CN101166823A

CN101166823A - Combinatorial interleukin-2 muteins

Info

Publication number: CN101166823A
Application number: CNA2005800070728A
Authority: CN
Inventors: L·奥克曼; K·德尼斯-米泽; C·海斯; D·门尼泽斯; S·E·威尔森
Original assignee: Chiron Corp
Current assignee: NOVARTIS VACCINES and DIAGNOSTIC Inc
Priority date: 2004-03-05
Filing date: 2005-03-07
Publication date: 2008-04-23
Also published as: CN1930300A; CN101426916A

Abstract

Novel human interleukin-2 (IL-2) muteins or variants thereof, and nucleic acid molecules and variants thereof are provided. Methods for producing these muteins as well as methods for stimulating the immune system of an animal are also disclosed. In addition, the invention provides recombinant expression vectors comprising the nucleic acid molecules of this invention and host cells into which expression vectors have been introduced. Pharmaceutical compositions are included comprising a therapeutically effective amount of a human IL-2 mutein of the invention and a pharmaceutically acceptable carrier. The IL-2 muteins have lower toxicity than native IL-2 or Proleukin (R) IL-2, while maintaining or enhancing NK cell-mediated effects, and can be used in pharmaceutical compositions for use in treatment of cancer, and in stimulating the immune response.

Description

Combinatorial interleukin-2-2 mutain

Invention field

The present invention relates to have human interleukin-2 (IL-2) mutain of the result of treatment of raising.Also provide preparation to be used for the treatment of cancer and the recruit of stimulation immune system and the method for pharmaceutical preparation.

Background of invention

Interleukin-2 (IL-2) is the strong activator (Morgan etc. of natural killer details (NK) and T-cell proliferation and function, Sience 193:1007-11,1976) proved that this spontaneous cytokine has various malignant diseases of antagonism when oozing out lymphocyte (TIL) associating separately or with the killer cell (LAK) of lymphokineactivation or tumour anti-tumor activity (for example sees, Rosenberg etc., N.Engl.J.Med.316:889-97,1987; Rosenberg, Ann.Surg.208:121-35,1988; Topalian etc., J.Clin.Oncol.6:839-53,1988; Rosenberg etc., N.Engl.J.Med.319:1676-80,1988; Weber etc., J.Clin.Oncol.10:33-40,1992).Yet the high dosage IL-2 that adopts for tumor growth is obtained positive treatment result usually causes severe side effect, comprising that heating, shiver with cold, ypotension and capillary vessel seepage (vascular leak syndrome or VLS) and neuroscience change (for example sees, Duggan etc., J.Immunotherapy 12:115-22,1992; Gisselbrecht etc., Blood 83:2081-85,1994; Sznol and Parkinson, Blood 83:2020-22,1994).

Though IL-2 induces the accurate mechanism of toxicity and VSL it be unclear that, but the data of accumulation prompting IL-2 inductive kills and wounds the toxicity (DLT) that (NK) cell has triggered dose limitation, the result has excessively produced pro-inflammatory cytokine, comprises IFN-α, IFN-γ, TNF-α, TNF-β, IL-β and IL-6.These cytokines have activated monocyte/macrophage and have induced NO production, cause subsequently endothelial cell damage (Dubinett etc., Cell Immunol157:170-80,1994; Samlowski etc., J.Immunother.Emphasis Tumor Immunol.18:166-78,1995).These discoveries cause other people according to hypothesis: the IL-2 acceptor (IL-2R) that the T cell selective has been expressed high-affinity has been developed the T cell but not the NK cell has preferentially optionally the IL-2 mutain (for example see, BAY50-4798, sophisticated human IL-2's N88R IL-2 mutain, be disclosed in international monopoly WO 99/60128 and Shanafelt etc., Nat.Biotechnol.18:1197-202,2000).

The multiple NK cells effector functions, as natural killer (NK), LAK and antibody rely on (ADCC) molten cell killing, and production of cytokines depend on propagation and activate different NK intracellular signal conduction path specificity intermediate products.Importantly, evidence suggests that interactional selectivity adjusting can influence downstream NK-and the cell-mediated multiple effector functions of T-to IL-2-IL-2R, produce and molten cell killing (Sauve etc., Proc.Natl.Acad.Sci.U.S.A.88:4636-40,1991 as propagation, cytokine; Heaon etc., Cancer Res.53:2597-2602,1993; Eckenberg etc., J.Exp.Med.191:529-40,2000).

IL-2 (the human IL-2's mutain rIL-2 that contains reorganization; Chiron Corporation, Emeryville, California) FDA approval is used for the treatment of metastasis melanin tumor and kidney.And just studying and be used for other clinical disease, comprise non Hodgkin lymphoma, HIV and mammary cancer.Yet, because the relevant toxic side effect of IL-2 needs toxicity to lower the IL-2 mutain so that this interleukin has better treatment application.Have the tolerance of raising and/or the NK cell of enhanced IL-2 mediation or the IL-2 mutain of T cytological effect device function and will have purposes widely, be particularly suitable for cancer therapy and regulate immune response.

Summary of the invention

The invention relates to interleukin-2 (IL-2) mutain of estimating that toxicity reduces and function improves.The coding human IL-2 is provided the nucleic acid molecule and the isolating polypeptide that contains these sudden changes of mutain.With take off alanyl-1, C125S human IL-2 or C125S human IL-2 mutain and compare, this mutain induces the NK cell to produce the pro-inflammatory cytokine of lower level, keep or strengthened the propagation of NK cell simultaneously, kept the cell-mediated NK of NK, LAK and the molten cell function of ADCC.Also set forth the clinical application of these improved human IL-2's mutain treatment cancers and adjusting immune response.

On the one hand, isolated nucleic acid molecule provided by the invention contains the nucleotide sequence of coding human IL-2 mutain.In some embodiments, this nucleic acid molecule encoding contains the human IL-2's mutain that is selected from SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 aminoacid sequence.

The isolated nucleic acid molecule of the IL-2 mutain that the present invention includes in some embodiments, contains and is selected from nucleotide sequence shown in the SEQID NO:9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69 and 71.

In some embodiments, the isolated nucleic acid molecule that the present invention includes contains the nucleotide sequence of coding human IL-2 mutain, and wherein the aminoacid sequence of this mutain comprises the residue 2-133 that is selected from SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 sequence.

In some embodiments, isolated nucleic acid molecule of the present invention comprises the nucleotide sequence that contains the Nucleotide 4-399 that is selected from SEQ ID NO:9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69 and 71 sequence.

In some embodiments, described hereinly also can comprise replacement, wherein the be encoded codeword triplet of L-Ala of SEQ ID NO:9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69 and 71 Nucleotide 373-375 replaces.

In some embodiments, nucleic acid molecule described herein also can comprise replacement, and wherein be encoded three sons of halfcystine of SEQ ID NO:9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69 and 71 Nucleotide 373-375 replace.

In some embodiments, nucleic acid molecule as herein described is modified and optimized expression.In the nucleotide sequence that these nucleic acid comprise, one or more codons of this mutain of encoding are expressed in host cell interested through optimizing.The exemplary nucleic acid that contains optimizing codon includes but not limited to: contain the sequence that is selected from SEQ ID NO:73, the Nucleotide 4-399 of SEQ ID NO:73, SEQ ID NO:74 and the Nucleotide 4-399 of SEQ ID NO:74.

The present invention comprises also and is used for the carrier of expressing at selected host cell that described carrier contains one or more nucleic acid of the present invention.In this class expression vector, described nucleic acid operability is connected with and expresses compatible controlling element in selected host cell.Many expression regulation elements are well known by persons skilled in the art, include but not limited to following: transcripting promoter, transcriptional enhancer element, transcription termination signal, polyadenylic acid sequence, optimize sequence, translation termination sequence that starting is translated.Exemplary transcripting promoter includes but not limited to the promotor derived from polyomavirus, adenovirus 2, cytomegalovirus and simian virus 40.

On the other hand, cell provided by the invention contains carrier of the present invention, and nucleotide sequence wherein (as coding human IL-2 mutain) operability is connected in the compatible controlling element of expression in selected cell.In one embodiment, described cell is a mammalian cell.Exemplary mammalian cell includes but not limited to Chinese hamster ovary cell (CHO) or COS cell.Can be used for implementing other cell of the present invention, cell type, types of organization etc. includes but not limited to available from following cell: insect (as powder mosquito pretty young woman at night (Trichoplusia ni) (Tn5) and Sf9), bacterium, yeast, plant, antigen presenting cell (as scavenger cell, monocyte, dendritic cell, B-cell, T-cell, stem cell and their progenitor cell), primary cell, immortality cell, tumour derived cell.

On the other hand, the present invention is for being useful on composition and the host cell that reorganization produces the expression vector of human IL-2's mutain of the present invention containing.This composition can contain acceptable vehicle on the studies of the Hongloumeng.

Also having on the one hand, the invention provides the isolated polypeptide that contains human IL-2's mutain.In some embodiments, the present invention includes the isolating polypeptide that is selected from SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 aminoacid sequences that contains.

In some embodiments, the present invention includes the isolating polypeptide that contains the amino-acid residue 2-133 that is selected from SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 aminoacid sequences.

In some embodiments, aforementioned polypeptides also contains replacement.Wherein replaced serine residue at SEQ ID NO:10,125 alanine residues of 12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72.

In some embodiments, aforementioned polypeptides also contains replacement, has wherein replaced serine residue at SEQ ID NO:10,125 cysteine residues of 12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72.

In some embodiments, the present invention includes isolated polypeptide contain human IL-2's mutain, this mutain contains aminoacid sequence shown in the SEQ ID NO:4,125 Serines at SEQ ID NO:4 have replaced halfcystine, has other aminoacid replacement of two places among the SEQ ID NO:4 at least, compare with take off alanyl-1, C125S human IL-2 or the C125S of similar quantity, this mutain: 1) kept or strengthened the propagation and 2 of natural killer (NK) cell) the pro-inflammatory cytokine level that causes the NK cell to produce reduces.Exemplary combination replacement includes but not limited to: 19D40D, 19D81K, 36D42R, 36D61R, 36D65L, 40D36D, 40D61R, 40D65Y, 40D72N, 40D80K, 40G36D, 40G65G, 80K36D, 80K65Y, 81K36D, 81K42E, 81K61R, 81K65Y, 81K72N, 81K88D, 81K91D, 81K107H, 81L107H, 91N95G, 107H36D, 107H24E, 107H65Y, 107R36D, 107R72N, 40D81K107H, 40G81K107H and 91N94Y95G.In embodiment, this mutain also comprises the L-Ala disappearance of SEQ ID NO:4 position 1.

Available NK-92 biological test detects the raising of natural killer (NK) cell proliferation and the reduction that the NK cell produces the pro-inflammatory cytokine level.Can equate that comparing aforementioned polypeptides under the test conditions produces the effect of pro-inflammatory cytokine and the effect of taking off alanyl-1, C125S human IL-2 or C125S human IL-2 of similar quantity to NK cell proliferation and NK cell.In some embodiments, can be under the test conditions that equates, prove with the NK-92 biological test, with similar quantity take off alanyl-1, C125S human IL-2 or the C125S human IL-2 is observed compares, aforementioned polypeptides inductive pro-inflammatory cytokine TNF-alpha levels reduces.

In some embodiments, equating under the test conditions, prove with NK3.3 cytotoxicity biological test, with similar quantity take off alanyl-1, C125S human IL-2 or the C125S human IL-2 is observed compares, aforementioned polypeptides has kept or has strengthened the natural killer cell toxicity of NK cells of human beings mediation, the cytotoxicity of killing and wounding (LAK) cytotoxicity or ADCC mediation of lymphokineactivation.

In some embodiments, equating under the test conditions, this mutain inductive NK cell proliferation than similar quantity to take off alanyl-1, C125S human IL-2 or C125S human IL-2 inductive high by 150%.

In some embodiments, to take off alanyl-1, C125S human IL-2 or C125S human IL-2 inductive high by 170% for this mutain inductive NK cell proliferation ratio.

In some embodiments, this mutain inductive NK cell proliferation is for taking off alanyl-1, C125S human IL-2 or the about 200-250% of C125S human IL-2 inductive

In some embodiments, equating under the test conditions that what this mutain inductive NK cell proliferation was higher than similar quantity takes off alanyl-1, C125S human IL-2 or C125S human IL-2 institute inductive at least 10%.

In some embodiments, this mutain inductive NK cell proliferation is higher than and takes off alanyl-1, C125S human IL-2 or C125S human IL-2 institute inductive at least 15%.

In some embodiments, equating under the test conditions that this mutain induces the pro-inflammatory cytokine of generation to be lower than similar quantity to take off alanyl-1, C125S human IL-2 or C125S human IL-2 institute inductive 100%.

In some embodiments, this mutain induces the pro-inflammatory cytokine of generation to be lower than to take off alanyl-1, C125S human IL-2 or C125S human IL-2 institute inductive 70%.

In some embodiments, the isolated polypeptide that the present invention includes contains human IL-2's mutain, wherein this mutain contains aminoacid sequence shown in the SEQ ID NO:4,125 halfcystines of SEQ ID NO:4 have replaced Serine, has other aminoacid replacement of two places among the SEQ ID NO:4 at least, equating under the test conditions, the ratio of this sudden change egg-products IL-2 inductive NK cell and IL-2 inductive TNF-α generation, than similar quantity to take off alanyl-1, C125S human IL-2 or C125S human IL-2 mutain observed high at least 1.5 times.Available NK-92 biological test is measured the NK cell proliferation of 0.1nM mutain and the TNF-α output of 1.0nM mutain.In some embodiments, to take off alanyl-1, C125S human IL-2 or C125S human IL-2 observed high at least 2.5 times for above-mentioned ratio ratio.In some embodiments, to take off alanyl-1, C125S human IL-2 or C125S human IL-2 observed high at least 3.0 times for this ratio ratio.

In some embodiments, the isolated polypeptide that the present invention includes contains the aminoacid sequence of human IL-2's mutant, wherein this mutain contains aminoacid sequence shown in the SEQ ID NO:4,125 Serines of SEQ ID NO:4 have replaced halfcystine, with have other aminoacid replacement of two places at least, the residue position of other replacement is selected among the SEQ ID NO:4: 19,36,40,42,61,65,72,80,81,88,91,95 and 107.Exemplary combination replacement includes but not limited to: 19D40D, 19D81K, 36D42R, 36D61R, 36D65L, 40D36D, 40D61R, 40D65Y, 40D72N, 40D80K, 40G36D, 40G65Y, 80K36D, 80K65Y, 81K36D, 81K42E, 81K61R, 81K65Y, 81K72N, 81K88D, 81K91D, 81K107H, 81L107H, 91N95G, 107H36D, 107H42E, 107H65Y, 107R36D, 107R72N, 40D81K107H, 40G81K107H and 91N94Y95G.In some embodiments, this polypeptide also contains 1 L-Ala disappearance in SEQ ID NO:4.

On the other hand, the invention provides the method that produces human interleukin-2 (IL-2) mutain, this method comprises: with the expression vector transformed host cell that contains above-mentioned nucleic acid molecule; Under expressing the condition that this nucleic acid molecule becomes polypeptide, cultivate described host cell in the cell culture medium; With separate this polypeptide.In some embodiments, under similar test conditions, take off alanyl-1, C125S human IL-2 or C125S human IL-2's reference product IL-2 mutain compares with being selected from of similar quantity, this human interleukin-2 (IL-2) mutain can keep or strengthen the propagation of NK cell, can also induce the NK cell to produce the promotion inflammatory cytokine of lower level, wherein measure the propagation of NK cell and the pro-inflammatory cytokine of generation with the NK-92 biological test.

On the other hand, the invention provides the composition that contains one or more aforementioned polypeptides for the treatment of significant quantity, said composition contains human IL-2's mutain.This composition also can contain pharmaceutically acceptable vehicle.

On the other hand, the invention provides the method that stimulates immune system, this method comprises the human IL-2's mutain that gives Mammals treatment significant quantity, equating under the test conditions, take off alanyl-1, C125S human IL-2 or C125S human IL-2's reference product IL-2 mutain compares with being selected from of similar quantity, described mutain is induced the NK cell to produce low-level pro-inflammatory cytokine and maintenance or has been strengthened the propagation of NK cell, wherein adopts the NK-92 biological test to measure the propagation of described NK cell and the pro-inflammatory cytokine of described generation.In some embodiments, described Mammals is the people.

In some embodiments, the human IL-2's mutain that is used for stimulating immune system contains and is selected from SEQ IDNO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 aminoacid sequence.

In some embodiments, be used for the residue 2-133 that aminoacid sequence that human IL-2's mutain of stimulating immune system contains is selected from SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 aminoacid sequences.

In some embodiments, the human IL-2's mutain that is used for stimulating immune system also contains replacement, has wherein replaced serine residue at SEQ ID NO:10,125 alanine residues of 12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72.

In some embodiments, the human IL-2's mutain that is used for stimulating immune system also contains, and has wherein replaced serine residue at SEQ IDNO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,125 cysteine residues of 70 and 72.

On the other hand, the invention provides the method for treatment mammalian cancer, this method comprises the human IL-2's mutain that gives Mammals treatment significant quantity, equating under the test conditions, take off alanyl-1, C125S human IL-2 or C125S human IL-2's reference product IL-2 mutain compares with being selected from of similar quantity, this mutain is induced the NK cell to produce the pro-inflammatory cytokine and the maintenance of lower level or has been strengthened NK cell proliferation, wherein adopts the NK-92 biological test to measure the propagation of described NK cell and the pro-inflammatory cytokine of described generation.In some embodiments, described Mammals is the people.

In some embodiments, the human IL-2's mutain that is used for the treatment of cancer contains and is selected from SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 aminoacid sequence.

In some embodiments, the human IL-2's mutain that is used for the treatment of cancer contains SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 2-133 residue aminoacid sequence.

In some embodiments, the human IL-2's mutain that is used for the treatment of cancer also contains replacement, has wherein replaced serine residue at SEQ IDNO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,125 alanine residues of 70 and 72.

In some embodiments, the human IL-2's mutain that is used for the treatment of cancer also contains replacement, has wherein replaced serine residue at SEQ IDNO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,125 cysteine residues of 70 and 72.

On the other hand, the invention provides the method that in giving the patient of IL-2, reduces interleukin-2 (IL-2) inductive signs of toxicity as treatment plan.This methods of treatment comprises the IL-2 that gives as the IL-2 mutain.

In some embodiments, the IL-2 mutain that is used for the treatment of contains and is selected from SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 aminoacid sequence.

In some embodiments, the IL-2 mutain that is used for the treatment of contains the 2-133 residue that is selected from SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 aminoacid sequences.

In some embodiments, the IL-2 mutain that is used for the treatment of also contains replacement, has wherein replaced serine residue at SEQ IDNO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,125 alanine residues of 70 and 72.

In some embodiments, the IL-2 mutain that is used for the treatment of also contains replacement, has wherein replaced serine residue at SEQ IDNO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,125 cysteine residues of 70 and 72.

The accompanying drawing summary

Fig. 1 has summarized with the IL-2 mutain stimulates separation to produce test from the human PBMC's of normal people's donor the propagation/pro-inflammatory cytokine of uniting.

Fig. 2 has shown the propagation of 40D72N IL-2 mutain mediation among the human PBMC and the generation of TNF-α.

Fig. 3 has shown the propagation of 40D61R IL-2 mutain mediation among the human PBMC and the generation of TNF-α.

Fig. 4 shows that the IL-2 activated separates LAK and the ADCC activity that has kept people NK-mediation from the human PBMC of normal people's donor.

Detailed Description Of The Invention

The present invention relates to new human interleukin-2 (IL-2) mutain, have improved result of treatment owing to its toxicity reduction and/or NK or T cytological effect device function improve.Above-mentioned human IL-2's mutain and biological activity variant thereof are compared with the reference product IL-2 mutain that takes off alanyl-1, C125S human IL-2 or C125S human IL-2, induce the pro-inflammatory cytokine of generation to reduce the propagation that has kept or strengthened natural killer (NK) cell simultaneously." pro-inflammatory cytokine " refers to the cytokine of energy stimulating immune system.This cytokine includes but not limited to: IFN-α, IFN-γ, TNF-α, TNF-β, IL-β and IL-6.

Term " mutain " refers to contain the protein of the aminoacid sequence of the sudden change different with the protein amino acid sequence of natural generation owing to aminoacid deletion, replacement or the two.Human IL-2's mutain of the present invention is different with sophisticated human IL-2 to be, locate serine residue 125 of sophisticated human IL-2's sequences and replaced cysteine residues (being C125S), also contain at least two place's aminoacid replacement, and also contain a place or many places aminoacid deletion with respect to sophisticated human IL-2's sequence, as L-Ala (Ala) disappearance of 1 N-end of sophisticated human IL-2's albumen.In another embodiment, human IL-2's mutain of the present invention has kept the cysteine residues at 125 places of sophisticated human IL-2's sequence, but has other two places aminoacid replacement at least, and also contain a place or many places aminoacid deletion with respect to sophisticated human IL-2's sequence, lack as 1 N-terminal alanine of sophisticated human IL-2's albumen (Ala).These human IL-2's mutains can be glycosylated or nonglycosylated, depend on the host expression system that produces them.The mutain that above-mentioned concrete replacement causes producing has kept required activity, promptly use above-mentioned NK-92 test cell line, compare with taking off alanyl-1, C125S human IL-2 or C125S human IL-2, it induces the pro-inflammatory cytokine of generation to reduce the propagation that has kept or strengthened the NK cell simultaneously.Cause toxigenicity to reduce and/or the position of NK cell proliferation enhanced IL-2 variant and the relevant replacement of these positions in the identifier IL-2 sequence, within the technology of this area, can obtain these required active human IL-2's mutain variants these human IL-2's mutain variants as herein described that also kept as herein described by other residue in the change human IL-2 sequence and also belong to the present invention, also comprise following.

The human IL-2 translates into the precursor polypeptide shown in the SEQ ID NO:2 earlier, its nucleotide sequence coded by shown in the SEQ ID NO:1.The residue 1-20 that this precursor polypeptide comprises SEQ ID NO:2 is a signal sequence.Term " sophisticated human IL-2 " refers to aminoacid sequence shown in the SEQ ID NO:4, and it is by nucleotide sequence coded shown in the SEQ ID NO:3.Term " C125S human IL-2 mutain " or " C125S human IL-2 " refer to sophisticated human IL-2's mutain, and it has kept 125 of 1 N-terminal alanine residues of sophisticated human IL-2 and sophisticated human IL-2's sequences to replace halfcystine for Serine.C125S human IL-2 mutain has aminoacid sequence shown in the SEQ ID NO:6, and it is by nucleotide sequence coded shown in the SEQ ID NO:5.Term " takes off alanyl-1C125S human IL-2 " and " taking off alanyl-1 Serine-125 human IL-2 " refers to sophisticated human IL-2's mutain, for having replaced halfcystine 125 of sophisticated human IL-2's sequences for Serine and at the terminal Serine of N-of 1 of the sophisticated human IL-2's sequence of disappearance (be SEQ ID NO:4 1).Take off alanyl-1C125S human IL-2 and have the aminoacid sequence shown in the SEQ ID NO:8, it is by nucleotide sequence coded shown in the SEQ ID NO:7.Intestinal bacteria reorganization produce the alanyl-1C125S human IL-2 mutain that takes off be called " rIL-2 ", the preparation commodity that can buy from the market are by name

IL-2 (Chiron Coporation, Emeryville, California).For purpose of the present invention, take off alanyl-1C125S human IL-2 and C125S human IL-2 mutant, be as measuring the active human IL-2's mutant of the shown ideal of human IL-2's mutain of the present invention reference product.Therefore under similar test conditions, mensuration is taken off the pro-inflammatory cytokine amount that propionamido--1, C125S human IL-2 or C125S human IL-2 mutain induce the NK cell to produce with respect to isodose, the required activity of human IL-2's mutain that the present invention is suitable has reduced pro-inflammatory cytokine, particularly the TNF-α of Chan Shenging that IL-2 induces the NK cell to produce.Similarly, under similar test conditions, mensuration is taken off the amount that propionamido--1, C125S human IL-2 or C125S human IL-2 mutain are induced NK cell proliferation with respect to isodose, and human IL-2's mutain that the present invention is suitable required active keeps or improved IL-2 inductive NK cell proliferation.

The isolating nucleic acid of the coding human IL-2 mutain that provides and biological activity variant thereof contain aminoacid sequence and other aminoacid replacement of at least two places that takes off propionamido--1, C125S human IL-2 (SEQ ID NO:8) or C125S human IL-2 (SEQ ID NO:6), when comparing with two kinds of reference IL-2 mutains, the pro-inflammatory cytokine that it can induce the NK cell to produce lower level keeps simultaneously or strengthens NK cell proliferation.The polypeptide of isolating nucleic acid molecule encoding of the present invention also is provided.

Human IL-2's mutain of the present invention comprises the mutain shown in the SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72, is also referred to as " even numbers SEQ ID NO:10-72 sequence ".The present invention also provides the nucleotide sequence of these mutains of coding, the encoding sequence shown in SEQ ID NO:9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69 and 71 difference.The mutain that these encoding sequences this paper is also referred to as " odd number SEQ ID NO:9-71 sequence " these above-mentioned aminoacid sequences comprises C125S human IL-2 aminoacid sequence and other replacement of at least two places, these other be substituted in the following table 1 to replace and represent with combination.One group of two place or many places residue that " combination replace " refers to occur in human IL-2's mutain sequence replace.Therefore for example, the combination that is called " 19D40D " replaces and refers to that human IL-2's mutain of the present invention contains two places and replaces, one 19 asparagicacid residues (being D) that are in corresponding to sophisticated human IL-2 have replaced leucine residue (seeing SEQ ID NO:4), and 40 asparagicacid residues (being D) that are in corresponding to sophisticated human IL-2 have replaced leucine residue (seeing SEQ IDNO:4).Similarly, the combination that is called " 40D81K107H " replaces and refers to that human IL-2's mutain of the present invention contains following three places and replaces: 40 asparagicacid residues (being D) corresponding to sophisticated human IL-2 have replaced leucine residue, replaced arginine residues and replaced tyrosine residues corresponding to 81 lysine residues (being K) of sophisticated human IL-2 corresponding to sophisticated human IL-2's 107 hyte propylhomoserin residues (being H).In other embodiments, human IL-2's mutain of the present invention has 1 original alanine residue of these sequential amino acid deletions, therefore comprise and take off other the replacement of propionamido--1, C125S human IL-2 aminoacid sequence and at least two places, these other be substituted in the following table 1 and represent with the combination replacement.The aminoacid sequence of these mutains comprises the residue 2-133 of sequence shown in the SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 or 72.The present invention also provides the nucleotide sequence of these mutains of coding, the sequence of the Nucleotide 4-399 of sequence shown in the SEQ ID NO:9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69 or 71 that for example encodes.

The biologic activity variant of human IL-2's mutain of the present invention described herein also is provided, comprises that it has the fragment and the clipped form of required human IL-2's mutain function.For example, the available well known in the art and above-mentioned recombinant DNA technology of this paper is by removing fragment or the clipped form that amino-acid residue in the total length human IL-2 mutain aminoacid sequence produces above-mentioned human IL-2's mutain.The suitable variant of inventor IL-2 mutain has and is similar to the shown biologic activity of this Novel Human IL-2 mutain itself, promptly use the IL-2 molecule of biological test described herein and reference, promptly take off propionamido--1, C125S human IL-2 or C125S human IL-2 when comparing, they have the hypotoxicity (pro-inflammatory cytokine promptly low or that reduce produces) of this Novel Human IL-2 mutain and can keep or strengthen NK cell proliferation.Think certain given Novel Human IL-2 mutain and observed the comparing of Novel Human IL-2 mutain of the present invention that this paper identifies, can have same anything but particular biological activity, as long as when comparing with human IL-2's mutain of reference, it has kept required biologic activity such as toxicity attenuating, promptly induce the NK cell to produce low-level pro-inflammatory cytokine, and/or strengthen NK cell proliferation.

Table 1 human IL-2's mutain of the present invention example, it contains C125S human IL-2 (SEQ ID NO:6) or takes off propionamido--1, C125S human IL-2's (SEQ ID NO:8) aminoacid sequence, other replaces at least two places, and the combination that this kind replacement is selected from the following stated replaces.Its residue position is with respect to the position of sophisticated human IL-2's sequence shown in the SEQ ID NO:4.The position of these residues is also corresponding to the position in the C125S human IL-2 sequence shown in the SEQ ID NO:6.Abridge for the amino acid single-letter that has replaced this position natural residue behind each residue position.

19D40D 19D81K 36D42R 36D61R 36D65L 40D36D 40D61R 40D65Y 40D72N 40D80K 40G36D 40G65Y 80K36D 80K65Y 81K36D 81K42E

81K61R 81K65Y 81K72N 81K88D 81K91D 81K107H 81L197H 91N95G 107H36D 107H42E 107H65Y 107R36D 107R72N 40D81K107H 40G81K107H 91N94Y95G

Composition of the present invention also comprises to be used to recombinate and produces the carrier and the host cell of inventor's IL-2 mutain or its biological activity variant.In addition, also provide and contain the human IL-2's mutain described herein for the treatment of significant quantity or the pharmaceutical composition of its biologic activity variant and pharmaceutically acceptable vehicle.

The present invention includes with reference product IL-2 mutain and compare, can induce the NK cell to produce the production method of human IL-2's mutain of the short inflammation product of lower level and maintenance or enhancing NK cell proliferation.These methods comprise the expression vector transformed host cell with the nucleic acid molecule that contains code book invention Novel Human IL-2 mutain or its biologic activity variant of encoding, under the condition that allows coded expression of polypeptides, in substratum, cultivate this host cell and isolated polypeptide product.

The method that stimulates animal immune system or treatment mammalian cancer also is provided, but this method comprises its biologic activity variant of inventor IL-2 mutain that gives the treatment of animals significant quantity, wherein measure with the biological test of this paper the following stated, compare with taking off propionamido--1, C125S human IL-2 or C125S human IL-2, this IL-2 mutain or its variant inductive NK cell produce the lower level pro-inflammatory cytokine and can keep or strengthen NK cell proliferation.

The present invention also provides and reduces the method give the signs of toxicity that IL-2 caused as interleukin-22 (IL-2) among the patient of treatment plan.This method comprises and gives IL-2 mutain of the present invention, promptly measure with the biological test of this paper the following stated, compare with taking off propionamido--1, C125S human IL-2 or C125S human IL-2, induce the NK cell to produce lower level pro-inflammatory cytokine and the IL-2 mutain that can keep or strengthen NK cell proliferation.

Term " nucleic acid molecule " this paper comprises dna molecular (as cDNA or genomic dna) and RNA molecule (as mRNA) and the DNA or the RNA congener that produce with the Nucleotide congener.Nucleic acid molecule can be strand or two strands, but preferred double-stranded DNA.Bright nucleic acid isolating or purifying basically or the protein composition of comprising." isolating " or " purifying " nucleic acid molecule or protein or its biologic activity part should not accompany or interactional component with it in the naturally occurring environment of this nucleic acid molecule or protein basically usually.Therefore, the nucleic acid molecule or the protein of isolating or purifying do not have other cellular material or medium component basically when producing with recombination method, maybe do not have precursor thing or other chemical substance when producing with chemical synthesis basically.Isolating nucleic acid should not contain the sequence (promptly being positioned at this nucleic acid 5 ' and 3 ' terminal sequence) (preferred protein encoding sequence) that produces natural this nucleic acid of side joint in this biological nucleic acid body genomic dna.For example, in various embodiments, what isolated nucleic acid molecule can contain natural this nucleic acid of side joint in the cell genomic dna that produces this nucleic acid is less than about 5kb, 4kb, 3kb, 2kb, 1kb, the nucleotide sequence of 0.5kb or 0.1kb.The protein of essentially no cellular material comprises that contaminating protein is lower than the protein product of about 30%, 20%, 10%, 5% or 1% (dry weight).When protein of the present invention or its biologic activity variant produced by reorganization, wherein the precursor composition of substratum or nonprotein chemical ingredients interested should be lower than about 30%, 20%, 10%, 5% or 1% (dry weight).

The biologic activity of Novel Human IL-2 mutain

With take off propionamido--1, the C125S human IL-2 compares, or compares with the C125S human IL-2, Novel Human IL-2 mutain of the present invention has the therapeutic index of raising.The first two is planted mutain and is referred to herein as " reference IL-2 mutain ", because in come the given comparison that classification is carried out to mutain of the present invention with similar protein concentration and equal test conditions, novel mutation albumen of the present invention is comparable to the biology performance of human IL-2's mutain that these two kinds previous characterized cross on biology performance.When the raising of mutain therapeutic index of the present invention is reflected in and compares with the toxicity of one of these two kinds of reference IL-2 mutains and NK and/or T cytological effect device function, its toxicity improves that (promptly this mutain induces the NK cell to produce the pro-inflammatory cytokine of lower level) and NK and/or T cytological effect device function are improved and toxicity does not improve, or the toxicity improvement of these mutains and NK and/or T cytological effect device function all improve.

The mutain that utilizes following three kinds of function terminal points to select therapeutic index to improve: (1) with take off propionamido--1, C125S human IL-2 or compare, can induce the NK cell to produce the lower level pro-inflammatory cytokine; (2) compare with taking off propionamido--1, C125S human IL-2 or C125S human IL-2, can keep or strengthen IL-2 inductive NK and T cell proliferation but do not improve the pro-inflammatory cytokine that the NK cell produces; (3) compare with taking off propionamido--1, C125S human IL-2 or C125S human IL-2, can keep or improve the molten cell killing of (promptly strengthening) NK-mediation.The molten cell killing of NK-mediation comprises: NK mediates, lymphokineactivation kills and wounds molten cell killing (LAK)-mediation and cytotoxicity (ADCC)-mediation that antibody relies on.

The maximum therapy index that above-mentioned Novel Human IL-2 mutain shows improves and belongs to three kinds of function types estimating that clinical efficacy improves.Should notice that all these mutains demonstrations can both keep or strengthen the propagation of T cell and the dissolved cell activity of NK mediation.The first kind functional character of this mutain is, with reference IL-2 mutain, promptly taking off propionamido--1, C125S human IL-2 or C125S human IL-2 compares, preferred mutain can make IL-2 inductive NK cell reduce the generation of pro-inflammatory cytokine, keeps IL-2 inductive NK cell proliferation simultaneously.The second class function of this mutain is that enhancing IL-2 inductive NK cell proliferation is stronger than two reference IL-2 mutain inductive, but does not have the undesirable action that two reference IL-2 mutain institutes inductive pro-inflammatory cytokines produce.The 3rd class function of this mutain comprises " difunctional " of this mutain, promptly compare with these two kinds of reference IL-2 mutain inductive activity levels, IL-2 inductive NK cell pro-inflammatory cytokine is produced reduce, strengthen IL-2 inductive NK cell proliferation simultaneously.

It is well known in the art measuring the IL-2 inductive NK cell proliferation of fresh separated NK cell and the test of pro-inflammatory cytokine generation.For example see Perussia, Methods 9:370,1996 and Baume etc., Eur.J.Immunol.22:1-6,1996.The phenotype and the functional performance that have the NK cell according to previous report NK-92 clone, comprise the propagation (Gong etc., Leukemia 8:652,1994) when having IL-2 however when having IL-2, seldom or not produce TNF-α (Nagashima etc., Blood 91:3850,1998).The active IL-2 biological test of screening people NK that has developed and T cell function is described in this paper following examples chapters and sections.Though can adopt the test that detects NK cell proliferation and pro-inflammatory cytokine generation, reaches T cytological effect Lu function, preferably screen interested IL-2 mutain to determine whether they have kept the required feature of mutain described herein with IL-2 biological test as herein described.Interested especially is that can they reduce NK cell induction TNF-α generation.Therefore, in one embodiment, NK cell proliferation of IL-2 inductive and TNF-alpha factor produce available this paper people NK-92 described below clone (ATCC CRL-2407, CMCC ID#11925) IL-2 biological test and measure.Gong etc. is seen in description for NK-92 clone, and Leukemia 8 (4): 652-658,1994.For the purposes of the present invention, this test is called " NK-92 biological test ".

" reduction " or " minimizing " pro-inflammatory cytokine produces the IL-2 mutain that refers to reference, promptly taking off propionamido--1, C125S human IL-2 or C125S human IL-2 mutain compares, the level that human IL-2's mutain of the present invention induces the NK cell to produce inflammatory cytokine reduces, and particularly the TNF-α of NK cell generation reduces.Though under comparable test conditions, human IL-2's mutain of the present invention can make TNF-α that the NK cell produces minimum level than similar quantity to take off propionamido--1, C125S human IL-2 or C125S human IL-2 inductive low at least by 20%, but the highest level that mutain of the present invention induces the NK cell to produce TNF-α depends on the function type under this mutain.

Therefore, for example in some embodiments, should select usually and can maximum strengthen inducing of NK cell proliferation and induce the NK cell to produce the mutain that TNF-α does not have undesirable action (being the second class function of mutain) IL-2.In these embodiments, as personnel selection NK-92 clone (ATCC CRL-2407, CMCC ID#11925) (promptly uses above-mentioned NK-92 biological test) and each human IL-2's mutain of 0.1nM or 100pM (being 0.1nM) concentration when measuring the TNF-α that produces, human IL-2's mutain of the present invention lures that the NK cell produces level and the reference IL-2 mutain inductive similar (promptly ± 10%) of TNF-α into, or preferably is lower than reference IL-2 mutain inductive 90%.In other embodiment of the present invention, under comparable test conditions, when each human IL-2's mutain of personnel selection NK-92 clone (promptly using above-mentioned NK-92 biological test) and 0.1nM concentration is measured the TNF-α that produces, human IL-2's mutain of the present invention lure into TNF-alpha levels that the NK cell produces be lower than similar quantity take off propionamido--1, C125S human IL-2 or the inductive TNF-α of C125S human IL-2 institute amount 90%, be preferable over 85%, even more preferably less than 80%.In some embodiments, when each human IL-2's mutain of personnel selection NK-92 clone (promptly using above-mentioned NK-92 biological test) and 0.1nM concentration was measured the TNF-α that produces, human IL-2's mutain of the present invention induced the TNF-α ratio of generation to take off propionamido--1, C125S human IL-2 or C125S human IL-2 inductive to hang down 20% to 60% at least.Compare with reference IL-2 mutain, this mutain that can keep or strengthen IL-2 inductive NK cell proliferation belongs to the IL-2 mutain of first kind function.

" maintenance " refers under comparable test conditions, human IL-2's mutain inductive level of biological activity of the present invention be similar quantity take off at least 70%, preferably at least 75%, more preferably at least 80% of propionamido--1, C125S human IL-2 or C125S human IL-2 finding activity level, most preferably at least 85% and high to 100% (equivalence).Therefore, when required activity is when inducing NK cell proliferation, when under comparable test conditions, measuring NK cell proliferation with same biological test (being these IL-2 mutains of above-mentioned NK-92 biological test and similar quantity), IL-2 mutain inductive NK cell proliferation level of the present invention be similar quantity take off at least 70%, preferably at least 75%, more preferably at least 80% of propionamido--1, C125S human IL-2 or C125S human IL-2 inductive proliferation activity, most preferably at least 85%, 90%, 95% higher 100% (± 5%) that comprises.

" enhancing " or " raising " or " improvement " refer under comparable test conditions, the required level of biological activity of human IL-2's mutain inductive than similar quantity to take off propionamido--1, C125S human IL-2 or C125S human IL-2 viewed higher.Therefore, when required biologic activity is when inducing NK cell proliferation and adopting same NK cell proliferation test (as above-mentioned NK-92 biological test), the IL-2 mutain inductive NK cell proliferation level that the present invention is suitable, take off at least 105%, 110%, 115% of propionamido--1, C125S human IL-2 or the observed NK cell-proliferation activity of C125S human IL-2 for similar quantity, more preferably at least 120%, even more preferably at least 125%, most preferably at least 130%, 140% or 150%.

The test of measuring NK cell proliferation is well known in the artly (for example to see Baume etc., Eur.J.Immuno.22:1-6,1992; Gong etc., Leukemia 8 (4): 652-658,1994 and above-mentioned NK-92 biological test).The preferred NK-92 cell that adopts detects the pro-inflammatory cytokine that IL-2 induces generation, particularly the propagation of the generation of TNF-α and NK cell (being above-mentioned NK-92 biological test).The suitable concn of the used human IL-2's mutain of NK cell proliferation test comprises about 0.005nM (5pM)-1.0nM (1000pM), comprises other numerical value between 0.005nM, 0.02nM, 0.05nM, 0.1nM, 0.5nM, 1.0nM and the 0.005nM-1.0nM.In the following preferred implementation of this paper, adopt human IL-2's mutain of NK-92 cell and about 0.1nM-1.0nM concentration to carry out the NK cell proliferation test.

Because human IL-2's mutain of the present invention can reduce inducing that pro-inflammatory cytokine produces, with maintenance or enhancing IL-2 inductive NK cell proliferation, the ratio that their IL-2 inductive NK cell proliferation: IL-2 induces NK cell pro-inflammatory cytokine to produce, be better than taking off propionamido--1, C125S human IL-2 or C125S human IL-2 inductive, equal protein concentration and biological test condition are adopted in these determinations of activity of every kind of mutain.When the pro-inflammatory cytokine of being measured is TNF-α, under similar biological test condition and protein concentration, suitable inventor IL-2 mutain is IL-2 inductive NK cell proliferation when the 0.1nM mutain: IL-2 induces the NK cell to produce the ratio of TNF-α when the 1.0nM mutain, at least be with taking off 1.5 times that propionamido--1, C125S human IL-2 or C125S human IL-2 are obtained, being more preferably at least 1.75 times, 2.0 times, 2.25 times even more preferably at least 2.75 times, 3.0 times, 3.25 times with reference IL-2 mutain.In some embodiments, under similar biological test condition and protein concentration, human IL-2's mutain of the present invention is IL-2 inductive NK cell proliferation when the 0.1nM mutain: IL-2 induces the NK cell to produce the ratio of TNF-α when the 1.0nM mutain, at least be with taking off 3.5 times that propionamido--1, C125S human IL-2 or C125S human IL-2 are obtained, 3.75 times, 4.0 times, 4.5 times or even 5.0 times.

Under similarly biological test condition and protein concentration, with respect to taking off propionamido--1, C125S human IL-2 or C125S human IL-2 finding, the mutain of invention also can improve the survival of (i.e. increase) NK cell.The available test known in the art of the survival of NK cell comprises that test described herein measures.For example, can the survival of NK cell can be blocked the apoptosis of glucocorticosteroid and induce NK cell expressing BCL-2 to measure and (for example see Armant etc. by detecting the IL-2 mutain when having IL-2 mutain interested, Immunology85:331,1995).

The invention provides the test of monitoring IL-2 to the influence of NK cell survival.In one embodiment, by detecting mutain at NK3.3 cell (CMCC ID#12022 with pAKT ELISA, see Kornbluth, J.Immunol.129 (6): 2831-37,1982) can inducing cell in the reaction of survival signal transduction cascade, the survival of NK cell when measuring interested human IL-2's mutain and existing.In this way, utilize interested human IL-2's mutain to raise the index of the AKT phosphorylation of NK cell as the NK cell survival.

The IL-2 mutain that adopts in the inventive method will activate and/or amplifying natural killer (NK) cell and mediate the cytotoxicity (ADCC) that lymphokineactivation kills and wounds (LAK) active and antibody dependence.Static (non-activated) NK cell can mediate spontaneity or the natural cytotoxicity that some is called the target cell (as human erythroleukemia K562 clone) of " NK-cellular sensitivity target ".Activated back NK cell by IL-2 and obtained the LAK activity.For example, IL-2 activated NK cell can be detected and various tumour cells and other " LAK-sensitivity/NK-is insensitive " target cell can be killed and wounded, as Daudi B-cell lymphoma system (solvency action that it can resist static (promptly unactivated) NK cell usually), measure this LAK activity.Similarly, when having the relevant tumor cell specific antibody of optimal concentration, can can dissolve " the responsive NK-of LAK is insensitive " target cell by detecting the IL-2 active cells, as DAUDI B-cell lymphatic system, or not diffluent other target cell of static (promptly non-activated) NK cell is measured the ADCC activity.The method that produces and measure the cytotoxic activity of NK/LAK cell and ADCC is known in the art.See for example " newly organized immunological experiment method: human immunity research " (Current Protocols in Immunology:Immunologic Studies in Humans), supplementary issue 17,7.7, Unit 7.18 and 7.27 (John Wiley ﹠amp; Son, Inc.1996).In one embodiment, adopting phenotype and functional character is the ADCC activity that the clone of the NK3.3 of peripheral blood NK cell detects IL-2 mutain of the present invention.For purpose of the present invention, this test is called " NK3.3 cytotoxicity biological test ".

Under similar biological test condition and protein concentration, to compare with taking off propionamido--1, C125S human IL-2 or C125S human IL-2 finding, human IL-2's mutain of the present invention also can keep or strengthen IL-2 inductive T cell proliferation.Cell proliferation test is well known in the art.In one embodiment, utilizing the human T-cell is Kit225 (CMCCID#11234; Hori etc., Blood 70 (4): 1069-72,1987) detect the T cell proliferation of the above-mentioned test of this paper.

As mentioned above, the candidate guide human IL-2 mutain (those novel mutation albumen that promptly have the most improved therapeutic index) of this paper evaluation belongs to three kinds of function types.First kind of function type is included in similar biological test condition and protein concentration 1.0nM when measuring all mutains, the TNF-alpha levels of inducing the NK cell to produce is lower, be about and take off propionamido--1, C125S human IL-2 or C125S human IL-2 inductive 60% or lower, and with take off propionamido--1, C125S human IL-2 or C125S human IL-2 and compare those mutains that keep or strengthened NK cell proliferation.These mutains can further be divided into two subclass: (1) is when when similarly biological test condition and about 1.0nM protein concentration detect these mutains, compare with human IL-2's mutain of reference, can strengthen (promptly greater than 100%) IL-2 inductive NK cell proliferation, but when about 0.1nM or lower concentration,, reduce human IL-2's mutain of (promptly being lower than 100%) NK cell-proliferation activity with observed the comparing of human IL-2's mutain of reference; (2) when similarly condition and the about 1.0-0.05 of protein concentration (promptly about 50pM) detect these mutains down, with the human IL-2's mutain or observed the comparing of reference, can improve (promptly greater than 100%) or keep (promptly at least about 70-100%) IL-2 to induce those human IL-2's mutains of NK cell proliferation.In one embodiment, the NK cell proliferation of IL-2 inductive and TNF-α produce with NK-92 cell (promptly with the test of NK-92 cell biological) and detect, wherein the MTT reducing dyes test kit (CellTiter that buys with commercialization of NK cell proliferation

The non-radioactive cell proliferation test kit available from Promega Corp Madison, Wisconsin) detects, and calculates stimulation index according to the colorimetric reader; TNF-α ELISA test kit (the BioSource Cytoscreen that buys with commercialization ^TMHumanTNF-ELISA test kit; Camarillo, California) quantitative TNF-α.The human IL-2's mutain that belongs to the subclass (1) of first kind of function type comprises containing to take off propionamido--1, C125S human IL-2 (SEQ ID NO:8) or C125S human IL-2 (SEQ ID NO:6) aminoacid sequence and contain with respect to sophisticated human IL-2's sequence (promptly with respect to SEQID NO:4) residue position (promptly 19,36,40,61,65,81 or 91) being selected from those mutains that 19D40D, 36D61R, 36D65L, 40D61R, 40D65Y, 40G65Y, at least a combination of 81K91D replace.See following examples 2 and table 3.The human IL-2's mutain that belongs to the subclass (2) of first kind of function type comprises containing to take off propionamido--1, C125S human IL-2 (SEQ ID NO:8) or C125S human IL-2 (SEQ ID NO:6) aminoacid sequence and contain with respect to sophisticated human IL-2's sequence (promptly with respect to SEQ ID NO:4) residue position (promptly 40,42,65,72,80,81,88 or 107) being selected from those mutains that 40D72,80K65Y, 81K88D, 81K42E, 81K72N, 107H65Y, at least a combination of 107R72N replace.See following examples 2 and table 4.

Human IL-2's mutain of second kind of function type comprises and can strengthen NK cell proliferation strongly but induce the NK cell to produce those mutains that TNF-α does not have undesirable action to IL-2.The mutain that belongs to this function type will meet three choice criteria: (1) induces the level of NK cell proliferation greater than taking off propionamido--1, C125S human IL-2 or C125S human IL-2 institute inductive about 200% when one or more concentration of the human IL-2's mutain that is selected from 0.005nM (being 5pM), 0.02nM (being 20pM), 0.05nM (being 50pM), 0.1nM (even 100pM) or 1.0nM (being 1000pM) IL-2; (2) when detection be selected from 0.005nM (being 5pM), 0.02nM (being 20pM), 0.05nM (being 50pM), 0.1nM (even 100pM) or at least two kinds of concentration of 1.0nM (being 1000pM) human IL-2's mutain the time, IL-2 induces the level of NK cell proliferation greater than taking off propionamido--1, C125S human IL-2 or C125S human IL-2 institute inductive about 150%; (3) when detecting the TNF-α of 1.0nM (being 1000pM) or 0.1nM (being 100pM) concentration mutain generation, IL-2 induces the level of NK cell generation TNF-α similar to reference IL-2 mutain inductive, preferably is lower than 90%.In one embodiment, IL-2 induces the NK cell to produce TNF-α and IL-2 induces NK cell proliferation to adopt NK-92 cell (promptly with above-mentioned NK-92 cell biological test) to detect, wherein IL-2 induces the TNF-α of generation to detect with ELISAS, and IL-2 inductive NK cell proliferation detects with above-mentioned MTT test.The human IL-2's mutain that belongs to this second kind of function type comprises containing to take off propionamido--1, C125S human IL-2 (SEQ ID NO:8) or C125S human IL-2 (SEQ ID NO:6) aminoacid sequence and contain with respect to sophisticated human IL-2's sequence (promptly with respect to SEQ ID NO:4) residue position (promptly 19,36,40 or 81) and is selected from those mutains that 19D81K, 40G36D and at least a combination of 81K36D replace.See following examples 3 and table 5.

Human IL-2's mutain of the third function type comprises having bifunctional those mutains, compares with the IL-2 mutain of reference, and they can be induced NK cell proliferation to strengthen and reduce the NK cell and produce TNF-α.The sudden change egg-products that belong to this third function type should meet following standard: (1) when detecting with any mutain that is selected from 0.005nM (being 5pM), 0.02nM (being 20pM), 0.05nM (being 50pM), 0.1nM (even 00pM) or 1.0nM (being 1000pM) concentration, inductive NK cell proliferation level is that to take off propionamido--1, C125S human IL-2 or C125S human IL-2 observed at least about 150%; (2) when detecting with about 1.0nM concentration mutain, the level of inducing the NK cell to produce TNF-α is lower than takes off propionamido--1, C125S human IL-2 or C125S human IL-2 inductive about 75%.In one embodiment, IL-2 induces the NK cell to produce TNF-α and IL-2 induces NK cell proliferation to adopt NK-92 cell (promptly with above-mentioned NK-92 cell biological test) to detect, wherein IL-2 induces the TNF-α of generation to detect with ELISAS, and IL-2 inductive NK cell proliferation detects with above-mentioned MTT test.The human IL-2's mutain that belongs to this third function type comprises containing and takes off propionamido--1, C125S human IL-2 (SEQ IDNO:8) or C125S human IL-2 (SEQ ID NO:6) aminoacid sequence and with respect to sophisticated human IL-2's sequence (promptly with respect to SEQ ID NO:4) residue position (promptly 36,40,42,61,80,81,91,94,95 or 107) contain and be selected from 36D42R, 36D80K, 40D80K, 81K61R, 91N95G, 107H36D, those mutains that at least a combination of 107R36D and 91N94Y95G replaces.See following examples 4 and table 6.

The present invention also provides and meets other choice criteria, has human IL-2's mutain of improved therapeutic index for taking off propionamido--1, C125S human IL-2 or C125S human IL-2.Therefore for example, in another embodiment, human IL-2's mutain provided by the invention the NK cell proliferation of IL-2 inductive and 0.1nM mutain IL-2 when the 0.1nM mutain induce the NK cell to produce the ratio of TNF-α, be higher than and take off observed at least 1.25 times, 1.5 times, 1.75 times of propionamido--1, C125S human IL-2 or C125S human IL-2s, preferably high at least 2.0 times, 2.5 times 3.0 times, more preferably high 3.5 times, 3.75 times, 4.0 times 4.5 times and up to 5.0 times.The mutain that meets these standards comprises all mutains shown in the table 1, exception be to contain human IL-2's mutain that 19D40D combination replaces.This index improves indication enhancing NK cytological effect device function and the toxic useful clinical effect of reduction improves to some extent.

The biologic activity variant of Novel Human IL-2 mutain

The variant that the present invention also provides the biology performance of above-mentioned Novel Human IL-2 mutain to improve than reference IL-2 molecule performance, promptly use biological test described herein, when with the IL-2 molecule of reference, promptly taking off propionamido--1, C125S human IL-2 or C125S human IL-2 compares, it can be induced the NK cell to reduce or reduce the biologic activity variant that pro-inflammatory cytokine produced and can keep strengthening NK cell proliferation.As previously mentioned, think and observed the comparing of Novel Human IL-2 mutain of the present invention, certain given Novel Human IL-2 mutain that this paper identifies can have same anything but particular organisms activity, as long as when IL-2 molecule with above-mentioned biological test and reference, promptly taking off propionamido--1, C125S human IL-2 or C125S human IL-2 compares, it has desired characteristic with respect to reference IL-2 molecule, promptly induces the toxicity of pro-inflammatory cytokine to reduce, and/or can strengthen the propagation of NK cell.

" variant " refers to similar basically sequence.Above-mentioned Novel Human IL-2 mutain can be (as mutain) nucleic acid or the aminoacid sequence that natural generation (as the allele variant of IL-2 locus) or reorganization produce.Polypeptide variants can be the fragment of above-mentioned Novel Human IL-2 mutain, or their are in have a place or many places other aminoacid replacement or disappearance or aminoacid insertion different with this Novel Human IL-2 mutain, as long as having kept the specific amino acids interested that exists in the above-mentioned Novel Human IL-2 mutain, variant polypeptide replaces.Therefore, suitable polypeptide variants comprises the those polypeptides that contains the C125S replacement corresponding to 125 of sophisticated human IL-2's sequences (being SEQ ID NO:4), they are that a kind of combination that this paper identifies replaces, and have other aminoacid replacement or disappearance or aminoacid insertion of the improvement therapeutic index (being that combination replaces shown in the above table 1) of Novel Human IL-2 mutain of the present invention and a place or many places.Therefore for example, as shown in table 1, when containing, Novel Human IL-2 mutain takes off propionamido--1, a kind of combination shown in C125S human IL-2 (SEQID NO:8) or C125S human IL-2's (SEQ ID NO:6) aminoacid sequence and the table 1 replaces, the suitable biologic activity variant of these Novel Human IL-2 mutain also contains the combination shown in C125S replacement and the table 1 and replaces, but be other the replacement of a place or many places with various Novel Human IL-2 mutain differences, insert or disappearance, as long as when (being the C125S human IL-2 or taking off propionamido--1 with the IL-2 molecule of reference, the C125S human IL-2) compare, this variant polypeptide (is the IL-2 mutain C125S human IL-2 of reference or takes off propionamido--1 with respect to reference IL-2 molecule, the C125S human IL-2) having toxicity reduces, pro-inflammatory cytokine produces and reduces, and/or NK cell proliferation enhanced desired characteristic.When mensuration sequence homogeny per-cent as mentioned below, the aminoacid sequence of this variant and each Novel Human IL-2 mutain, SEQ ID NO:10 for example, 12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68, it is 70% identical that the aminoacid sequence of the Novel Human IL-2 mutain shown in 70 or 72 has at least, usually at least 75%, 80%, 85%, 90% is identical, and preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% is identical.

In other embodiments, when mensuration sequence homogeny per-cent as mentioned below, the aminoacid sequence of this biologic activity variant and SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68, it is 70% identical that aminoacid sequence shown in 70 or 72 the residue 2-133 has at least, usually at least 75%, 80%, 85%, 90% is identical, and preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% is identical.

In some embodiments of the present invention, the C125S that human IL-2's mutain biological activity variant of the present invention contains with other natural amino acid such as L-Ala replaces, and this does not influence the functional performance of required human IL-2's mutain.Therefore for example, this variant has replaced serine residue at SEQ ID NO:10,125 alanine residues of 12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 or 72.In another embodiment, human IL-2's mutain biological activity variant of the present invention contains SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 or 72 residue 2-133 and 125 alanine residues of these sequences have replaced serine residue.

In other embodiment of invention, human IL-2's mutain biological activity variant of the present invention contains SEQ IDNO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 or 72 aminoacid sequence and 125 cysteine residues of these sequences and has replaced serine residue.In other embodiments, human IL-2's mutain biological activity variant of the present invention contains SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 or 72 residue 2-133 and 125 cysteine residues of these sequences have replaced serine residue.

Nucleic acid " variant " refer to the to encode polynucleotide of Novel Human IL-2 mutain of the present invention, but because its nucleotide sequence of degeneracy of gene codon is different from the proteic sequence of above-mentioned novel mutation.The codon of natural amino acid is well known in the art, comprises that the specific host biology is used for those the most frequently used codons of express recombinant protein.Those sequences shown in the attached sequence table of nucleotide sequence this paper of above-mentioned IL-2 mutain of encoding, and be different from the nucleotide sequence of above-mentioned sequence owing to the gene codon degeneracy.

Therefore for example, when IL-2 mutain of the present invention contains aspartic acid (being D) replacement, for example at C125S or take off and contain 19D40D, 19D81K, 36D42R, 36D61R, 36D65L, 40D36D, 40D61R, 40D65Y, 40D72N, 40D80K, 49D36D, 80K36D, 81K36D, 81K88D, 81K91D, 107H35D, 107R36D or 40D81K107H combination in propionamido--1, the C125S mutain and replace, the nucleotide sequence of the asparagicacid residue that coding replaces can be selected from two kinds of general codeword triplets of aspartic acid, i.e. GAC and GAT.Replace the replacement that comprises two kinds of similar residues when this combination, when containing 19D40D or 40D36D combination replacement as this mutain, the residue of this replacement can be by identical universal code coding (promptly being replaced by GAC or GAT coding two), or can be by another universal code coding (promptly a place replaces the coding by GAC, and another place replaces the coding by GAT).Similarly, when containing glycine (being G), IL-2 mutain of the present invention replaces, as at C125S or take off when containing 40G36D, 40G65Y, 91N95G, 40G81K107H or 91N94Y95G combination in propionamido--1, the C125S mutain and replacing, the nucleotide sequence of the glycine residue that coding replaces can be selected from 4 general codeword triplets of glycine, i.e. GGT, GGC, GGA and GGG.

When IL-2 mutain of the present invention contains Methionin (being K) replacement, for example at C125S or take off and contain 19D81K, 40D80,8036D, 8065,81K36D, 81K42E, 81K61R, 81K65Y, 81K72N, 81K88D, 81K9ID, 81K107H, 40D81K107H or 40D81K107H combination in propionamido--1, the C125S mutain and replace, the nucleotide sequence of the lysine residue that coding replaces can be selected from two kinds of general codeword triplets of Methionin, i.e. AAA and AAG.Similarly, when IL-2 mutain of the present invention contains leucine (being L) replacement, for example at C125S or take off and contain 36D65L or 81K107H combination in propionamido--1, the C125S mutain and replace, the nucleotide sequence of the leucine residue that coding replaces can be selected from leucic six kinds of general codeword triplets, i.e. TTA, TTG, CTT, CTC, CTA and CTG.

When IL-2 mutain of the present invention contains l-asparagine (being N) replacement, for example at C125S or take off and contain 40D72N, 81K72N, 91N95G, 107R72N in propionamido--1, the C125S mutain or the 91N94Y95G combination replaces, the nucleotide sequence of the asparagine residue that coding replaces can be selected from two kinds of general codeword triplets of l-asparagine, i.e. GAT and GAG.Similarly, when IL-2 mutain of the present invention contains Histidine (being H) replacement, for example at C125S or take off and contain 81K107H, 81L107H, 107H36D, 107H42E, 107H65Y, 40D81K107H or 40G81K107H combination in propionamido--1, the C125S mutain and replace, the nucleotide sequence of the histidine residues that coding replaces can be selected from two kinds of general codeword triplets of Histidine, i.e. CTA and CAC.

When IL-2 mutain of the present invention contains arginine (being R) replacement, for example at C125S or take off and contain 36D42R, 36D61R, 40D61R, 81K61R, 107R36D or 107R72N combination in propionamido--1, the C125S mutain and replace, the nucleotide sequence of the arginine residues that coding replaces can be selected from arginic six kinds of general codeword triplets, i.e. CGT, CGC, CGA, CGG, AGA and AGG.Similarly, when IL-2 mutain of the present invention contains tyrosine (being Y) replacement, for example at C125S or take off and contain 40D65Y, 40G65Y, 80K65Y, 81K65R, 107H65Y or 91N94Y95G combination in propionamido--1, the C125S mutain and replace, the nucleotide sequence of the tyrosine residues that coding replaces can be selected from two kinds of general codeword triplets of tyrosine, i.e. TAT and TAC.When IL-2 mutain of the present invention contains L-glutamic acid (being E) replacement, for example at C125S or take off contain in propionamido--1, the C125S mutain 81 cheat 2 or 107H42 combination replace, the nucleotide sequence of the glutaminic acid residue that coding replaces can be selected from two kinds of general codeword triplets of L-glutamic acid, i.e. GAA and GAG.

Though above-mentioned nucleic acid variant list has been described universal code that the specific residue that this paper identified of can be used to encode replaces, should think all the nucleic acid variants that cause owing to the gene codon degeneracy that the present invention includes coding human IL-2's mutain described herein.

Natural human IL-2's natural allele variant can be identified with the Protocols in Molecular Biology of knowing, as polymerase chain reaction (PCR) and hybridization technique, can be used as the guide that adds sudden change it is joined this paper human IL-2 of institute mutain and does not influence the required therapeutic index of these Novel Human IL-2 mutain.The variant nucleotide sequence also comprises the mutain derived from synthetic Nucleotide, and is for example as described below, the mutain of the above-mentioned Novel Human IL-2 of coding that produces by site-directed mutagenesis.Usually when mensuration sequence homogeny per-cent as mentioned below, the nucleotide sequence of nucleotide sequence variant of the present invention and they Novel Human IL-2 mutain separately, SEQ ID NO:9 for example, 11,13,15,17,19,21,23,25,27,29,31,33,35,57,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67, the Nucleotide 4-399 of encoding sequence shown in 79 or 71 has at least 70%, usually have at least 75%, 80%, 85%, 90% sequence is identical, and preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence is identical.

Term " gene " and " recombination " this paper are used in reference to the nucleic acid molecule that contains code book invention IL-2 mutain open reading frame.Phrase " allele variant " refers to be positioned at the nucleotide sequence of IL-2 locus or refers to the polypeptide that this is nucleotide sequence coded.This natural allele variant can cause the nucleotide sequence of IL-2 gene that the 1-5% variation takes place usually.Any He all nucleotide variants in the IL-2 sequence and the amino acid polymorphism that is produced be natural allelic variation institute extremely, can not change the functionally active of Novel Human IL-2 mutain of the present invention, comprise the sequence that produces by the prominent Beijing Opera of the present invention, the sequence of all these generations all belongs to scope of the present invention.

For example, can in the cloned dna sequence of encoding novel IL-2 mutain, make the aminoacid sequence variant that sudden change comes above-mentioned Novel Human IL-2 mutain, not replace as long as these sudden changes do not change the combination of table 1 evaluation.The method of mutagenesis and change nucleotide sequence is well known in the art, for example see, Walker and Gaasta compile, and (1983) " Protocols in Molecular Biology " Techniques in Molecular Biology (MacMillan Publishing Company, NewYork); Kunkel (1985) Proc.Natl.Acad.Sci.USA 82:488-92; Kunkel etc., (1987) MethodsEnzymol.154:267-82; Sambrook etc. (1989) " molecular cloning: laboratory manual " (Molecular Cloning:ALaboratory Manual) (the 2nd edition, Cold Spring Harbor Laboratory Press, Plainview, NewYork), United States Patent (USP) 4,873,192 and the reference quoted of this paper.Can be at Dayhoff about the suitable aminoacid replacement guide of the required biologic activity that do not influence the IL-2 mutain (promptly reducing the NK cell produces pro-inflammatory cytokine, estimates to reduce toxicity and maintenance or strengthen NK cell proliferation) etc., " peptide sequence and structure atlas " Atlas ofPolypeptide Sequence and Structure (Natl.Biomed.Res.Found., Washington finds in model DC).

When designing the biologic activity variant of above-mentioned human IL-2's mutain, preferential conservative property replaces, and has the aminoacid replacement of similar performance as an amino acid with another." conservative amino acid replacement " is that wherein amino-acid residue replaces with the amino-acid residue with similar side chain.This field has defined the amino-acid residue family with similar side chain.These families comprise and contain basic side chain (as Methionin, arginine, Histidine), acid side-chain is (as aspartic acid, L-glutamic acid), no charge polarity side chain is (as glycine, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), non-polar sidechain is (as L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), β-branched building block is (as tyrosine, phenylalanine, tryptophane, Histidine) amino acid.For example see Bowie etc., Science 247:1306,1990.The example that conservative property replaces includes but not limited to:

With

This replacement should not contain conserved cysteine residue, the cysteine residues that adjoins as N-terminal.

About in human IL-2's albumen can by replace, disappearance or to insert the zone that required replacement that this paper identifies change be that this area is known.For example see Bazan, Science 257:410-12,1992; McKay, Science 257:412; Theze etc., Immunol.Today 17:481-86,1996; Buchli and Ciardelli, Biochem.Biophys307:411-15,1993; Collins etc., Proc.Natl.Acad.Sci.USA.85:7709-13,1988; Kunkel etc., J.Immunol.150:5731,1993; Eckenberg etc., Cytokine 9:488-98,1997.

When making up Novel Human IL-2 mutain variant of the present invention, make variant stop the sustainable required activity that has the nucleotide sequence of modifying this variant of coding more.Obviously, any sudden change of in coding variant polypeptide DNA, carrying out must this sequence not placed frame outside, the preferred complementation district that may cause secondary mRNA structure that do not produce.Polypeptide variants can have few to 1-15 amino acid whose difference, and is individual few to 5 as 6-10, few to 4,3,2, even 1 amino-acid residue difference.The nucleotide sequence variant can have few to 1-30 Nucleotide difference, and is few to 5 as 6-25, few to 4,3,2, even 1 Nucleotide difference.

The biologic activity variant of human IL-2's mutain of the present invention comprises the fragment of these mutains." fragment " refers to the part of coding nucleotide sequence or the part of aminoacid sequence.About encoding sequence, the fragment codified of human IL-2's mutain nucleotide sequence has kept the mutain fragment of the required biologic activity of Novel Human IL-2 mutain.The fragment of above-mentioned Novel Human IL-2 mutain can be grown the total length that 15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130 amino acid merits reach this Novel Human IL-2 polypeptide.The fragment of coding nucleotide sequence can be the whole nucleotide sequence of 45,60,75,90,105,120,135,150,165,180,195,210,225,240,255,270,285,300,315,330,345,360,375,390 Nucleotide and this Novel Human IL-2 mutain of nearly encoding at least.

Can further modify above-mentioned human IL-2's mutain and biologic activity variant thereof, as long as they are with respect to C125S human IL-2 mutain or take off propionamido--1C125S human IL-2 mutain and have desired characteristic, promptly toxicity reduces and/or strengthens NK cell proliferation.Other modification includes but not limited to: the replacement of phosphorylation, alpha-non-natural amino acid congener etc.Can cause its expression in vivo to prolong, therefore improve the IL-2 mutain of the effectiveness of IL-2 mutain pharmaceutical preparation and modified glycosylation or the PEGization that comprises protein molecular.Natural is that nonglycosylated proteinic glycosylation is often undertaken by the glycosyl site of inserting the N-connection in molecule.Can come the half life of protein such as IL-2 mutain in this way.In addition, available this method is not covered the immunogenicity epi-position, improves proteinic stability, reduces cohesion and improves expression and purity output.

In case obtain the variant of above-mentioned human IL-2's mutain, estimate that disappearance, insertion and the replacement in this human IL-2's mutain sequence can not produce radical change to the characteristic of concrete human IL-2's mutain.Yet when doing definite influence of replacement difficult to calculate, disappearance or insertion so in advance, those skilled in the art will know that available conventional shaker test estimates this effect.Promptly use standard cell lines proliferation test well known by persons skilled in the art, comprise that above-mentioned test estimates IL-2 inductive NK cell or T cell-proliferation activity.Available cytokine specific ELISA, for example above-mentioned TNF-alpha specific ELISA detects IL-2 inductive pro-inflammatory cytokine and produces.NK cell survival signal can detect (for example seeing test described below) with pAKT ELISA.The cell-mediated available test known in the art of dissolved cell activity (being cytotoxicity) of NK detects (for example, NK-as herein described is that mediate, the LAK mediation or the dissolved cell activity of ADCC mediation).

Above-mentioned human IL-2's mutain and biologic activity variant thereof can be built into and contain IL-2 and merge or coupling protein, during it contains contained IL-2 mutain (or above-mentioned biologic activity variant) be blended in second albumen or covalent coupling in polyproline or water-soluble polymers to reduce administration number of times or further to improve tolerance to IL-2.For example, available means known in the art are blended in human albumin or albumin fragment (seeing for example WO 01/79258) with human IL-2's mutain (or its above-mentioned biologic activity variant).Perhaps, can adopt means known in the art, in polyproline or polyoxyethylene glycol homopolymer and polyoxyethylene poly alcohol, wherein said available alkyl of homopolymer one end and polyalcohols do not replace or replace with human IL-2's mutain (or its above-mentioned and biologic activity variant) covalent coupling.(see for example United States Patent (USP) 4,766,106; 5,206,344 and 4,894,226).

" sequence homogeny " refers to when the specific continuous section of arranging nucleotide sequence or aminoacid sequence, when the nucleotide sequence of itself and reference sequence or aminoacid sequence are made comparisons, the identical Nucleotide or the amino-acid residue that in variant sequence and reference sequence, find.The method of homogeny is that this is well known in the art between series arrangement contrast and mensuration sequence.See that for example, Ausubel etc. write, (1995) " newly organized molecular biology experiment guide " (Current Protocol inMolecular Biology), the 9th chapter (Greene Publishing and Wiley-Interscience, New York); With the ALIGN program (Dayhoff, 1978, see " peptide sequence and structure atlas 5 ": supplementary issue 3 (National BiomedicalResearch Foundation, Washington, D.C).For the optimal arrangement contrast of dinucleotides sequence, the continuous section of this variant nucleotide sequence is compared with the reference nucleotide sequence, can add Nucleotide or disappearance Nucleotide.Equally, for optimal arrangement contrast diamino acid sequence, the section that adjoins of this variant aminoacid sequence is compared with the reference aminoacid sequence, can add amino-acid residue or disappearance amino-acid residue.Being used for adjoining section and should containing at least 20 contiguous nucleotides or amino-acid residue with reference nucleotide sequence or reference aminoacid sequence are made comparisons, can be 30,40,50,100 or more Nucleotide or amino-acid residue.Can comprise the space in this variant nucleotide sequence or aminoacid sequence for improving the sequence homogeny, this can correct by the space point penalty is set.The correlated method of series arrangement is well known in the art.

Available mathematical algorithm carries out the mensuration of homogeny per-cent between two sequences.For purpose of the present invention, adopt the sequence homogeny per-cent of the homology searching algorithm mensuration aminoacid sequence of Smith-Waterman, adopt the retrieval of affine 6 spaces, space point penalty 12, point penalty 2, BLOSUM matrix 62 are extended in the space.The homology searching algorithm of Smith-Waterman is explained in Smith-Waterman (1981) Adv.Appl.Math 2:482-89.Perhaps, with the homology searching algorithm of Smith-Waterman, adopt space point penalty 25, point penalty 5 is extended in the space, measures homology of nucleotide sequence per-cent.Therefore, the DeCyoher Hardware Accelerator of available for example Time Logic carries out the mensuration of sequence homogeny.

Recognize further that when considered amino acid homogeny per-cent it is different that some amino acid may replace (can not influence the polynucleotide function characteristic) because of conservative amino acid.At this moment, the amino acid similarity that can consider the conservative property replacement is come correction sequence homogeny per-cent.This bearing calibration is well known in the art.For example see Meyers etc., ComputerApplic.Biol. Sci.4:11-17,1988.

Recombinant expression vector and host cell

Usually, human IL-2's mutain carrier of the present invention, preferred expression vector expression.Carrier can be used for self-replication in host cell maybe can be integrated into host cell gene group (as non-episome Mammals carrier) by importing host cell.Expression vector can instruct the encoding sequence that links to each other with its operability to express.Usually the expression vector of using in the recombinant DNA technology is plasmid (carrier) form.Yet, the present invention includes the expression vector of other type, as virus vector (as replication defect type retrovirus, adenovirus and adeno associated virus).

Expression constructs of the present invention or carrier contain the nucleic acid molecule of inventor IL-2 mutain, and its form is suitable for expressing this nucleic acid molecule in host cell.Interested encoding sequence can be by following recombinant DNA technology, as Taniguchi etc., Nature 302:305-310,1983 and Devos, Nucleic Acids Research 11:4307-4323 or employing Wang etc., Science 224:1431-33, the IL-2 that 1984 described sudden changes change prepares.Recognize that encoding sequence shown in the odd number SEQ ID NOS:9-71 starts from the codon (i.e. the codon of 1 L-Ala) of sophisticated first residue of human IL-2's sequence SEQ ID NO:4, but not the methionine(Met) codon, it is the translation initiation codon ATG in the messenger RNA(mRNA) normally.These disclosed nucleotide sequences also lack 399 Nucleotide translation stop codon afterwards of odd number SEQ ID NOS:9-71.When the 4-399 position nucleotide sequence that adopts these sequences or contain odd number SEQ ID NOS:9-71 is expressed inventor IL-2 mutain, should know that the expression constructs that contains these human IL-2's mutain encoding sequences also should and contain in the correct frame of human IL-2's mutain encoding sequence in the upstream comprises translation initiation codon, as the ATG codon.Translation initiation codon can utilize translation initiation codon, as ATG, upstream position in human IL-2's mutain encoding sequence initiator codon provides, ATG may be present in the sequence that comprises human IL-2's mutain encoding sequence, or can be by external source, as the plasmid that is used to express provides, as long as translation initiation codon is arranged in human IL-2's mutain encoding sequence initiator codon before first in the correct frame that contains human IL-2's mutain encoding sequence initiator codon.Similarly, going up this human IL-2's mutain encoding sequence is one or more translation stop codon afterwards, as TGA, makes terminal last amino acid for sequence shown in the SEQ ID NOS:10-72 of human IL-2's mutain product.

The contained one or more adjusting series of operations selected according to host cell of recombinant expression vector are connected in the nucleotide sequence of being expressed." operability link to each other " refers to that interested nucleotide sequence (being the sequence of code book contriver IL-2 mutain) is connected in described adjusting sequence, its mode can make this nucleotide sequence express (as in in-vitro transcription/translation system maybe when this carrier is imported in the host cell in host cell)." regulating and controlling sequence " comprises promotor, enhanser and other expression controlling elements (as the polyadenylic acid signal).For example see, Goeddel (1990), " gene expression technique: Enzymology method " (Gene Expression Technology:Methods in Enzymology) 185 (AcademicPress, San Diego, California).Regulating and controlling sequence comprises those sequences (as the tissue specificity regulating and controlling sequence) that can instruct the nucleotide sequence constructive expression and only instruct nucleotide sequence to express in many type host cells in certain host cell.Many factors are depended in the design that those skilled in the art will know that expression vector, as desired level of selected host cell to be transformed, protein expression etc.Expression constructs of the present invention can be imported in the host cell and produce above-mentioned human IL-2's mutain, or produce its biologic activity variant.

Expression constructs of the present invention or carrier design are used at protokaryon or eukaryotic host cell expressing human IL-2 mutain or its variant.The most frequently used carrier marking protein in the protokaryon Bacillus coli cells that contains composing type or inducibility promotor.The method of express recombinant protein matter or referring to for example at utmost in intestinal bacteria, Gottesman (1990), " gene expression technique: Enzymology method " 185 (Academic Press, San Diego, CA) 119-128 page or leaf and Wada etc., Nucleic Acid Res 20:2111-18,1992.Cultivate, collect, break or describe substantially as seen from the method for exquisiteness extraction human IL-2's mutain or its variant, for example United States Patent (USP) 4,604, and 377; 4,738,927; 4,656,132; 4,569,790; 4,748,234; 4,530,7874,572,798:4,748,234 and 4,931,543.

Recombinant human il-2's mutain or its biologic activity variant also can be at eukaryotic cells, as preparing in yeast or the people's cell.Suitable eukaryotic host cell comprises the insect cell (insect cell of example for utilizing baculovirus vector cultivating, as the sf9 cell, comprise pAc series (Smith etc., Mol.Cell.Biol.3:2156-65,1983) and pVL series (Lucklow and Summers, Virology 170:31-39,1989) middle marking protein); (example of expression vector comprises pYepSecl (Baldari etc., EMBO J.6:229-234,1987) to yeast cell in the S. cervisiae, pMFa (Kurjan and Herskowitz, Cell 30:933-43,1982), pJRY88 (Schultz, Gene 54:113-123,1987), pYES2 (Invitrogen Corporation, San Diego, CA), and pPicZ (Invitrogen Corporation, San Diego, CA)); Or mammalian cell (mammalian expression vector comprises pCDM8 (Seed, Nature 329:840,1987) and pMT2PC (Kaufman etc., ENBO J.6:187-195,1987)).Suitable mammalian cell comprises Chinese logical sequence mouse gonad cell (CHO) or COS cell.In the mammalian cell, often provide the controlled function of expression vector by viral controlling element.For example, Chang Yong promotor derives from polyomavirus, adenovirus 2, cytomegalovirus and Sendai virus 40.The suitable expression system of other of eucaryon and prokaryotic cell prokaryocyte can be referring to (1989) such as Sambrook, " molecular cloning: laboratory manual " (the 2nd edition, Cold Spring Harbor Laboratory Press, Plainview, New York) 16 and 17 chapters are seen Goeddel (1990), " gene expression technique: zymetology works " 185 (AcademicPress, San Diego, California).

Can optimize and in host cell interested, express human IL-2's mutain encoding sequence of the present invention.To certain given cell host, can regulate G-C content in this sequence as calculated to mean level (ML) with reference to the known of in this host cell, expressing.Can optimize indivedual codons, for example carry out residue and replaced, the codon that other combination replaces shown in C125S replacement, C125A replacement and/or table 1.Perhaps, can optimize other codon in human IL-2's mutain encoding sequence improving host cell expression, as having optimized 1%, 5%, 10%, 25%, 50%, 75% in this codon sequence or up to 100% codon in particular host cell, expressing.For example see SEQ IDNO:73 and 74 disclosed human IL-2's mutain sequences.Wherein be at expression in escherichia coli, optimized the codon that 36D61R and 107R36D combination replaces respectively.

Term " host cell " or " recombinant host cell " are used interchangeably in this article.Should understand this term not only refers to concrete object cell but also refers to the filial generation of these cells or possible daughter cell.Because some variation may take place in sudden change or its offspring of environmental influence.These filial generations are in fact inequality with parental cell, but this still belongs to the category of this term.

Available conventional conversion or rotaring dyeing technology import carrier DNA in protokaryon or the eukaryotic cell.Term " conversion " and " transfection " this paper are used in reference to and various exogenous nucleic acid (as DNA) are imported technology known in the art in the host cell, comprise calcium phosphate-calcium chloride co-precipitation, the DEAE-dextran-transfection of mediation, lipofection, particle is robbed or electroporation.(1989) such as the visible Sambrook of appropriate method of conversion or transfectional cell, " molecular cloning: laboratory manual " (the 2nd edition, Cold Spring Harbor Laboratory Press, Plainview, New York) and the molecular biology experiment handbook of other standard.

In suitable medium, cultivate (1989) such as the protokaryon be used to produce IL-2 mutain of the present invention and its biologic activity variant and the general visible Sambrook of eukaryotic cell, " molecular cloning: laboratory manual " (the 2nd edition, ColdSpring Harbor Laboratory Press, Plainview, New York) description in.

Pharmaceutical composition

Produce and purifying human IL-2 mutain or its variant after, they can be incorporated into and be used for people and veterinary treatment, in the pharmaceutical composition for the treatment of as cancer therapy, immunotherapy and transmissible disease.Therefore, human IL-2's mutain or its biologic activity variant can be mixed with pharmaceutical composition and be used for various treatments.As composition, human IL-2's mutain or its biologic activity variant with this field currently known methods through the parenteral administration object.Described object comprise Mammals as; Primate, people, dog, ox, horse etc.These pharmaceutical compositions can contain other compound that can improve inventor IL-2 mutain drug effect or improve required quality.This pharmaceutical composition must safety by selected administration, and they must sterilization, retains biological activity, they must stably dissolve human IL-2's mutain or its biologic activity variant.Depend on the processing of preparation, IL-2 mutain pharmaceutical composition of the present invention can liquid form, in atmospheric environment, refrigeration or freezing or drying agent form, preserve as lyophilized powder, can be redeveloped into liquor, suspension or emulsion, again with one of the whole bag of tricks, comprise oral or gi tract external administration approach gives.

This pharmaceutical composition contains at least a human IL-2's mutain, its biologic activity variant or its combination and pharmaceutically acceptable vehicle usually.The method that preparation is used for the inventor IL-2 mutain of administration is that these those skilled in the art know.For example see, and Gennaro (volume) (1995) " Lei Mingdun pharmaceutical science with put into practice " (Remington:The Science and Practice of Pharmacy) (the 19th edition, Mack PublishingCompany, Easton, PA).

Statement " pharmaceutically acceptable vehicle " this paper be used in reference to comprise any He all solvent compatible, dispersion agent, Drug coating, antibiotic and anti-mycotic agent with administration, etc. blend absorption delay agent etc.This media and the preparation that can be used for pharmaceutically active substances are that this field is known.Except known and the inconsistent any conventional media of this active compound and preparation so far, this class medium all can be used in human IL-2's mutain pharmaceutical preparation of the present invention.Also can in said composition, add the active compound that adds.

Can be mixed with the IL-2 mutain pharmaceutical composition that contains human IL-2's mutain of the present invention compatible with its route of administration.Can be different according to required route of administration as a result.But medication group's formulation, continuous infusion or CI (in short-term, as 1-6 hour infusion) administration of human IL-2 mutain pharmaceutical composition.But in the oral administration, nose, gi tract comprise intravenously, subcutaneous, intraperitoneal, intramuscular etc. outward, intracutaneous, transdermal (part), saturating mucous membrane and rectal administration, or lung sucks administration of human IL-2 mutain pharmaceutical composition.

Gi tract are outer, the solution or the suspension of intracutaneous or subcutaneous application can comprise following composition: sterilization diluent such as water for injection, salt solution, nonvolatile oil, polyoxyethylene glycol, glycerine, propylene glycol or other synthetic; Antiseptic-germicide such as benzylalcohol or methyl p-Hydroxybenzoate; Antioxidant such as xitix or S-WAT; Sequestrant such as EDTA; Tensio-active agent such as Polysorbate 80; SDS; The preparation of buffer reagent such as acetate, Citrate trianion or phosphoric acid salt and adjustment of tonicity such as sodium-chlor or dextran.Usable acid or alkali, example hydrochloric acid or sodium hydroxide are regulated pH.The outer goods of gi tract can be encapsulated in glass or plastic ampoule, one-shot injector or the multi-agent bottle.

The pharmaceutical composition of suitable injection comprises sterile water solution (water-soluble) or dispersion liquid and prepares the sterile powder of sterilization Injectable solution or suspension at once.Should reduce the formation of protein condenses thing in the preparation process, the suitable vehicle of intravenous administration comprises physiological saline, bacteriostatic water, Cemophor EL as far as possible ^TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS).In all cases, said composition must be sterilized, and should be the liquid that is easy to be contained in the syringe.Should be stable under operation and storage requirement, must anticorrosionly prevent microorganism, as the contamination of bacterium and mould.Vehicle can be solvent or dispersion agent, for example comprises water, ethanol, polyvalent alcohol (as glycerine, propylene glycol and liquid macrogol etc.) and suitable mixture thereof.For example, can adopt Drug coating such as lecithin, keep suitable flowability by keeping required granular size (adopting tensio-active agent during dispersion liquid).Available various antiseptic-germicide and anti-mycotic agent wait the effect of prophylaxis of microbial as p-Hydroxybenzoate, butylene-chlorohydrin, phenol, xitix, Thiomersalate.Should comprise isotonic agent in this composition under many situations, as sugar, polyvalent alcohol such as N.F,USP MANNITOL, sorbyl alcohol, sodium-chlor.Can in this composition, comprise the preparation that can postpone absorption, realize that as aluminum monostearate and gelatin the prolongation of Injectable composition absorbs.

As needs, can in suitable solvent, mix aequum a kind of active compound or with the combination of mentioned component, filtration sterilization then prepares aseptic injectable solution.Usually in containing the aseptic vehicle of basic dispersion medium and required above-mentioned other composition, add active compound and prepare dispersion liquid.With regard to the sterilized powder of preparation sterile injectable solution, vacuum-drying and freeze-drying in the preferred manufacturing procedure, the powder that produces activeconstituents adds other required composition of previous sterile filtration solution.

Oral compositions comprises inert diluent or edible vehicle usually.They can be encapsulated in the gelatine capsule or be pressed into tablet.Be oral administration, can pass through currently known methods, medicine is made the enteric form avoid by gastric digestion or further dressing or mix to discharge at the digestive tube privileged site.For oral therapeutic administration, can in active compound, add vehicle and adopt tablet, spindle agent or capsule.Also available liquid vehicle prepares oral compositions, as collutory.Wherein compound is with the oral suction of liquid vehicle, expectoration or swallow.Can comprise the tackiness agent of pharmaceutically compatible, and/or assist a ruler in governing a country the part of material as composition.Tablet, pill, capsule, lozenge etc. can contain the compound of following composition or similar performance: tackiness agent such as Microcrystalline Cellulose, xanthan gum or gelatin; Vehicle such as starch or lactose; Disintegrating agent such as Lalgine, Primogel or W-Gum; Lubricant such as magnesium stearate or hydrogenated vegetable oil (Sterote); Lubricant such as colloidal silicon dioxide; Sweetener such as sucrose or asccharin; Or seasonings such as peppermint, wintergreen oil or orange flavor.

Also can be by saturating mucous membrane or the administration of transdermal means whole body.For saturating mucous membrane or transdermal administration, can in preparation, adopt the permeate agent that is fit to through barrier.This permeate agent for example comprises by being that this area is known, the washing composition of transmucosal administration, cholate and fudidic acid derivative.

In one embodiment, prepare this active compound,, comprise the system that transports of implantation and microcapsule parcel as controlled release preparation with the vehicle that can protect this compound opposing to be eliminated fast by body.Can adopt biodegradable, biocompatible polymer, as ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen, poe and poly(lactic acid).The method for preparing these preparations is that these those skilled in the art know.Also can be from Alza Corporation and novaPharmaceutical, Inc. buys these materials.Also can adopt liposome suspension (comprising the liposome that falls the antibody target cells infected with antiviral antigenic Dan Ke) as pharmaceutically acceptable vehicle.These can as United States Patent (USP) 4,522, prepare described in 811 by the known method of these those skilled in the art.

Preferred especially preparation is easy to the composition of the oral of administration and dosage homogeneous or gi tract other unit formulation.Unit dosage this paper is used in reference to the unitary dose of the suitable treatment target that physically separates; Constituent parts contains the active compound that can produce required result of treatment with required medicine athletic meeting blended predetermined amount as calculated.Specification sheets has illustrated that unit dosage of the present invention depends on that directly the special performance of this active compound and accessible particular treatment effect and this specialty are to the individual inherent limitations of this active compound treatment.

Human IL-2's mutain of the present invention or its biologic activity variant, available human IL-2's compound method preparation known in the art.Visible various patents of the appropriate formulation that adopts in the current method and publication, for example United States Patent (USP) 4,604, described in 377, they have shown the IL-2 that contains therapeutic dose, but do not have non-IL-2 albumen and intracellular toxin basically, can accept water-soluble vehicle and dissolving IL-2R q.s tensio-active agent on the physiology, as the preferred IL-2 preparation of sodium laurylsulfonate.Can include other composition has, for example armpit.United States Patent (USP) 4,766,106 have shown the IL-2 preparation that contains polyoxyethylene glycol (PEG) modification.European patent application publication No.268,110 demonstrations contain the IL-2 preparation that is selected from polyethylene sorbitan fatty acid ester (Tween-80), polyoxyethylene glycol single-hard ester acid ester and octylphenoxy polyethoxy ethanol compound various nonionogenic tensides such as (Triton X405).United States Patent (USP) 4,992,271 disclose the IL-2 preparation that contains human serum albumin.United States Patent (USP) 5,078,997 disclose and have contained human serum albumin and amino acid whose IL-2 preparation.United States Patent (USP) 6,525,102 disclose and contain amino acid matrix and stablize this polypeptide as the IL-2 preparation of the main stablizer of this polypeptide and acid and/or its salt form solution is buffered in acceptable pH scope.Pending U.S. Patent Application No.10/408,648 disclose the IL-2 preparation that suitable lung is carried.

Therepic use

Contain available from the inventor IL-2 mutain of these human IL-2's mutains or the pharmaceutical preparation of its biologic activity variant and can be used for stimulating immune system, the treatment cancer, as present IL-2 or The cancer of IL-2 treatment.Human IL-2's mutain of the present invention and its suitable biologic activity variant advantage are to reduce the generation of pro-inflammatory cytokine, estimate that toxicity is lower, simultaneously can keep or strengthen required functionally active, as NK cell proliferation, survival, cell-mediated cytotoxicity (NK, LAK and ADCC) and the T cell proliferation of NK-.

Owing to estimate that toxicity is lower, need in the clinical disease of high dosage IL-2 at those, can give with natural IL-2 or IL-2 inventor IL-2 similar or more high dosage mutain and its biologic activity variant still have minimum toxic action.Therefore, the invention provides and reduce the method for experience IL-2 administration as treatment plan object interleukin-2 (IL-2) inductive signs of toxicity, this method comprises and gives above-mentioned IL-2 mutain as IL-2.And human IL-2's mutain of the present invention and its suitable biologic activity variant have better other advantage of result of treatment, therefore than these human IL-2's mutains of low dosage can provide than natural IL-2 or

The better result of treatment of IL-2.

Give the pharmaceutically IL-2 mutain pharmaceutical composition of the present invention of significant quantity of object." pharmaceutically significant quantity " refers to can be used for treating, preventing or diagnose the amount of certain disease or illness." object " refers to Mammals, as primate, people, dog, cat, ox, horse, pig, sheep etc.The preferred people of object with pharmaceutical preparation treatment of the present invention.

During for the therapeutic purpose administration, administration can be preventative or therapeutic.When preventative providing, the symptom prerequisite is appearring for medicine.The effect of preventive administration is prevention or alleviates follow-up symptom.When therapeutic provides, when paresthesia epilepsy, provide medicine.It is to alleviate existing symptom that therapeutic gives drug effect.

Therefore for example, the preparation that contains the significant quantity pharmaceutical composition of inventor IL-2 mutain or its biologic activity variant can be used for treating, preventing and diagnoses many to the disease of clinical response symptom is arranged with the IL-2 treatment.Can prepare inventor IL-2 mutain and its biologic activity variant be used for the treatment of with the IL-2 of native sequences or

The same disease that IL-2 treated.Therefore, the preparation that contains inventor IL-2 mutain or its biologic activity variant can be used for diagnosis, prevention and treatment (part or whole body) bacterium, virus, parasite, protozoon and fungal infection; Strengthen cell-mediated cytotoxicity; What stimulate lymphokineactivation kills and wounds (LAK) cytoactive; The recovery of mediation lymphocyte immunologic function; Promote the allogenic antigen reaction; Promote after the radiotherapy or the immunologic reconstitution of (merit combines) cancer patient after marrow or self stem cell transplantation; Promote the immunologic function of acquired immunodeficiency state to recover; Rebuild the normal immunologic function of old man and aged animal; Other method of exploitation diagnostic test IL-2 level when utilizing enzymatic amplification, radio-labeled, radiation developing and monitor disease states known in the art; Promotion is used for the treatment of the T cell in-vitro growth with diagnostic purpose; The acceptor site of blocking-up lymphokine; Use with various other therapeutic, diagnostic and research.Investigated the various treatments and the diagnostic use of human IL-2 or its variant such as human IL-2's mutain, seen Rosenberg etc., N.Engl.J.Med.316:889-97,1987; Rosemberg, Ann.Surg.208:121-135,1988; Topalian, J.Clin.Oncol.6:839-53,1988; Rosenberg etc., N.Engl.J.Med.319:1676-80,1988; W eber etc., J.Clin.Oncol.10:33-40,1992; Grimm etc., Cell Immunol.70 (2): 248-59,1982; Mazumder, Cancer J.Sci.Am.3 (supplementary issue 1): S37-42; Mazumder and Rosenberg, J.Exp.Med.159 (2): 495-507,1984; With Mazumder etc., CancerImmunol.Immuther.15 (1): 1-10,1983.The preparation that contains inventor IL-2 mutain or its biologic activity variant can be used as single therapeutic activity medicine, or can with other immunology relevant cell or other medicine coupling.The example of relevant cell has B or T cell, NK cell, LAK cell etc., and the exemplary medicine that can be used for IL-2 or its variant coupling is various Interferon, rabbit, particularly gamma-interferon, B-cell growth factor, IL-1 and antibody, and Anti-HER 2 for example, as

(Trastuzumab; Genentech, Inc., South SanFrancisco, California) or anti-CD 20 antibodies, as

(Rituximab; IDEC-C2B8; BiogenIDEC Pharmaceutical Corp., San Diego California).

The dosage range of administration of human IL-2 mutain or its biologic activity variant can be between 0.1-15mIU/m2.Treatment significant quantity and concrete treatment plan that the antibody coupling falls in IL-2 immunotherapy and anticancer disease Dan Ke are that this area is known.For example see, exercise question is the United States Patent (USP) 20030235556 that awaits the reply of " overexpression HER2 receptor protein is that the cancer IL-2/ of feature resists-the antibody combined treatment of HER2 (CombinationIL-2/Anti-HER2 Antibody Therapy for Cancers Characterized by Overexpression of theHER2Receptor Protein) " for pending U.S. Patent Application 2003-0185796 number and the exercise question of " methods of treatment of non_hodgkin lymphoma (Methods of Therapy forNon-Hodgkin ' s Lymphoma) ", with exercise question is the pending U.S. Patent Application No.60/491 of " methods of treatment of chronic lymphocytic leukemia ", proxy's p.m.entry No.59516-278 that on July 31st, 371,2003 submitted to.For disease such as renal cell carcinoma and metastasis melanin tumor, but administration of human IL-2 mutain or its biologic activity variant 300, and 000-800 did high dosage vein medicine group and injects in 000IU/kg/8 hour.See the IL-2 immunotherapy recommended dose of B-cell lymphoma in the above-mentioned U.S. Patent application, HER2+ cancer, mammary cancer and CLL.

Adopting the IL-2 immunotherapy to treat the HIV infection also is that this area is known.For example see U.S. Patent No. 6,579, this clinical disease is recommended in 521 dosage and treatment plan.

Therefore, the invention provides the method for treatment target cancer or controlled plant immunne response, this method comprises and gives IL-2 mutain or its biologic activity variant that the present invention treats significant quantity." treatment significant quantity " refers to be enough to induce required biological results but the dosage level of not inducing unacceptable toxic action.The amount that gives according to the activity of the concentration of human IL-2's mutain in this pharmaceutical composition or its biologic activity variant, needs, treated mammiferous disease condition, formulation, medication and patient's factor such as age, sex and disease seriousness and different.Thinking to provide the treatment of broad range concentration significant quantity, as needs can give that object treats repeatedly that significant quantity alleviates and/or relief of symptoms, Signs or as described in the cause of disease of disease, or carry out other required change of the system of biosystem.Usually, the IL-2 mutain pharmaceutical composition of the present invention concentration range that contains human IL-2's mutain or its variant is higher than

The scope that IL-2 is used.Because dosage ratio The IL-2 height should be paid close attention to object and occur to define the nontoxicity side effect.It is well known to those skilled in the art that this class clinical experiment is analyzed, and for example has been used to determine

The dosage that IL-2 immunomodulatory and cancer therapy are currently used.

The biological test of monitoring human IL-2 mutain functionally active

The present invention also provides monitoring IL-2 inductive NK cell proliferation and produces the biological test method of cell-mediated cytotoxicity, the T cell proliferation of IL-2 inductive and IL-2 inductive NK cell survival of TNF-α, IL-2 inductive NK.Developed the required function characteristic that these test methods are screened candidate IL-2 mutain, as the generation (particularly TNF-α) that reduces pro-inflammatory cytokine improves tolerance and improves the cell-mediated function of NK, as is reflected in this mutain and keeps or strengthen NK cell and/or T cell proliferation, maintenance or strengthen the cell-mediated cytotoxicity (NK, LAK and ADCC) of NK and keep or strengthen the survival of NK cell.

These tests before were also referred to as " NK-92 biological test " in this article, were used to monitor TNF-α and the IL-2 inductive NK cell proliferation that IL-2 induces generation.This biological test adopts people's NK-92 clone (ATCCCRL-2407, CMCC ID#11925).NK-92 clone is at first by Gong etc., and leukemia 8 (4): 652-58,1994 reports show phenotype and functional performance with activation NK cell.The propagation of NK-92 is that IL-2 relies on, if cultivate 72 hour cells with death in the substratum that lacks IL-2.But this clone also produced the TNF-α of detection level within behind the contact IL-2 48-72 hour.

According to method of the present invention, can screen with this NK-92 cell biological experiment sieving IL-2 mutain and induce the relative capacity that produces TNF-α and inductive NK cell proliferation.In this method, with the NK-92 cell cultures in the perfect medium of forming by α-MEM, 12% heat-inactivated fetal bovine serum (FBS), 8% hot deactivation horse serum, 0.02mM folic acid, 0.2mM inositol, 2mM 1-glutamine and 0.1mM beta-mercaptoethanol (NK-92 substratum).With least density 1-3 * 10 ⁵Cells/ml inoculation culture thing, the reference recombinant human il-2 mutain (for example being called the C125S human IL-2 mutain that takes off propionamido--1, C125S human IL-2 or reference) of interpolation 1000IU/ml.Prepare this test, place fresh NK-92 substratum to be used for test at least in 48 hours then in cell.Test the day before yesterday, washing NK-92 cell three times places the NK-92 substratum not add IL-2 in 24 hours.Eccentric cell is suspended in (no IL-2) in the NK-92 substratum, with 4 * 10 ⁴Cells/well density, 200 microlitres that dilute with the NK-92 substratum contain different concns reference IL-2 mutain, as take off propionamido--1, C125S human IL-2 or C125S human IL-2; Or the IL-2 mutain of waiting to screen functional performance interested of different concns, be seeded in the 96 hole flat undersides.37 ℃, 5%CO ₂Cultivate after 72 hours, it is frozen to get 100 microlitre equivalent culture supernatant, subsequently the TNF-α ELISA test kit of buying with commercialization (BioSource Cytoscreen for example ^TMHumanTNF-ELISA test kit; Camarillo, california) detection by quantitative TNF-α.For the cell of survival in cultivating, the MTT dye reagent box of buying with commercialization (

Non-radioactive cell proliferation test test kit (Wisconsin) breed and calculate stimulation index according to the colorimetric reading for Promega Corp., madison by mensuration.

Above-mentioned second kind of IL-2 biological test provides screening IL-2 candidate mutain to induce the cell-mediated cytotoxicity method of NK.This biological test is called " NK-92 cytotoxicity biological test ", has adopted people NK3.3 clone.Phenotype and functional performance (Kornbluth, J Immunol129 (6): 2831-37,1982) that NK3.3 clone has shown peripheral blood NK cell can pass through Fc acceptor (CD16Fc γ RIIIA) mediate antibody dependent cellular cytotoxicity (ADCC).Below table 2 brief summary of experiment chapters and sections detect the biologic activity of NK3.3 cell with this kind IL-2 biological test.

According to method of the present invention, available NK3.3 cytotoxicity biological test screening has the candidate IL-2 mutain of cytotoxic activity.In this method, amplification NK3.3 cell and maintaining be added with 15% heat-inactivated fetal bovine serum, 25mM hepes 2mM L-glutaminate and 20% people T-Stim ^TMIn the RPMI-1640 substratum of w/PHA as the IL-2 source.Prepare this test, when no IL-2 (" hunger "), cultivated the NK3.3 cell 24 hours.This test is included at the bottom of the U-shaped and inoculates 5 * 10 in the 96-orifice plate ⁴Individual " hungry " the NK3.3 cell, make the reference IL-2 mutain of its contact different concns, as take off propionamido--1, C125S or C125S human IL-2 mutain, or the candidate IL-2 mutain interested of different concns, cumulative volume 100 microlitres.Cultivate after 18 hours IL-2 activated NK3.3 cytological effect cell and 5 * 10 ³The target cell of the target cell (K562 or Daudi) of individual fluorexon (calcein) AM-mark or the fluorexon AM-mark of antibody sandwich (being coated with the Daudi cell of the rituximab monoclonal antibody of ultimate density 2 μ g/ml) is cultivated altogether, obtain final effect-target than 10: 1, final volume is 200 microlitres.After effect-target cell is cultivated 4 hours altogether, simple centrifugal 96 orifice plates; Get 100 microlitre culture supernatant and place of the release of flat 96 orifice plates of black transparent with photofluorometer quantitative assay fluorexon AM.The scale of measuring is shown SL per-cent, calculates with following formula: % SL==100 * [(experimental port mean value-spontaneous release mean value)/(the maximum mean value-spontaneous release mean value that discharges)]; Mensuration contains underlined target cell but does not have the spontaneous release of effect cell hole and measure the maximum release that contains underlined sour cell and 1%Triton X-100 hole.

Above-mentioned the third IL-2 biological test provides the method for screening IL-2 candidate mutain inducing T cell multiplication capacity.In this method, T-cell proliferation IL-2 biological test adopts people T-clone Kit225 (CMCC ID#11234), and (Hori etc., Blood 70 (4): 1069-72) derived from a chronic T-Lymphocytic leukemia patient for they.α, β, the γ subunit of Kit225 cell energy constructive expression IL-2 receptor complex.Kit225 propagation is that IL-2 relies on, if cultivate the medium-term and long-term IL-2 of shortage cell with death.

According to the present invention, when being included in no IL-2, this test cultivated the Kit225 cell 24 hours, the cell of given number and the reference IL-2 mutain of different concns are gone in inoculation then, as take off propionamido--1, C125S or C125S human IL-2 mutain, or the candidate IL-2 mutain interested of different concns, cultivate after 48 hours, the MTT reducing dyes test kit of buying with the commercialization of standard detects propagation, calculates stimulation index according to the colorimetric reading.

The 4th kind of biological test of the present invention provides screening IL-2 candidate mutain to promote the method for NK cell survival ability.In this method, the screening mutain is induced the ability of NK cell survival signal. IL-2 (promptly containing the preparation that takes off propionamido--1, C125S human IL-2 mutain) can induce the AKT phosphorylation in the NK3.3 cell of prior IL-2 hunger, think that this is " a survival signal ".According to this biological test, amplification NK3.3 cell also maintains and is added with 15% heat-inactivated fetal bovine serum, 25mM HEPES, 2mM L-glutaminate and 20% people T-Stim ^TMIn the RPMI-1640 substratum of w/PHA as the IL-2 source.Prepare this test, when no IL-2 (" hunger "), cultivated the NK3.3 cell 24 hours.As the cell survival signal, by adding 2nM reference IL-2 mutain, as take off propionamido--1, C125S or C125S human IL-2 mutain, or 2nM candidate IL-2 interested mutain, stimulate " hungry " NK3.3 cell (2 * 10 ⁶) 30 minutes.With phosphate-buffered saline (PBS) washed cell secondary.Contain the cell extraction buffer dissolved cell group of proteinase inhibitor with 50 microlitres, carry out taking turns freeze molten.4 ℃, the centrifugal extract of 13000rpm 10 minutes.The clarification lysate equal portions of dilution in 1: 10 are added AKT[pS473] ^*In the hole of immunity test test kit (BioSource Internatioal).In operation sequence, detect the level of phosphorylation AKT with quantitative ELISA.

The present invention also provides the biological test with human peripheral blood single nucleus cell (PBMC) screening IL-2 mutain functional performance.One of these tests are that the propagation/pro-inflammatory cytokine of associating produces biological test.When contact IL-2, the human PBMC breeds and relies on the mode secrete cytokines with dosage.Design this kind Combined Trials and assess, or interested candidate IL-2 mutain stimulates after 72 hours the level that propagation and cytokine produce with reference IL-2 mutain (as taking off propionamido--1, C125S mutain or C125S mutain).PBMC with one of separation of density gradient centrifugation (as using ACDA Vacutainer CPT test tube) or several normal donors.Complete RPMI substratum (RPMI in 96 hole tissue culture treated plates, 10% hot deactivation people AB serum, 25mM HEPES, 2mM glutamine, penicillin/streptomycin/amphotericin) in, IL-2 with different concns (0.039nM-10nM), or there is not IL-2 as negative control, or 37 ℃ of the candidate IL-2 mutains interested of different concns, 7%CO ₂, cultivate 200000 cells in every hole.Cultivate that to get 100 microlitre equivalent cell culture supernatants after 66 hours frozen, detect cytokine subsequently.With 1 μ Ci ³H-thymidine pulse cell 6 hours, collecting cell is measured Nucleotide and is mixed level (as using Wallac Trilux microbeta plate reader) then, as the measurement of cell proliferation.The ELISA test kit that useful commercial is buied (as available from BioSource International) is pressed the TNF-alpha levels in the business directory detection cell culture supernatant.To different donors, carry out this test repeatedly as the complete group of 6,8 or 10 donors, the feature that can provide " normal population " that representativeness propagation and the cytokine response of IL-2 are reacted.Then can analytical data as shown in table 1, this will further describe in following examples 10.

Can utilize second kind of PBMC biological test to screen the Cytotoxic ability of IL-2 candidate mutain mediation effector cell.In this test, with the human PBMC of density gradient centrifugation separating whole blood.Stimulation PBMC produced the LAK activity in 3 days under 10nM IL-2 contrast or the IL-2 mutain interested existing, as present common enforcement the in this area, (for example see, (the active generation of the separation of NK cells of human beings and LAK " (Isolation of Human K Cellsand Generation of LAK activity), be selected from " compiling the immunological experiment guide " (Current protocol inImmunology); 1996John Wiley﹠amp; Son, Inc.).The gained cell mass contains " effect " cell, can be categorized as NK or LAK, and it can kill and wound K562 and Daudi tumour target cell respectively.These effector cells also can mediate ADCC, can discern the Fc part of the specific antibody that is incorporated into the Daudi target cell because of the effector cell.In one embodiment, can be in conjunction with the antibody of Daudi target cell

(rituximab).

According to method of the present invention, will be with the target cell of interested candidate IL-2 mutain activated human PBMC (effector cell) and fluorexon AM-mark with different effects: target is than (E: the T ratio) cultivate 4 hours altogether.Detect in the culture supernatant cytotoxicity amount with respect to fluorexon AM.Measured according to spontaneous release and the maximum contrast amount that discharges, be expressed as each E: the SL per-cent during the T ratio.This biological test can detect following biologic activity: nature/spontaneous cell toxicant (NK), and wherein target cell is the K562 cell; The the killing and wounding of lymphokine mediation (LAK), wherein target cell is the Daudi cell; The cell toxicant (ADCC) that relies on antibody, wherein target cell be antibody sandwich the Daudi cell (as

The Daudi cell of bag quilt).

Obtain the data that luminoscope records, be expressed as relative fluorescence (intensity) unit (rfu).The contrast of this biological test comprises only markd target cell (minimum) and dissolves the labels targets cell of measurement and the Triton X-100 (maximum) of ultimate density 1% as 100%.Calculate the minimum of this test validity of measurement (as＞30% invalidate the test) and the percentage of maximum ratio with following formula:

In case should test confirm effectively to calculate the mean value and the standard deviation of triplicate sample, go out SL per-cent with following formula from the mean value calculation of triplicate sample then:

Data report is a SL per-cent; In addition, available candidate IL-2 mutain determines with respect to the ratio of IL-2 reference contrast (as taking off propionamido--1, C125S mutain or C125S mutain) whether the cytotoxicity horizontal plane remains in the level that the IL-2 reference contrasts in the mixing human PBMC group of people's donor.

Can utilize above-mentioned test to screen in the IL-2 candidate mutain storehouse and have required function characteristic person, wherein interested functionally active comprise following one or more: the generation of IL-2 inductive pro-inflammatory cytokine (particularly TNF-α and/or INF-γ), IL-2 inductive NK cell and/or T cell proliferation, cell-mediated cell toxicant (NK, LAK and ADCC) and the IL-2 inductive NK cell survival of IL-2 inductive NK.

It is unrestricted for elaboration giving following examples.

Embodiment

The treatment of IL-2 is used and is subjected to the obstruction xicity related with its administration, comprises heating, shiver with cold, ypotension and vascular leak syndrome.Have improved tolerance and allow to give similar therapeutic dose that can better tolerate or higher dosage, therefore improved the potentiality of the better curative effect of this albumen with the NK of IL-2 mediation and the IL-2 mutain of T cytological effect function.The whole strategy of this research is to select Novel Human IL-2 mutain, the NK-cell that utilizes one group of comprehensive specialized output appropriateness shows that for the immunity test screening system on basis it has following functional performance: can reduce the generation of pro-inflammatory cytokine (particularly TNF-α), thereby improved tolerance, with improved the cell-mediated function of NK, be reflected in this albumen and can keep or strengthen the propagation, maintenance of NK cell and/or T cell or strengthen the cell-mediated cell toxicant (NK, LAK and ADCC) of NK and keep or the survival of enhancing NK cell.

In order to identify suitable IL-2 mutain, the biologic activity of recombinant human il-2's mutain of candidate is compared with these biologic activity that propionamido--1, C125S human IL-2 (in following examples with its abbreviation " Pro ") and C125S human IL-2 (in following examples with its abbreviation " Ala-Pro ") show of taking off that are called reference IL-2 mutain with required treatment characteristic.RIL-2 is that propionamido--1, C125S human IL-2 mutain are taken off in the reorganization that intestinal bacteria produce, and the goods trade mark of putting on market is by name

IL-2 (ChironCorporation, Emeryville, California).

IL-2 is a kind of special freeze-dried preparation, is the non-glycosylated form of this mutain, produces with intestinal bacteria, rebuilds with distilled water when using in following biological test.In the C125S human IL-2 mutain preliminary screening experiment of adopting reorganization to produce, adopted AME mammalian expression system DirectAME ^TMAnd ExpressAME ^TM(Applied Molecular Evolution, Inc., San Diego, California).

Following human IL-2's mutain is expressed in host mammal 293T cell.Wherein reference IL-2 mutain is the C125S human IL-2, and host cell transforms with the expression constructs that the operability that contains the C125S sudden change is connected with the natural human IL-2 encoding sequence of Pro-1 promotor.The real IL-2 signal sequence that contains the human IL-2 and the encoding sequence of N-terminal alanine codon (being the Nucleotide 1-63 of SEQ ID NO:1) on the encoding sequence that takes off propionamido--1, C125S human IL-2 (being SEQ IDNO:7), have been merged.This protein is expressed as GSHis-tailing albumen in 293T cell mammalian expression system, with NI-NTA pearl purifying.

Embodiment 1 preliminary screening human IL-2 mutain

Utilize codon induced-mutation technique platform (Applied Molecular Evolution, Inc., San Diego California) has made up and has contained 2508 all possible single amino acids mutation variants of C125 human IL-2 molecule (present embodiment is called " Ala-Pro ").Commodity are buied

Usedly in the IL-2 product take off propionamido--1, C125S human IL-2 mutain is different with Ala-Pro is: the terminal Ala residue of ripe human IL-2 N-that has kept natural generation in the C125S human IL-2 mutain.When recombinant production Ala-Pro mutain, adopted AME mammalian expression system DirectAME ^TMAnd ExpressAME ^TM(Applied Molecular Evolution, Inc., San Diego, California).

The test of preliminary screening personnel selection NK-92 clone functional immunity is carried out, and personnel selection NK3.3 clone has been tested generation, NK cell proliferation and the molten cell killing of NK (NK, LAK and ADCC) and the cell survival (pAKT) of pro-inflammatory cytokine (TNF-α).The preliminary function terminal point of selecting comprises: (1) and Ala-Pro IL-2 (being C125S human IL-2 mutain) or Observed the comparing of IL-2 (promptly taking off propionamido--1, C125S human IL-2 mutain), the short inflammation TNF-α that has reduced NK-92 clone produces; (2), keep or strengthened the propagation of people NK-92 clone with these two kinds of observed comparing of reference IL-2 mutain; (3) with these two kinds of observed comparing of reference IL-2 mutain, keep or strengthened NK, the LAK of people NK3.3 clone mediation and the molten cell killing of ADCC-mediation.Second kind of function terminal point be, in NK3.3 clone, with Ala-Pro IL-2 (being C125S human IL-2 mutain) or

Observed the comparing of IL-2 (promptly taking off propionamido--1, C125S human IL-2 mutain) keeps or strengthened the T cell proliferation of people Kit225T clone.

This preliminary screening method identifies 168 monamino acids replacements and (sees that exercise question is the pending U.S. Patent Application No.60/550 of " improved interleukin-2 muteins (Improved Interleukin-2 Muteins) ", 868, attorney No.PP20354.001 (035784/261164, on March 5th, 2004 submitted to), wherein three of the required function characteristic combination extortions being incorporated in these 168 monamino acid variants that are designed for associating C125S human IL-2 mutain of C125S human IL-2 mutain are reined in the storehouse, to seek other tolerance with raising (promptly reducing IL-2 induces the NK cell to produce TNF-α) and to keep or the IL-2 mutain of enhancing NK cytological effect function.Combinatorial library is made up of 753 IL-2 mutains, and they contain a plurality of aminoacid replacement, and scope is (promptly except that the C125S of ripe human IL-2's mutain of natural generation replaces) from 1 replacement to 6 possible aminoacid replacement nearly.

Except that these three combinatorial library, (developing the pro-inflammatory cytokine (TNF-α) that this system is used for people NK that quantitative IL-2 relies on and T cell line proliferation, NK cell produces for the immunity test screening system on basis to utilize the NK of one group of comprehensive specialized output appropriateness and T cell, the dissolved cell activity cell-mediated (NK/LAK/ADCC)), identify and have the required function characteristic 32 associativity mutains (associativity that sees Table 1 these mutains replaces) of (with taking off propionamido--1, C125S human IL-2 mutain or C125S human IL-2 and comparing) with NK.The screening data of all 32 mutains is seen shown in the following table 7.

The dose response that enlarges is adopted in the analysis that screens behind these mutains, causes identifying specific mutant albumen, and it contains can predict whether improved three kinds of difference in functionalitys of clinical effectiveness, sees described in following examples 2-4.When taking off propionamido--1, C125S or C125S human IL-2 mutain and compare, all IL-2 mutains of selecting have kept the molten cell function (NK/LAK/ADCC) of NK cell.Adopted following scheme in the screening process.

NK cell proliferation/TNF-α produces

The IL-2 biological test that natural killer (NK) cell proliferation and TNF-α produce adopts people's NK-92 clone (ATCC CRL-2407, CMCC ID#11925).By Gong etc., Leukemia 8 (4): 652-658, the NK-92 clones of 1994 reports have shown the phenotype and the functional performance of activation NK cell at first.The propagation of NK-92 is that IL-2 relies on; If culturing cell can be not dead when having IL-2.Behind the contact IL-2 in 48-72 hour, but this clone also produces the TNF-α of detection level.

With the NK-92 cell cultures in the perfect medium of forming by α-MEM, 12% heat-inactivated fetal bovine serum (FBS), 8% hot deactivation horse serum, 0.02mM folic acid, 0.2mM inositol, 2mM 1-glutamine and 0.1mM beta-mercaptoethanol (NK-92 substratum).With least density 1-3 * 10 ⁵Cells/ml inoculation culture thing, add 1000IU/ml reference recombinant human il-2 mutain (take off propionamido--1, C125S human IL-2 (be rIL-2 or

IL-2, Chiron Corporation Emeryville, california) or C125S human IL-2 mutain (in above-mentioned AME mammalian expression system reorganization produce).Prepare this test, place fresh NK-92 substratum to be used for test at least in 48 hours then in cell.Test the day before yesterday, washing NK-92 cell three times places the NK-92 substratum not add IL-2 in 24 hours.Eccentric cell is suspended in (no IL-2) in the NK-92 substratum, with 4 * 10 ⁴Cells/well density, the different concns that 200 microlitres that dilute with the NK-92 substratum contain as reference IL-2 molecule takes off propionamido--1, C125S or C125S human IL-2 mutain; Or the IL-2 mutain of the present invention of different concns, be seeded in the 96 hole flat undersides.37 ℃, 5%CO ₂Cultivate after 72 hours, it is frozen to get 100 microlitre equivalent culture supernatant, subsequently TNF-α ELISA test kit (the BioSource Cytoscreen that buys with commercialization ^TMHumanTNF-ELISA test kit; Camarillo, California) detection by quantitative TNF-α.For the cell of survival in cultivating, the MTT dye reagent box of buying with commercialization ( Non-radioactive cell proliferation test test kit (Wisconsin) breed and calculate stimulation index according to the colorimetric reading for Promega Corp., Madison by mensuration.

The cell toxicant that NK is cell-mediated

The cell-mediated cytotoxic IL-2 biological test of natural killer (NK) adopts people NK3.3 clone.Phenotype and functional performance (Kornbluth, J.Immunol.129 (6): 2831-2837,1982) that NK3.3 clone has shown peripheral blood NK cell can pass through Fc acceptor (CD16 Fc γ RIIIA) mediate antibody dependent cell poison (ADCC).This clone is available from Jackie doctor Kornbluth, and limiting permission uses in the upright university in Lu Yisi continent, is stored in CMCC (ID 12022).

Table 2 biological activity of the Nk3.3 cell of IL-2 biological test detection

Amplification NK3.3 cell also maintains and is added with 15% heat-inactivated fetal bovine serum, 25mM HEPES, 2mM L-glutaminate and 20% people T-Stim ^TMIn the RPMI-1640 substratum of w/PHA as the IL-2 source.Prepare this test, when no IL-2 (" hunger "), cultivated the NK3.3 cell 24 hours.This test is included at the bottom of the U-shaped and inoculates 5 * 10 in the 96-orifice plate ⁴Individual " hungry " NK3.3 cell makes its contact take off propionamido--1, C125S or C125S human IL-2, or the IL-2 mutain of the present invention of different concns, cumulative volume 100 microlitres as the different concns of reference IL-2 molecule.Cultivate after 18 hours IL-2 activated NK3.3 cytological effect cell and 5 * 10 ³The target cell of the fluorexon AM-mark of target cell of individual fluorexon AM-mark (K562 or Daudi) or antibody sandwich (being coated with the Daudi cell of the rituximab monoclonal antibody of ultimate density 2 μ g/ml) is cultivated altogether, obtain final effect-target than 10: 1, final volume is 200 microlitres.After effect-target cell is cultivated 4 hours altogether, simple centrifugal 96 orifice plates; Get 100 microlitre culture supernatant and place of the release of flat 96 orifice plates of black transparent with photofluorometer quantitative assay fluorexon AM.The scale of measuring is shown SL per-cent, calculates with following formula: % SL==100 * [(experimental port mean value-spontaneous release mean value)/(the maximum mean value-spontaneous release mean value that discharges)]; Mensuration contains underlined target cell but does not have the spontaneous release of effect cell hole and measure the maximum release that contains underlined target cell and 1%Triton X-100 hole.

T cell proliferation

It is Kit225 (CMCC ID#11234) that the IL-2 biological test of T cell proliferation adopts the human T-cell, and (Hori etc., Blood 70 (4): 1069-72) derived from a chronic T-Lymphocytic leukemia patient for they.α, β, the γ subunit of Kit225 cell energy constructive expression IL-2 receptor complex.Kit225 propagation is that IL-2 relies on, if cultivate the medium-term and long-term IL-2 of shortage cell with death.This test comprises cultivates the Kit225 cell 24 hours when the no IL-2, what the cell of given number and different concns were gone in inoculation then takes off propionamido--1, C125S or C125S human IL-2 mutain as reference IL-2 molecule, or the IL-2 mutain of the present invention of different concns, cultivate after 48 hours, the MTT reducing dyes test kit of buying with the commercialization of standard detects propagation, calculates stimulation index according to the colorimetric reader.

NK cell survival signal

Screen human IL-2's mutain storehouse subgroup and induced the ability of NK cell survival signal.

IL-2 (promptly containing the preparation that takes off propionamido--1, C125S human IL-2 mutain) can induce the AKT phosphorylation in the NK3.3 cell of prior IL-2 hunger, think that this is " a survival signal ".According to this biological test, amplification NK3.3 cell also maintains and is added with 15% heat-inactivated fetal bovine serum, 25mM HEPES, 2mM L-glutaminate and 20% people T-Stim ^TMIn the RPMI-1640 substratum of w/PHA as the IL-2 source.Prepare this test, when no IL-2 (" hunger "), cultivated the NK3.3 cell 24 hours.For producing the cell survival signal, by adding 2nM reference IL-2 mutain, as take off propionamido--1, C125S or C125S human IL-2 mutain, or 2nM candidate IL-2 interested mutain, stimulate " hungry " NK3.3 cell (2x10 ⁶) 30 minutes.With phosphate-buffered saline (PBS) washed cell secondary.Contain the cell extraction buffer dissolved cell group of proteinase inhibitor with 50 microlitres, carry out taking turns freeze molten.4 ℃, the centrifugal extract of 13000rpm 10 minutes.The clarification lysate equal portions of dilution in 1: 10 are added AKT[pS473] ^*In the hole of immunity test test kit (BioSource Internatioal).In operation sequence, detect the level of phosphorylation AKT with quantitative ELISA.

Embodiment 2: evaluation can reduce the useful sudden change that the NK cell produces TNF-α

First kind of function of mutain estimates it is when detecting with 1.0nM concentration, and it makes the TNF-α of NK cell induction be produced as C125S human IL-2 mutain (hereinafter referred to as " Ala-Pro ") observed＜60% and has improved tolerance.The mutain that belongs to this kind function has two classes:

(1) when being 1.0nM (being 1000pM), concentration can induce the NK cell to produce low TNF-α and maintenance NK cell proliferation, but those mutains that proliferation activity reduces when lower concentration, comprise and take off propionamido--1, C125S or C125S IL-2 mutain, they contain 19D40D, 36D61R, 36D65L, 40D61R, 40D65Y, 40G65Y or 81K91D combination and replace in the residue position (promptly 19,36,40,61,65,81 or 91) with respect to sophisticated human IL-2's sequence (promptly with respect to SEQ IDNO:4), see the following form shown in 3.

(2) those mutains that can induce during for 50pM the NK cell to produce low TNF-α and keep NK cell proliferation not reduce in concentration, in addition, the TNF-α that produces of NK cell must be lower than 0.05nM (being 50pM) and o.lnM (being 100pM) the C125S human IL-2 80%; This subclass comprises and takes off propionamido--1, C125S or C125SIL-2 mutain, they contain 40D72N, 80K65Y, 81K88D, 81K42E, 81K72N, 107H65Y or 107R72N combination and replace in the residue position (promptly 40,42,65,72,80,81,88 or 107) with respect to sophisticated human IL-2's sequence (promptly with respect to SEQ ID NO:4), see the following form shown in 3.

Having that table 3 identifies reduces the IL-2 mutain of inducing the NK cell to produce TNF-α.The TNF-α that the NK cell produces different concns IL-2 mutain be expressed as with the C125S human IL-2 (: Ala-Pro) observed ratio.The NK cell proliferation (NK-92-MTT) of different concns IL-2 mutain is expressed as the observed ratio with C125S human IL-2 (Ala-Pro).

Having that table 4 identifies reduces other IL-2 mutain of inducing the NK cell to produce TNF-α.The TNF-α that the NK cell produces different concns IL-2 mutain be expressed as with the C125S human IL-2 (: Ala-Pro) observed ratio.The NK cell proliferation (NK-92-MTT) of different concns IL-2 mutain is expressed as the observed ratio with C125S human IL-2 (Ala-Pro).

Embodiment 3: the preferred sudden change that can strengthen NK cell proliferation is identified

Second kind of function of human IL-2's mutain is breeding ratio C125S human IL-2 height＞200% that strengthens the NK cell when one or more test concentrations (5pM, 20pM, 50pM, 100pM and 1000pM), and TNF-α is produced have no adverse effects (be lower than the observed TNF-α of concentration 100pM or 1nM reference IL-2 mutain and produce 100%).In addition, choice criteria is included under two kinds of test concentrations proliferation index at least greater than reference IL-2 mutain, i.e. C125S human IL-2 (Ala-Pro) observed 150%.The mutain of this kind function comprises and takes off propionamido--1, C125S or C125S human IL-2 mutain that they contain 19D81K, 40G36D in the residue position (promptly 19,36,40 or 81) with respect to sophisticated human IL-2's sequence (promptly with respect to SEQ ID NO:4) or 81K36D replaces.See the following form 5.

Table 5 identifies has and can strengthen NK cell proliferation and the NK cell is produced the IL-2 mutain that TNF-α has no adverse effects.The TNF-α that the NK cell produces different concns IL-2 mutain be expressed as with the C125S human IL-2 (: Ala-Pro) observed ratio.The NK cell proliferation (NK-92-MTT) of different concns IL-2 mutain is expressed as the observed ratio with C125S human IL-2 (Ala-Pro).

Embodiment 4: the evaluation of " difunctional " sudden change

The 3rd function of human IL-2's mutain shows as proliferation activity that strengthens the NK cell and the generation that reduces TNF-α, wherein TNF-α produces and is lower than any test concentrations (5pM, 20pM, 50pM, 100pM and 1000pM) time C125S human IL-2 mutain viewed 150%. these groups comprise and take off propionamido--1, C125S human IL-2 mutain or C125S human IL-2 mutain, they are with respect to the residue position of sophisticated human IL-2's sequence (promptly with respect to SEQID NO:4) (promptly 36,40,42,61,80,81,91,94,95 or 107) in contain 36D42R, 36D80K, 40D80K, 81K61R, 91N95G, 107H36D, 107R36D or 91N94Y95G replace, and see the following form 6.

Table 6 identifies has to strengthen and induces NK cell proliferation and the NK cell is produced the difunctional IL-2 mutain that TNF-α has no adverse effects.The TNF-α that the NK cell produces different concns IL-2 mutain be expressed as with the C125S human IL-2 (: Ala-Pro) observed ratio.The NK cell proliferation (NK-92-MTT) of different concns IL-2 mutain is expressed as the observed ratio with C125S human IL-2 (Ala-Pro).

Table 7 brief summary the functional performances that replace of identify 32 kinds combinations of this screening process

Embodiment 5: can reduce normal people's peripheral blood mononuclear cell pro-inflammatory cytokine and produce

Keep or strengthen the evaluation of the preferred IL-2 mutain of its propagation and cell toxicant level simultaneously

From the associativity aminoacid replacement of above-mentioned 32 kinds of IL-2 mutains shown in the table 8, select 18 kinds of IL-2 mutains to carry out small-scale expression/purifying.Tested these IL-2 mutains and can cause separating peripheral blood mononuclear cell (PBMC) tolerance from several normal everybody donors and improve and keep active similar functions characteristic, contrast with relevant IL-2 (take off propionamido--1, C125S human IL-2 mutain (is present in Among the IL-2)) and yeast expressed take off propionamido--1, C125S human IL-2 mutain (being called Y-Pro in following data) is made comparisons.Specifically, by stimulating the IL-2 mutain that screens purifying derived from the PBMC of one group of normal everybody donor, measure the generation of propagation and pro-inflammatory cytokine (TNF-α), and can be, or the cytotoxicity (ADCC) that relies on of antibody and killing tumor cells target cell by kill and wound (LAK) of nature/spontaneous cell toxicant (NK), lymphokineactivation.

Table 8 screening has the active human IL-2's mutain that contains the C125S human IL-2 (SEQ ID NO:6) of following combination replacement or take off propionamido--1, C125S human IL-2 (SEQ ID NO:8) aminoacid sequence in the human PBMC. ¹

19D40D	80K65Y	91N95G	40D72N	81K61R
19D40D	80K65Y	91N95G	40D72N	81K61R	40G36D	81K91D	40D65Y	81K42E	107R72N
81K88D	40D61R	81K36D	107R36D		40G36D	81K91D	40D65Y	81K42E	107R72N
81K88D	40D61R	81K36D	107R36D		36D42R	80K36D	107H36D	40D80K

¹By identifying the IL-2 mutain with the relevant amino acid position of sophisticated human IL-2 (SEQ ID NO:4) and the aminoacid replacement of this position

Adopted following major function terminal point:

1) compare with relevant human IL-2's mutain contrast, IL-2 mutain activated human PBMC produces the pro-inflammatory cytokine (TNF-α) of reduction;

2) compare with relevant human IL-2's mutain contrast, can keep or strengthen IL-2 inductive human PBMC propagation, but the generation that does not improve pro-inflammatory cytokine;

3) compare with relevant human IL-2's mutain contrast, can keep or strengthen external use IL-2 mutain activated human PBMC's NK, the LAK and the molten cell killing of ADCC mediation.

The test explanation

Unite propagation/pro-inflammatory cytokine and produce the method for test

Behind the contact IL-2, the human PBMC breeds and secrete cytokines in dosage dependence mode.For at utmost improving the data output efficiency, designed Combined Trials and assessed with reference IL-2 mutain or human IL-2's mutain interested and irritate propagation and cytokine generation level after 72 hours.Test setting comprises: with the PBMC of one of density gradient centrifugation (ACDAVacutainer CPT test tube) separation or several normal people's donors.Complete RPMI substratum (RPMI in 96 hole tissue culture treated plates, 10% hot deactivation people AB serum, 25mM HEPES, 2mM glutamine, penicillin/streptomycin/amphotericin) in, with the IL-2 of different concns (0.039nM-10nM), or there is not IL-2 as negative control, 37 ℃, 7%CO ₂, cultivate 200000 cells in every hole.Cultivate that to get the equivalent cell culture supernatant after 66 hours frozen, detect cytokine subsequently.With 1 μ Ci ³H-thymidine pulse cell 6 hours, collecting cell is measured Nucleotide and is mixed level (Wallac Trilux Microbeta plate reader) then, weighs cell proliferation.The ELISA test kit of buying with commodity (BioSource International) is pressed business directory and is detected TNF-alpha levels in the cell culture supernatant.6 different people donors of one close set are carried out this test repeatedly, provide " normal population " the representativeness propagation of IL-2 and the signature analysis of cytokine response reaction.

Data analysis

Duplicate inoculation PBMC sample is assessed the repeatability of (test) in different test panel.Analytical data is by deducting the mean value of background propagation (PBMC+ does not have IL-2) and the duplicate sample of calculating.From the test holes that contains PBMC, obtain the thin,tough silk culture supernatant, obtain the cytokine data after the merging, be the cytokine mean level (ML) in duplicate hole.According to the purifying TNF-α typical curve of adorning in the ELISA test kit, quantitatively the horizontal plane of TNF-α is represented with pg/ml.Further the data of one group 6 normal everybody donors of compilation are summarised in the synoptic diagram shown in Figure 1.

Cytotoxicity test (NK/LAK/ADCC)

In this test, with the PBMC of density gradient centrifugation separating whole blood.As carry out at present usually this area, as present common enforcement the in this area, (for example see that " the active generation of the separation of NK cells of human beings and LAK " is selected from " newly organized immunological experiment guide "; 1996 John Wiley﹠amp; Son Inc.), stimulates PBMC to produce the LAK activity when having 10nM IL-2 contrast or interested IL-2 mutain.The gained cell mass contains " effect " cell, can be categorized as NK or LAK, and it can kill and wound K562 and Daudi tumour target cell respectively.These effector cells also can mediate ADCC, and (this example is because of the effector cell can discern the specific antibody that is incorporated into the Daudi target cell

) the Fc part.This test comprises with different effects--target is cultivated the target cell 4 hours of effector cell and fluorexon AM-mark altogether than (E:T ratio).Detect in the culture supernatant cytotoxic activity amount with respect to fluorexon AM.Measured according to spontaneous release and maximum contrast amount, the SL per-cent when being expressed as each E:T ratio of discharging.This test of brief summary can detect following biologic activity:

Active	The effector cell	Target cell	Explanation
Active	The effector cell	Target cell	Explanation	NK	PBMC	K562	The nature cell poison
LAK	PBMC	Daudi	The IL-2 active cells	NK	PBMC	K562	The nature cell poison
LAK	PBMC	Daudi	The IL-2 active cells	ADCC	PBMC	Daudi+Rituxan	Antibody relies on

Data analysis

Data report is SL %; In addition, can utilize the IL-2 mutain and determine compared with the control in blended human PBMC group, whether kept cytotoxic activity with the ratio of relevant IL-2 contrast.

The result

Identify and in normal pbmc, can reduce by two preferably combination IL-2 mutains that pro-inflammatory cytokine produces maintenance simultaneously or strengthens propagation and cell toxicant level: 40D72N and 40D61R.For the following data that provide, only with relevant contrast, promptly in same Yeast system expression formula and purifying take off propionamido--1, C125S human IL-2 (being called Y-Pro) has tested the IL-2 mutain.In the secondary independent experiment, with propagation/pro-inflammatory cytokine generation test of associating, tested the dose response curve (39pM-10nM) of IL-2 mutain earlier, each PBMC with three normal people's donors does duplicate the test.Data analysis comprise people's donor individual overview, means standard deviation, with the variance analysis of internal reference IL-2 and cytokine production (pg/ml) by propagation (cpm) stdn, to obtain the cytokine relative quantity that each cell produces.Calculate at last with the IL-2 contrast and compare the percentage ratio that TNF-α output reduces.If kept multiplication capacity, 10, the IL-2 mutain of TNF-α output minimizing more than 25% is preferred really during 000pM concentration.Table 9 brief summary other amino acid combination in two kinds of preferably combination IL-2 mutain skeletons replace.Fig. 2 and 3 is presented at respectively among the human PBMC, and the propagation and the TNF-α of 40D72N and 40D61R mediation produce.

The reduction per-cent that table 9IL-2 contrast TNF-α produces ¹

ID	625pM	2500pM	10,000pM
ID	625pM	2500pM	10,000pM	40D72N	-31.53	-29.94	-26.89
40D61R	-19.62	-20.77	-27.69	40D72N	-31.53	-29.94	-26.89

¹Numerical value is represented the average per-cent that reduces of the Y-Pro contrast of one group 6 normal everybody donor PBMC.The cytokine data are by the propagation stdn

In case identify two kinds of preferred IL-2 mutains, determine importantly whether this IL-2 mutain activated PBMC has kept the ability by NK, LAK and ADCC active dissolution tumour target cell.As shown in Figure 4, observe between 40D72N IL-2 mutain or 40D61R IL-2 mutain and the relevant IL-2 contrast and on by NK, LAK and ADCC active dissolution tumour target cell ability, do not see difference.

Those skilled in the art will remember that above-mentioned of the present invention many modifications and other embodiment, these inventions have the advantage that the above and accompanying drawing show.Therefore should understand and the invention is not restricted to above-mentioned embodiment, various modifications and other embodiment are also included within this extortion and rein in the scope of described embodiment.Adopted some buzzwords though this extortion is reined in, they only use by general implication, rather than limit the scope of the invention.

Sequence table

＜110〉L. Ao Keman (Lea Aukerman)

K. De Nys-Mi Ze (Kimberly Denis-Mize)

C. Hai Si (Carla Heise)

B. Jie Laer (Bahija Jallal)

D. Mooney pool this (Daniel Menezes)

S.T. Wilson (Susan T.Wilton)

＜120〉combinatorial interleukin-2-2 mutain

<130>PP020568.0003

<140>60/585,980

<141>2004-07-07

<140>60/550,868

<141>2004-03-05

<140>60/646,095

<141>2005-01-21

<160>74

<170>FastSEQ for Windows Version 4.0

<210>1

<211>459

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(1)...(459)

<221>misc_feature

<222>(0)...(0)

＜223〉human IL-2's precursor

<400>1

atg tac agg atg caa ctc ctg tct tgc att gca cta agt ctt gca ctt 48

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

gtc gca aac agt gca cct act tca agt tct aca aag aaa aca cag cta 96

Val Ala Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

caa ctg gag cat tta ctg ctg gat tta cag atg att ttg aat gga att 144

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

aat aat tac aag aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt 192

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

50 55 60

tac atg ccc aag aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa 240

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

gaa gaa ctc aaa cct ctg gag gaa gtg cta aat tta gct caa agc aaa 288

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

85 90 95

aac ttt cac tta aga ccc agg gac tta atc agc aat atc aac gta ata 336

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

gtt ctg gaa cta aag gga tct gaa aca aca ttc atg tgt gaa tat gct 384

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

115 120 125

gat gag aca gca acc att gta gaa ttt ctg aac aga tgg att acc ttt 432

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

tgt cag agc atc atc tca aca ctg act 459

Cys Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210>2

<211>153

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Ala Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His LeuLeu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

50 55 60

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

85 90 95

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

115 120 125

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

Cys Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210>3

<211>399

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(1)...(399)

<221>misc_feature

<222>(0)...(0)

＜223〉sophisticated human IL-2

<400>3

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tgt cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>4

<211>133

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>4

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>5

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉C125S human IL-2 mutain

<400>5

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>6

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉C125S human IL-2 mutain

<400>6

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>7

<211>396

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(396)

＜223〉take off alanyl 1, C125S human IL-2 mutain

<400>7

cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat tta 48

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag aat 96

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag aag 144

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa cct 192

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

50 55 60

ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta aga 240

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta aag 288

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca acc 336

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc atc 384

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

tca aca ctg act 396

Ser Thr Leu Thr

130

<210>8

<211>132

<212>PRT

＜213〉artificial sequence

<220>

＜223〉take off alanyl 1, C125S human IL-2 mutain

<400>8

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

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

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210>9

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉19D40D, C125S human IL-2 mutain

<400>9

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg gat gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Asp Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg gac aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>10

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉19D40D, C125S human IL-2 mutain

<400>10

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

1 5 10 15

Leu Leu Asp Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>11

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉19D81K, C125S human IL-2 mutain

<400>11

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg gat gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Asp Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt csc tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ssr Thr Leu Thr

130

<210>12

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉19D81K, C125S human IL-2 mutain

<400>12

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

1 5 10 15

Leu Leu Asp Asp Leu Gln Met Ile Leu Ash Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>13

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉36D42R, C125S human IL-2 mutain

<400>13

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gac acc agg atg ctc aca aga aag ttt tac atg ccc aag 144

Asn Pro Lys Asp Thr Arg Met Leu Thr Arg Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>14

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉36D42R, C125S human IL-2 mutain

<400>14

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Lsu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Asp Thr Arg Met Leu Thr Arg Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>15

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉36D61R, C125S human IL-2 mutain

<400>15

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gac acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa aga gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>16

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉36D61R, C125S human IL-2 mutain

<400>16

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>17

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉36D65L, C125S human IL-2 mutain

<400>17

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gac acc agg atg ctc aca ttt aag ttt tac atg ccc aag 14

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

ttg ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Leu Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 331

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>18

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉36D65L, C125S human IL-2 mutain

<400>18

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Leu Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>19

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉40D36D, C125S human IL-2 mutain

<400>19

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gac acc agg atg gac aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Asp Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca sca ctg act 399

Ile Ser Thr Leu Thr

130

<210>20

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉40D36D, C125S human IL-2 mutain

<400>20

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Asp Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>21

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉40D61R, C125S human IL-2 mutain

<400>21

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg gac aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcG aca gaa ctg aaa cat ctt cag tgt cta gaa aga gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>22

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉40D61R, C125S human IL-2 mutain

<400>22

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>23

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉40D65Y, C125S human IL-2 mutain

<400>23

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg gac aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

tac ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Tyr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>24

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉40D65Y, C125S human IL-2 mutain

<400>24

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys ALa Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Tyr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>25

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉40D72N, C125S human IL-2 mutain

<400>25

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg gac aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat aac gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Asn Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>26

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉40D72N, C125S human IL-2 mutain

<400>26

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Asn Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>27

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉40D80K, C125S human IL-2 mutain

<400>27

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg gac aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac aag 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Lys

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>28

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉40D80K, C125S human IL-2 mutain

<400>28

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Lys

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>29

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉40G36D, C125S human IL-2 mutain

<400>29

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gac acc agg atg ggt aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Agp Thr Arg Met Gly Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>30

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉40G36D, C125S human IL-2 mutain

<400>30

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Asp Thr Arg Met Gly Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>31

<211>399

<212>DNA

＜213〉artificial sequence

<22D>

<221>CDS

<222>(1)...(399)

＜223〉40G65Y, C125S human IL-2 mutain

<400>31

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Ieu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ggt aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Gly Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

tac ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Tyr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>32

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉40G65Y, C125S human IL-2 mutain

<400>32

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Gly Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Tyr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>33

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉80K36D, C125S human IL-2 mutain

<400>33

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gac acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac aag 240

Pro Leu Glu Glu Va1 Leu Asn Leu Ala Gln Ser Lys Asn Phe His Lys

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>34

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉80K36D, C125S human IL-2 mutain

<400>34

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Lys

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>35

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉80K65Y, C125S human IL-2 mutain

<400>35

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

tac ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac aag 240

Tyr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Lys

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>36

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉80K65Y, C125S human IL-2 mutain

<400>36

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Tyr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Lys

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>37

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉81K36D, C125S human IL-2 mutain

<400>37

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gac acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>38

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉81K36D, C125S human IL-2 mutain

<400>38

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>39

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉81K42E, C125S human IL-2 mutain

<400>39

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca gaa aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Glu Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Va1 Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>40

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉81K42E, C125S human IL-2 mutain

<400>40

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Glu Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>41

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉81K61R, C125S human IL-2 mutain

<400>41

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa aga gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc aat atc aao gta ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>42

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉81K61R, C125S human IL-2 mutain

<400>42

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>43

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉81K65Y, C125S human IL-2 mutain

<400>43

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

tac ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Tyr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aGa ctg act 399

Ile Ser Thr Leu Thr

130

<210>44

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉81K65Y, C125S human IL-2 mutain

<400>44

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Tyr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>45

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉81K72N, C125S human IL-2 mutain

<400>45

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat aac gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Asn Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>46

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉81K72N, C125S human IL-2 mutain

<400>46

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Asn Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>47

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉81K88D, C125S human IL-2 mutain

<400>47

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

l 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc gat atc aac gta ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>48

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉81K88D, C125S human IL-2 mutain

<400>48

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>49

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉81K91D, C125S human IL-2 mutain

<400>49

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

l 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc aat atc aac gat ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Asp Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>50

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉81K91D, C125S human IL-2 mutain

<400>50

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Asp Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>51

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉81K107H, C125S human IL-2 mutain

<400>51

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa cac gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>52

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉81K107H, C125S human IL-2 mutain

<400>52

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>53

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉81L107H, C125S human IL-2 mutain

<400>53

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

ttg ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Leu Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa cac gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>54

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉81L107H, C125S human IL-2 mutain

<400>54

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Leu Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>55

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉91N95G, C125S human IL-2 mutain

<400>55

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac aac ata gtt ctg ggt cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Asn Ile Val Leu Gly Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>56

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉91N95G, C125S human IL-2 mutain

<400>56

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Asn Ile Val Leu Gly Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>57

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉107H36D, C125S human IL-2 mutain

<400>57

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gac acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc ace gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa cac gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>58

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉107H36D, C125S human IL-2 mutain

<400>58

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>59

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉107H42E, C125S human IL-2 mutain

<400>59

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca gaa aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Glu Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa cac gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>60

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉107H42E, C125S human IL-2 mutain

<400>60

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Glu Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>61

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉107H65Y, C125S human IL-2 mutain

<400>61

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

tac ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Tyr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa cac gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>62

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉107H65Y, C125S human IL-2 mutain

<400>62

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Tyr Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>63

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉107R36D, C125S human IL-2 mutain

<400>63

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gac acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa aga gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Arg Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>64

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉107R36D, C125S human IL-2 mutain

<400>64

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Arg Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>65

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉107R72N, C125S human IL-2 mutain

<400>65

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat aac gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Asn Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa aga gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Arg Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>66

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉107R72N, C125S human IL-2 mutain

<400>66

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Asn Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Arg Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>67

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉40D81K107H, C125S human IL-2 mutain

<400>67

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg gac aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa cac gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>68

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉40D81K107H, C125S human IL-2 mutain

<400>68

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Asp Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>69

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉40G81K107H, C125S human IL-2 mutain

<400>69

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ggt aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Gly Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aag ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa cac gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>70

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉40G81K107H, C125S human IL-2 mutain

<400>70

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Gly Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Lys Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>71

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉91N94Y95G, C125S human IL-2 mutain

<400>71

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac aac ata gtt tac ggt cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Asn Ile Val Tyr Gly Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>72

<211>133

<212>PRT

＜213〉artificial sequence

<220>

＜223〉91N94Y95G, C125S human IL-2 mutain

<400>72

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

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Asn Ile Val Tyr Gly Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>73

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉36D61R, C125S human IL-2 mutain contains 36D and the 61R codon optimized at expression in escherichia coli

<400>73

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gat acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa cgt gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

<210>74

<211>399

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(399)

＜223〉107R36D, C125S human IL-2 mutain contains and optimizes at expression in escherichia coli

107R and 36D codon

<400>74

gca cct act tca agt tct aca aag aaa aca cag cta caa ctg gag cat 48

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

1 5 10 15

tta ctg ctg gat tta cag atg att ttg aat gga att aat aat tac aag 96

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

aat ccc aaa gat acc agg atg ctc aca ttt aag ttt tac atg ccc aag 144

Asn Pro Lys Asp Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa 192

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta 240

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg gaa cta 288

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

aag gga tct gaa aca aca ttc atg tgt gaa cgt gct gat gag aca gca 336

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Arg Ala Asp Glu Thr Ala

100 105 110

acc att gta gaa ttt ctg aac aga tgg att acc ttt tct cag agc atc 384

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

atc tca aca ctg act 399

Ile Ser Thr Leu Thr

130

Claims

1. an isolated nucleic acid molecule is characterized in that, this nucleic acid molecule contains and is selected from following nucleotide sequence:

A) nucleotide sequence of coding human IL-2 mutain, described mutain contain and are selected from SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72 aminoacid sequence;

B) nucleotide sequence shown in the SEQ ID NO:9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69 or 71;

C) nucleotide sequence of coding human IL-2 mutain, described mutain contains the aminoacid sequence that is selected from sequence residue 2-133 shown in the SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 and 72;

D) contain the nucleotide sequence that is selected from sequence residue 4-399 shown in the SEQ ID NO:9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69 and 71;

E) a,), b,), c) or d) in arbitrary described nucleotide sequence, wherein said sequence comprises the be encoded codeword triplet of L-Ala of SEQ ID NO:9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69 or 71 Nucleotide 373-375 and replaces;

F) a,), b,), c) or d) in arbitrary described nucleotide sequence, wherein said sequence comprises the be encoded codeword triplet of halfcystine of SEQ ID NO:9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69 or 71 Nucleotide 373-375 and replaces; With

G) a), b), c) or d) in arbitrary described nucleotide sequence, the expression of the codon of wherein one or more described mutains of encoding in host cell interested is optimised.

2. isolated nucleic acid molecule as claimed in claim 1, it is characterized in that g) described nucleotide sequence is selected from the sequence of Nucleotide 4-399, SEQ ID NO:74 of sequence, SEQ ID NO:73 of SEQ ID NO:73 and the Nucleotide 4-399 of SEQ ID NO:74.

3. one kind contains the expression vector of nucleic acid molecule as claimed in claim 1 or 2.

4. one kind contains the host cell of nucleic acid molecule as claimed in claim 1 or 2.

5. an isolated polypeptide is characterized in that, this polypeptide contains and is selected from following aminoacid sequence:

A) aminoacid sequence shown in the SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 or 72;

B) contain the aminoacid sequence of SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 or 72 residue 2-133;

C) a) or b) described aminoacid sequence, wherein said sequence comprises the alanine residue that replaces SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 or 72 the 125th serine residues; With

D) a) or b) described aminoacid sequence, wherein said sequence comprises the cysteine residues that replaces SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 or 72 the 125th serine residues.

6. isolated polypeptide, it is characterized in that, this polypeptide contains human IL-2's mutain, wherein said mutain contains the aminoacid sequence shown in the SEQ ID NO:4,125 Serines at SEQ ID NO:4 have replaced halfcystine, with in SEQ ID NO:4, have other aminoacid replacement of two places at least, wherein said mutain: equating under the test conditions, compare 1 with take off alanyl-1, C125S human IL-2 or the C125S human IL-2 of similar quantity) can keep or strengthen the propagation of natural killer (NK) cell; 2) level that can cause the NK cell to produce pro-inflammatory cytokine reduces, and the propagation of wherein said NK cell and described NK cell produce pro-inflammatory cytokine and detect with the NK-92 biological test.

7. isolated polypeptide as claimed in claim 6 is characterized in that, described mutain also contains the L-Ala disappearance at 1 of SEQ ID NO:4.

8. isolated polypeptide as claimed in claim 6, it is characterized in that other replacement among the described SEQ ID NO:4 is selected from 19D40D, 19D81K, 36D42R, 36D61R, 36D65L, 40D36D, 40D61R, 40D65Y, 40D72N, 40D80K, 40G36D, 40G65Y, 80K36D, 80K65Y, 81K36D, 81K42E, 81K61R, 81K65Y, 81K72N, 81K88D, 81K91D, 81K107H, 81L107H, 91N95G, 107H36D, 107H42E, 107H65Y, 107R36D, 107R72N, 40D81K107H, 40G81K107H and 91N94Y95G combination replace.

9. isolated polypeptide as claimed in claim 8 is characterized in that, described mutain also contains the L-Ala disappearance at 1 of SEQ ID NO:4.

10. isolated polypeptide as claimed in claim 6 is characterized in that, described pro-inflammatory cytokine is TNF-α.

11. isolated polypeptide as claimed in claim 6, it is characterized in that, equating under the test conditions, with similar quantity take off alanyl-1, C125S human IL-2 mutain or the C125S human IL-2 is observed compares, the cytotoxicity that described mutain provides natural killer cell toxicity, the lymphokineactivation of maintenance or the mediation of enhanced NK cells of human beings to kill and wound (LAK) cytotoxicity or ADCC mediation, the cell-mediated cytotoxicity of wherein said NK detects with NK3.3 cell toxicant biological test.

12. isolated polypeptide as claimed in claim 6 is characterized in that, equating under the test conditions, described mutain inductive NK cell proliferation than similar quantity to take off alanyl-1, C125S human IL-2 or C125S human IL-2 inductive high by 150%.

13. isolated polypeptide as claimed in claim 12 is characterized in that, it is high by 170% that described mutain inductive NK cell proliferation ratio takes off alanyl-1, C125S human IL-2 or C125S human IL-2 inductive.

14. isolated polypeptide as claimed in claim 13 is characterized in that, described mutain inductive NK cell proliferation is for taking off alanyl-1, C125S human IL-2 or C125S human IL-2 inductive about 200% to about 250%.

15. isolated polypeptide as claimed in claim 6 is characterized in that, is equating under the test conditions, what described mutain inductive NK cell proliferation was higher than similar quantity takes off alanyl-1, C125S human IL-2 or C125S human IL-2 inductive at least 10%.

16. isolated polypeptide as claimed in claim 15 is characterized in that, described mutain inductive NK cell proliferation is higher than takes off alanyl-1, C125S human IL-2 or C125S human IL-2 inductive at least 15%.

17. isolated polypeptide as claimed in claim 16 is characterized in that, is equating under the test conditions, what described mutain induced that the pro-inflammatory cytokine of generation is lower than similar quantity takes off alanyl-1, C125S human IL-2 or C125S human IL-2 inductive 100%.

18. isolated polypeptide as claimed in claim 17 is characterized in that, the generation of described mutain inductive pro-inflammatory cytokine is lower than takes off alanyl-1, C125S human IL-2 or C125S human IL-2 inductive 70%.：

19. isolated polypeptide, it is characterized in that, this polypeptide contains human IL-2's mutain, wherein said mutain contains the aminoacid sequence shown in the SEQ ID NO:4,125 Serines at SEQ ID NO:4 have replaced halfcystine, with in SEQ ID NO:4, have other aminoacid replacement of two places at least, wherein equating under the test conditions, the ratio that the IL-2 inductive NK cell proliferation of described mutain and IL-2 inductive TNF-α produce, take off alanyl-1 than similar quantity, C125S human IL-2 mutain or C125S human IL-2 mutain are observed high at least 1.5 times, wherein detect the NK cytokine propagation of 0.1nM mutain and the TNF-α generation of 1.0nM mutain with the NK-92 biological test.

20. isolated polypeptide as claimed in claim 19 is characterized in that, it is observed high at least 2.5 times that described ratio ratio takes off alanyl-1, C125S human IL-2 or C125S.

21. isolated polypeptide as claimed in claim 19 is characterized in that, it is observed high at least 3.0 times that described ratio ratio takes off alanyl-1, C125S human IL-2 or C125S.

22. isolated polypeptide as claimed in claim 19 is characterized in that, described mutain also contains the L-Ala disappearance at 1 of SEQ IDNO:4.

23. isolated polypeptide, it is characterized in that, this polypeptide contains the aminoacid sequence of human IL-2's mutain, wherein said mutain contains the aminoacid sequence shown in the SEQ ID NO:4,125 Serines at SEQ ID NO:4 have replaced halfcystine, with have other aminoacid replacement of two places at least, wherein said other replaces residue and is arranged in SEQ ID NO:4 and is selected from following position: 19,36,40,42,61,65,72,80,81,88,91,95 and 107.

24. isolated polypeptide as claimed in claim 23 is characterized in that, described mutain also contains the L-Ala disappearance at 1 of SEQ IDNO:4.

25. isolated polypeptide as claimed in claim 23, it is characterized in that other replacement among the described SEQ ID NO:4 is selected from 19D40D, 19D81K, 36D42R, 36D61R, 36D65L, 40D36D, 40D61R, 40D65Y, 40D72N, 40D80K, 40G36D, 40G65Y, 80K36D, 80K65Y, 81K36D, 81K42E, 81K61R, 81K65Y, 81K72N, 81K88D, 81K91D, 81K107H, 81L107H, 91N95G, 107H36D, 107H42E, 107H65Y, 107R36D, 107R72N, 40D81K107H, 40G81K107H and 91N94Y95G combination replace.

26. isolated polypeptide as claimed in claim 25 is characterized in that, described mutain also contains the L-Ala disappearance at 1 of SEQ IDNO:4.

27. the method for manufacturer's interleukin-2 (IL-2) mutain, equating under the test conditions, take off the reference IL-2 mutain of alanyl-1, C125S human IL-2 or C125S compares with being selected from of similar quantity, this mutain can keep or strengthen NK cell proliferation and also can induce the NK cell to produce the lower level pro-inflammatory cytokine, wherein, the propagation and the pro-inflammatory cytokine that adopt the NK-92 biological test to detect described NK cell produce, and described method comprises:

A) with the expression vector transformed host cell that contains the described nucleic acid molecule of claim 1;

B) in cell culture medium, cultivate described host cell allowing described nucleic acid molecule to be expressed as under the condition of polypeptide; With

C) separate described polypeptide.

28. the method for manufacturer's interleukin-2 (IL-2) mutain, equating under the test conditions, take off the reference IL-2 mutain of alanyl-1, C125S human IL-2 or C125S compares with being selected from of similar quantity, this mutain can keep or strengthen NK cell proliferation and also can induce the NK cell to produce the lower level pro-inflammatory cytokine, wherein, the propagation and the pro-inflammatory cytokine that adopt the NK-92 biological test to detect described NK cell produce, and described method comprises:

A) usefulness contains the expression vector transformed host cell of the nucleic acid molecule of the described polypeptide of coding claim 23;

C) separate described polypeptide.

29. a pharmaceutical composition, described pharmaceutical composition contain described human IL-2's mutain of the claim 5 for the treatment of significant quantity and pharmaceutically acceptable vehicle.

30. a pharmaceutical composition, described pharmaceutical composition contain described human IL-2's mutain of the claim 6 for the treatment of significant quantity and pharmaceutically acceptable vehicle.

31. a pharmaceutical composition, described pharmaceutical composition contain described human IL-2's mutain of the claim 19 for the treatment of significant quantity and pharmaceutically acceptable vehicle.

32. a pharmaceutical composition, described pharmaceutical composition contain described human IL-2's mutain of the claim 23 for the treatment of significant quantity and pharmaceutically acceptable vehicle.

33. method that stimulates immune system, it is characterized in that, this method comprises the human IL-2's mutain that gives described Mammals treatment significant quantity, wherein, equating under the test conditions, take off alanyl-1, C125S human IL-2 or C125S with being selected from of similar quantity, reference IL-2 mutain compare, described mutain can induce the NK cell to produce the pro-inflammatory cytokine of lower level, with the propagation that keeps or strengthen the NK cell, wherein detect the generation of NK cell proliferation and described pro-inflammatory cytokine with the NK-92 biological test.

34. method as claimed in claim 33 is characterized in that, described Mammals is the people.

35. method as claimed in claim 33 is characterized in that, described human IL-2's mutain contains and is selected from following aminoacid sequence:

36. method for the treatment of mammalian cancer, it is characterized in that, this method comprises the human IL-2's mutain that gives described Mammals treatment significant quantity, wherein equating under the test conditions, take off the reference IL-2 mutain of alanyl-1, C125S human IL-2 or C125S compares with being selected from of similar quantity, described mutain can induce the NK cell to produce the pro-inflammatory cytokine of lower level, with the propagation that keeps or strengthen the NK cell, wherein detect the generation of NK cell proliferation and described pro-inflammatory cytokine with the NK-92 biological test.

37. method as claimed in claim 36 is characterized in that, described Mammals is the people.

38. method as claimed in claim 36 is characterized in that, described human IL-2's mutain contains and is selected from following aminoacid sequence:

39. method that alleviates experience IL-2 drug treatment solution object interleukin-2 (IL-2) inductive signs of toxicity, it is characterized in that, this method comprises and gives the IL-2 mutain as described IL-2, and wherein said IL-2 mutain contains and is selected from following aminoacid sequence:

40. the application of human IL-2's mutain in stimulating the immune system method, it is characterized in that, this method comprises the described human IL-2's mutain that gives described Mammals treatment significant quantity, wherein equating under the test conditions, take off alanyl-1 with being selected from of similar quantity, the reference IL-2 mutain of C125S human IL-2 or C125S is compared, described mutain can induce the NK cell to produce the pro-inflammatory cytokine of lower level, with the propagation that keeps or strengthen the NK cell, wherein detect the generation of NK cell proliferation and described pro-inflammatory cytokine with the NK-92 biological test.

41. the application of human IL-2's mutain in treatment mammalian cancer method, it is characterized in that, this method comprises the described human IL-2's mutain that gives described Mammals treatment significant quantity, wherein equating under the test conditions, take off the reference IL-2 mutain of alanyl-1, C125S human IL-2 or C125S compares with being selected from of similar quantity, described mutain can induce the NK cell to produce the pro-inflammatory cytokine of lower level, with the propagation that keeps or strengthen the NK cell, wherein detect the generation of NK cell proliferation and described pro-inflammatory cytokine with the NK-92 biological test.

42. the application of human IL-2's mutain in reducing experience IL-2 drug treatment solution object interleukin-2 (IL-2) inductive signs of toxicity method, it is characterized in that, this method comprises and gives described IL-2 mutain as described IL-2, and wherein said mutain contains and is selected from following aminoacid sequence:

D) have a) or b) described aminoacid sequence, wherein said sequence comprises the cysteine residues that replaces SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70 or 72 the 125th serine residues.