification does not affect the affinity of the targeting region, as compared to the molecule without disulfide modification (the sequence is identical to complex 2 except for disulfide modification).
EXAMPLE 5 disulfide engineering of other IL15Rα molecules
Through verification, the disulfide bond modification position IL15 (E90C)/IL 15Rα (P67C) can be successfully applied to the following IL15Rα structures with different lengths, and the specific sequences are shown in the following table:
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
SEQUENCE LISTING
<110> Guangdong Phepoaching pharmaceutical Co., ltd
<120> A polypeptide complex of Interleukin 15 and its receptor
<130> 2021
<160> 20
<170> PatentIn version 3.5
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile Arg Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Met Phe
50 55 60
Lys Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Ile His Asp Tyr Gly His Gly Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Thr Cys Pro Pro
130 135 140
Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu
145 150 155 160
Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala
165 170 175
Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val
180 185 190
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Cys Ala Leu
195 200 205
Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr Ala Gly Val
210 215 220
Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu Pro Ala Ala
225 230 235 240
Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala Ala Ile Val
245 250 255
Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr Gly Thr Thr
260 265 270
Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser Gln Thr Thr
275 280 285
Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln Pro Pro Gly
290 295 300
Val Tyr Pro Gln Gly His Ser Asp Thr Thr Glu Pro Lys Ser Ser Asp
305 310 315 320
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
325 330 335
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
340 345 350
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
355 360 365
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
370 375 380
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
385 390 395 400
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
405 410 415
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
420 425 430
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
435 440 445
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
450 455 460
Thr Cys Arg Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
465 470 475 480
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
485 490 495
Leu Lys Ser Asp Gly Ser Phe Phe Leu Ala Ser Lys Leu Thr Val Asp
500 505 510
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
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Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
530 535 540
Gly Lys
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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
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Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val
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Tyr Ala Ala Ser His Leu Pro Asp Gly Val Pro Ser Arg Phe Ser Gly
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Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro
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Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Arg
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Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
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Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
130 135 140
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
145 150 155 160
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
165 170 175
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
180 185 190
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
195 200 205
Thr Glu Ser Gly Cys Lys Glu Cys Glu Cys Leu Glu Glu Lys Asn Ile
210 215 220
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
225 230 235 240
Thr Ser
<210> 3
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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Thr Cys
130 135 140
Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr
145 150 155 160
Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg
165 170 175
Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr
180 185 190
Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Cys
195 200 205
Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr Ala
210 215 220
Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu Pro
225 230 235 240
Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala Ala
245 250 255
Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr Gly
260 265 270
Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser Gln
275 280 285
Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln Pro
290 295 300
Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Glu Pro Arg Gly
305 310 315 320
Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu
325 330 335
Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val
340 345 350
Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val
355 360 365
Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val
370 375 380
Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser
385 390 395 400
Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met
405 410 415
Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala
420 425 430
Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro
435 440 445
Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln
450 455 460
Val Thr Leu Trp Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr
465 470 475 480
Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Asp Asn Thr
485 490 495
Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Asp Leu
500 505 510
Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser
515 520 525
Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser
530 535 540
Arg Thr Pro Gly Lys
545
<210> 4
<211> 245
<212> PRT
<213> Artificial Sequence
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<223> Complex 2-2
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Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
85 90 95
Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu
130 135 140
Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser
145 150 155 160
Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu
165 170 175
Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
180 185 190
Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn
195 200 205
Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Cys Leu Glu Glu
210 215 220
Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met
225 230 235 240
Phe Ile Asn Thr Ser
245
<210> 5
<211> 544
<212> PRT
<213> Artificial Sequence
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<223> Complex 3-1
<400> 5
Gln Val Gln Leu Glu Ser Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ser Ser Leu Ser Ser Ser Tyr
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Ile Ile Gly Ser Asn Gly Asn Thr Tyr Tyr Ala Asn Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Glu Leu Lys Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly
85 90 95
Tyr Arg Thr Ser Gly Met Asp Pro Trp Gly Pro Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Ile Thr Cys Pro Pro Pro Met Ser
130 135 140
Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg
145 150 155 160
Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser
165 170 175
Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp
180 185 190
Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln
195 200 205
Arg Pro Ala Pro Pro Ser Thr Val Thr Thr Ala Gly Val Thr Pro Gln
210 215 220
Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu Pro Ala Ala Ser Ser Pro
225 230 235 240
Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala Ala Ile Val Pro Gly Ser
245 250 255
Gln Leu Met Pro Ser Lys Ser Pro Ser Thr Gly Thr Thr Glu Ile Ser
260 265 270
Ser His Glu Ser Ser His Gly Thr Pro Ser Gln Thr Thr Ala Lys Asn
275 280 285
Trp Glu Leu Thr Ala Ser Ala Ser His Gln Pro Pro Gly Val Tyr Pro
290 295 300
Gln Gly His Ser Asp Thr Thr Glu Pro Arg Gly Pro Thr Ile Lys Pro
305 310 315 320
Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser
325 330 335
Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu
340 345 350
Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro
355 360 365
Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala
370 375 380
Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val
385 390 395 400
Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe
405 410 415
Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr
420 425 430
Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu
435 440 445
Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Trp Cys
450 455 460
Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn
465 470 475 480
Asn Gly Lys Thr Glu Leu Asn Tyr Asp Asn Thr Glu Pro Val Leu Asp
485 490 495
Ser Asp Gly Ser Tyr Phe Met Tyr Ser Asp Leu Arg Val Glu Lys Lys
500 505 510
Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly
515 520 525
Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
530 535 540
<210> 6
<211> 245
<212> PRT
<213> Artificial Sequence
<220>
<223> Complex 3-2
<400> 6
Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Leu Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Glu Ser
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Glu His Ser Val Gly Asn
85 90 95
Val Asp Asn Val Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu
130 135 140
Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser
145 150 155 160
Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu
165 170 175
Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
180 185 190
Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn
195 200 205
Gly Asn Val Thr Glu Ser Gly Cys Lys Cys Cys Glu Glu Leu Glu Glu
210 215 220
Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met
225 230 235 240
Phe Ile Asn Thr Ser
245
<210> 7
<211> 546
<212> PRT
<213> Artificial Sequence
<220>
<223> Complex 4-1
<400> 7
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile Arg Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Met Phe
50 55 60
Lys Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Ile His Asp Tyr Gly His Gly Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Thr Cys Pro Pro
130 135 140
Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu
145 150 155 160
Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Cys Lys Ala
165 170 175
Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val
180 185 190
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu
195 200 205
Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr Ala Gly Val
210 215 220
Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu Pro Ala Ala
225 230 235 240
Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala Ala Ile Val
245 250 255
Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr Gly Thr Thr
260 265 270
Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser Gln Thr Thr
275 280 285
Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln Pro Pro Gly
290 295 300
Val Tyr Pro Gln Gly His Ser Asp Thr Thr Glu Pro Lys Ser Ser Asp
305 310 315 320
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
325 330 335
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
340 345 350
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
355 360 365
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
370 375 380
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
385 390 395 400
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
405 410 415
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
420 425 430
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
435 440 445
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
450 455 460
Thr Cys Arg Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
465 470 475 480
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
485 490 495
Leu Lys Ser Asp Gly Ser Phe Phe Leu Ala Ser Lys Leu Thr Val Asp
500 505 510
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
515 520 525
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
530 535 540
Gly Lys
545
<210> 8
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> Complex 4-2
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val
35 40 45
Tyr Ala Ala Ser His Leu Pro Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
130 135 140
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
145 150 155 160
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
165 170 175
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
180 185 190
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
195 200 205
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Cys Lys Asn Ile
210 215 220
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
225 230 235 240
Thr Ser
<210> 9
<211> 114
<212> PRT
<213> Artificial Sequence
<220>
<223> IL15(E90C)
<400> 9
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
1 5 10 15
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
20 25 30
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
35 40 45
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Cys Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser
<210> 10
<211> 175
<212> PRT
<213> Artificial Sequence
<220>
<223> IL15RA-175aa(P67C)
<400> 10
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg Asp Cys Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val
65 70 75 80
Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly
85 90 95
Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr
100 105 110
Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro
115 120 125
Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr
130 135 140
Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser
145 150 155 160
His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr
165 170 175
<210> 11
<211> 77
<212> PRT
<213> Artificial Sequence
<220>
<223> IL15RA-77aa(P67C)
<400> 11
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg Asp Cys Ala Leu Val His Gln Arg Pro Ala Pro Pro
65 70 75
<210> 12
<211> 73
<212> PRT
<213> Artificial Sequence
<220>
<223> IL15RA-73aa(P67C)
<400> 12
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg Asp Cys Ala Leu Val His Gln Arg
65 70
<210> 13
<211> 86
<212> PRT
<213> Artificial Sequence
<220>
<223> IL15RA-86aa(P67C)
<400> 13
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg Asp Cys Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val
65 70 75 80
Thr Thr Ala Gly Val Thr
85
<210> 14
<211> 102
<212> PRT
<213> Artificial Sequence
<220>
<223> IL15RA-102aa(P67C)
<400> 14
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg Asp Cys Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val
65 70 75 80
Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly
85 90 95
Lys Glu Pro Ala Ala Ser
100
<210> 15
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Complex 1-3/Complex 4-3
<400> 15
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Pro Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Gln Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Thr Met Ser Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Arg Ala Trp Ile Arg Thr Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 16
<211> 215
<212> PRT
<213> Artificial Sequence
<220>
<223> Complex 1-4/Complex 4-4
<400> 16
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser
20 25 30
Asn Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp
35 40 45
Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro
85 90 95
Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 17
<211> 445
<212> PRT
<213> Artificial Sequence
<220>
<223> Complex 2-3
<400> 17
Gln Val Gln Leu Glu Ser Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ser Ser Leu Ser Ser Ser Tyr
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Ile Ile Gly Ser Asn Gly Asn Thr Tyr Tyr Ala Asn Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Glu Leu Lys Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly
85 90 95
Tyr Arg Thr Ser Gly Met Asp Pro Trp Gly Pro Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro
115 120 125
Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val
130 135 140
Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser
145 150 155 160
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
165 170 175
Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser
180 185 190
Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
195 200 205
Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro
210 215 220
Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile
225 230 235 240
Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln
260 265 270
Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln
275 280 285
Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu
290 295 300
Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys
305 310 315 320
Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys
325 330 335
Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro
340 345 350
Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Arg Val Thr
355 360 365
Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys
370 375 380
Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Lys Ser Asp Gly
385 390 395 400
Ser Tyr Phe Met Ala Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val
405 410 415
Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn
420 425 430
His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
435 440 445
<210> 18
<211> 217
<212> PRT
<213> Artificial Sequence
<220>
<223> Complex 2-4
<400> 18
Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Leu Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Glu Ser
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Glu His Ser Val Gly Asn
85 90 95
Val Asp Asn Val Phe Gly Gly Gly Thr Glu Val Val Val Lys Arg Thr
100 105 110
Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu
115 120 125
Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro
130 135 140
Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn
145 150 155 160
Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His
180 185 190
Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile
195 200 205
Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215
<210> 19
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Complex 3-3
<400> 19
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser
180 185 190
Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile
210 215 220
Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile
245 250 255
Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp
260 265 270
Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His
275 280 285
Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg
290 295 300
Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys
305 310 315 320
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu
325 330 335
Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr
340 345 350
Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu
355 360 365
Thr Cys Arg Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp
370 375 380
Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val
385 390 395 400
Leu Lys Ser Asp Gly Ser Tyr Phe Met Ala Ser Lys Leu Arg Val Glu
405 410 415
Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His
420 425 430
Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro
435 440 445
Gly Lys
450
<210> 20
<211> 217
<212> PRT
<213> Artificial Sequence
<220>
<223> Complex 3-4
<400> 20
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
85 90 95
Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Arg Thr
100 105 110
Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu
115 120 125
Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro
130 135 140
Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn
145 150 155 160
Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His
180 185 190
Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile
195 200 205
Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215

Claims (9)

1. An IL15/IL15Rα polypeptide complex, wherein an unnatural inter-chain bond is formed between IL15 and IL15Rα, the unnatural inter-chain bond is formed between a first mutation residue of IL15 and a second mutation residue of IL15Rα, the first mutation residue is that E at position 90 of IL15 is mutated to C, and the amino acid sequence of the mutated IL15 is shown in SEQ ID NO. 9; the second mutation residue is that the P at the 67 th position of the total length of the extracellular region of the IL15Rα is mutated into C; the amino acid sequence of IL15Rα is the amino acid sequence from the 1 st amino acid to the nth amino acid from the N end of SEQ ID NO.10, and N is any integer between 73 and 175.
2. The IL15/IL15 ra polypeptide complex of claim 1, wherein the IL15 ra is the full length of the IL15 ra extracellular region or a portion of the IL15 ra extracellular region comprising the IL15 ra sushi domain.
3. The IL15/IL15 ra polypeptide complex of claim 2, wherein the amino acid sequence of the full length of the extracellular region of IL15 ra is as shown in SEQ ID No. 16.
4. The IL15/IL15 ra polypeptide complex of claim 1, wherein the amino acid sequence of IL15 ra is as shown in SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No. 14.
5. An isolated nucleic acid encoding the IL15/IL15 ra polypeptide complex of any one of claims 1-4.
6. A vector comprising the nucleic acid of claim 5.
7. A host cell comprising the nucleic acid of claim 5 or the vector of claim 6.
8. A pharmaceutical composition comprising the IL15/IL15 ra polypeptide complex of any one of claims 1-4, and a pharmaceutically acceptable carrier, excipient, or stabilizer.
9. Use of an IL15/IL15 ra polypeptide complex as claimed in any one of claims 1 to 4 in the manufacture of a medicament for the treatment of a disease.
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