Novel immunoactive interleukin-2 analog

Abstract

The present invention relates to interleukin-2 analogs with altered binding affinity to interleukin-2α receptors and increased binding affinity to interleukin-2β receptors.

Description

Technical Field

[0001] The present invention relates to a novel interleukin-2 analog.

Background Art

[0002] Interleukin 2 is an important immunostimulant with a molecular weight of approximately 15 kDa, composed of a total of 133 amino acid residues, and activates various cells of the immune system, including T cells and B cells. The high efficacy of interleukin 2 as an immunostimulant is useful for the treatment of various immune-related diseases such as cancer and AIDS (Patent Document 1). Currently, interleukin 2 (Proleukin (registered trademark)) is a pharmaceutical approved by the FDA for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, since high-dose interleukin 2 therapy is associated with serious toxicity, the applicable patients are limited, and this treatment therapy is actually carried out only for a small number of appropriate patients. Toxicities associated with interleukin 2 include vascular leak syndrome, which causes high fever, nausea, vomiting, vascular leak, severe hypotension, pulmonary edema, and liver damage.

[0003] The interleukin-2 receptor has three subunit receptors. These subunits are formed from an α chain (IL-2Rα, CD25), a β chain (IL-2Rβ or CD122), and a γ chain (IL-2Rγ or CD132). Interleukin-2 can exert various functions by binding to various combinations of these receptor subunits. A single interleukin-2α receptor is a low-affinity interleukin-2 receptor and is not involved in signal transduction. The interleukin-2β and γ receptor complex binds to interleukin-2 with moderate affinity. The interleukin-2α,β, and γ receptor complex binds to interleukin-2 with high affinity. The interleukin-2β and γ receptor complex is necessary for effective signal transduction through kinase activation in multiple signaling pathways. In particular, the interleukin-2β and γ receptor complex is prominent in CD8+ cells and natural killer (NK) cells. Furthermore, the high-affinity interleukin-2α,β, and γ receptor complex is commonly found on CD4 + It is observed not only in T regulatory cells (Tregs) but also, more recently, in activated T cells. The interleukin-2β receptor is CD8 + Because β-receptors are distributed to T cells or natural killer cells (NK cells) and are involved in the body's immune response, research is being conducted to develop therapeutic agents by improving their activity toward immune activation.

[0004] On the other hand, despite the potential of interleukin-2 as a treatment for various immune-related diseases, there are currently insufficient drugs that can reduce toxicity and side effects while also decreasing the required dosage. Therefore, further research into novel and improved drugs is needed. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Korean Published Patent No. 10-2017-0070091 [Non-patent literature]

[0006] [Non-licensed document 1] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed document 3] Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]: 2444

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

[0007] The present invention aims to provide an interleukin-2 analog.

[0008] Furthermore, the present invention aims to provide an isolated nucleic acid encoding the interleukin-2 analog, a recombinant expression vector containing the nucleic acid, and a transformant containing the vector.

[0009] Furthermore, the present invention aims to provide a method for manufacturing the aforementioned interleukin-2 analog.

[0010] Furthermore, the present invention aims to provide a method for increasing interleukin-2β receptor binding affinity, comprising the step of mutating at least one amino acid in natural interleukin-2. [Means for solving the problem]

[0011] One aspect of the present invention is a novel interleukin 2 analog (interleukin 2 analog or IL-2 analog). The interleukin 2 analog may be an interleukin 2 analog having an increased binding affinity for interleukin 2β receptor as compared to natural interleukin 2 or aldesleukin which is an interleukin 2 analog, and the interleukin 2 analog may include a sequence in which at least one amino acid is mutated in natural interleukin 2.

[0012] As another specific example, the interleukin 2 analog is characterized in that it includes a sequence in which at least one of the amino acids corresponding to the positions of the 1st, 12th, 18th, 19th, 20th, 22nd, 32nd, 35th, 38th, 42nd, 43rd, 45th, 48th, 49th, 61st, 68th, 69th, 74th, 76th, 80th, 81st, 82nd, 84th, 85th, 86th, 87th, 88th, 89th, 91st, 92nd, 94th, 95th, 96th, 125th, 126th and 133rd in natural interleukin 2 is mutated.

[0013] The interleukin 2 analog according to any of the above-described specific examples is characterized in that the binding affinity for interleukin 2α receptor is changed and the binding affinity for interleukin 2β receptor is increased as compared to natural interleukin 2 or aldesleukin.

[0014] The interleukin 2 analog according to any of the above-described specific examples is characterized in that at least one amino acid is added to the amino acid corresponding to the 133rd position.

[0015] The interleukin 2 analog according to any of the above-described specific examples is characterized in that it includes a sequence in which the 1st amino acid is deleted and the 125th amino acid is substituted with another amino acid in natural interleukin 2.

[0016] An interleukin-2 analog according to any of the above-mentioned specific examples is characterized by further comprising 1 to 10 amino acid substitutions.

[0017] An interleukin-2 analog according to any of the above-mentioned specific examples is characterized in that at least one amino acid corresponding to the 18th, 19th, 20th, 22nd, 38th, 42nd, 43rd, 45th, 61st, 68th, 69th, 74th, 80th, 81st, 84th, 85th, 86th, 88th, 89th, 91st, 92nd, 94th, and 96th positions is further substituted with another amino acid.

[0018] An interleukin-2 analog according to any of the above-mentioned specific examples is characterized in that at least one amino acid corresponding to the 18th, 19th, 22nd, 38th, 42nd, 43rd, 45th, 61st, 68th, 74th, 80th, 81st, 84th, 85th, 86th, 88th, 91st, 92nd, 94th, and 96th positions in natural interleukin-2 is further substituted with another amino acid.

[0019] The interleukin-2 analog according to any of the above-mentioned specific examples is characterized by being one of the following analogs. (a) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 32nd amino acids are substituted with other amino acids. (b) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 35th amino acids are substituted with other amino acids. (c) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 38th amino acids are substituted with other amino acids. (d) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 42nd amino acids are substituted with other amino acids. (e) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 43rd amino acids are substituted with other amino acids. (f) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 48th amino acids are substituted with other amino acids. (g) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 49th amino acids are substituted with other amino acids. (h) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 76th amino acids are substituted with other amino acids. (i) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, 92nd, 94th and 96th amino acids are substituted with other amino acids. (j) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 87th amino acids are substituted with other amino acids. (k) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, and 42nd amino acids are substituted with other amino acids. (l) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, and 80th amino acids are substituted with other amino acids. (m) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, and 84th amino acids are substituted with other amino acids. (n) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 19th, 38th, and 42nd amino acids are substituted with other amino acids. (o) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 12th, 38th, and 42nd amino acids are substituted with other amino acids. (p) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 61st amino acids are substituted with other amino acids. (q) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 84th amino acids are substituted with other amino acids. (r) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 88th amino acids are substituted with other amino acids. (s) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 89th amino acids are substituted with other amino acids. (t) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 91st amino acids are substituted with other amino acids. (u) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 94th amino acids are substituted with other amino acids. (v) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 126th amino acids are substituted with other amino acids. (w) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, and 84th amino acids are substituted with other amino acids. (x) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 94th, and 96th amino acids are substituted with other amino acids. (y) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 81st, and 92nd amino acids are substituted with other amino acids. (z) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 61st, 81st, and 92nd amino acids are substituted with other amino acids. (aa) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, and 92nd amino acids are substituted with other amino acids. (ab) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, and 92nd amino acids are substituted with other amino acids. (ac) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 81st, 84th, and 92nd amino acids are substituted with other amino acids. (ad) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 20th, 38th, 42nd, 81st, and 92nd amino acids are substituted with other amino acids. (ae) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, and 92nd amino acids are substituted with other amino acids. (af) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 74th, 81st and 92nd amino acids are substituted with other amino acids. (ag) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, 84th, and 92nd amino acids are substituted with other amino acids. (ah) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, 88th, and 92nd amino acids are substituted with other amino acids. (ai) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (aj) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, 85th, and 92nd amino acids are substituted with other amino acids. (ak) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, 86th, and 92nd amino acids are substituted with other amino acids. (al) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (am) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 45th, 74th, 81st, and 92nd amino acids are substituted with other amino acids. (an) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 74th, 80th, 81st and 92nd amino acids are substituted with other amino acids. (ao) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 84th, and 92nd amino acids are substituted with other amino acids. (ap) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 45th, 80th, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (aq) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (ar) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 42nd, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (as) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 61st, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (at) Interleukin-2 analogs in which the first amino acid is deleted and the 125th, 69th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (au) Interleukin-2 analogs in which the first amino acid is deleted from natural interleukin-2 and the 125th, 80th, 81st, 85th, 86th, 91st, and 92nd amino acids are substituted with other amino acids. (av) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (aw) Interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (ax) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 45th, 74th, 80th, 81st and 92nd amino acids are substituted with other amino acids. (ay) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 68th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (az) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 69th, 74th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (ba) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 84th, 85th, 86th, 91st and 92nd amino acids are substituted with other amino acids. (bb) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 85th, 86th, 92nd, 94th, and 96th amino acids are substituted with other amino acids. (bc) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 19th, 22nd, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bd) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 38th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (be) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 61st, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (bf) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 68th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bg) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and amino acids 125, 35, 38, 42, 80, 81, 85, 86 and 92 are substituted with other amino acids. (bh) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 45th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bi) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st and 92nd amino acids are substituted with other amino acids. (bj) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 92nd and 95th amino acids are substituted with other amino acids. (bk) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bl) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 43rd, 61st, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bm) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st, 92nd and 95th amino acids are substituted with other amino acids. (bn) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 82nd, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bo) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 86th, and 92nd amino acids are substituted with other amino acids. (bp) Interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 85th, and 86th amino acids are substituted with other amino acids.

[0020] An interleukin-2 analog according to any of the above-mentioned specific examples is characterized by containing at least one selected from the group consisting of the following amino acid substitutions. (a) Substitution of the 12th amino acid with valine or phenylalanine (b) Substitution of the 18th amino acid with arginine (c) Substitution of the 19th amino acid with tyrosine, valine, phenylalanine, or arginine. (d) Substitution of the 20th amino acid with valine or phenylalanine (e) Substitution of the 22nd amino acid with glutamic acid (f) Substitution of the 32nd amino acid with cysteine (g) Substitution of the 35th amino acid with cysteine ​​or glutamic acid (h) Substitution of the 38th amino acid with alanine or aspartic acid (i) Substitution of the 42nd amino acid with lysine, alanine, or tryptophan (j) Substitution of the 43rd amino acid with cysteine, glutamic acid, or glutamine (k) Substitution of the 45th amino acid with alanine (l) Substitution of the 48th amino acid with cysteine (m) Substitution of the 49th amino acid with cysteine (n) Substitution of the 61st amino acid with glutamine, arginine, or aspartic acid (o) Substitution of the 68th amino acid with aspartic acid or glutamine (p) Substitution of the 69th amino acid with glycine (q) Substitution of the 74th amino acid with histidine or alanine (r) Substitution of the 76th amino acid with cysteine (s) Substitution of the 80th amino acid with phenylalanine, tyrosine, valine, aspartic acid, or tryptophan (t) Substitution of the 81st amino acid with aspartic acid, glutamic acid, or asparagine (u) Substitution of the 82nd amino acid with glycine or valine (v) Substitution of the 84th amino acid with glutamic acid, valine, or phenylalanine (w) Substitution of the 85th amino acid with valine, alanine, glycine, tryptophan, tyrosine, threonine, isoleucine, glutamic acid, or phenylalanine. (x) Substitution of the 86th amino acid with valine, alanine, glycine, or leucine (y) Substitution of the 87th amino acid with cysteine (z) Substitution of the 88th amino acid with glutamine, valine, or phenylalanine (aa) Substitution of amino acid 89 with phenylalanine (ab) Substitution of the 91st amino acid with threonine, phenylalanine, or glutamic acid (ac) Substitution of the 92nd amino acid with phenylalanine, leucine, tyrosine, or tryptophan (ad) Substitution of the 95th amino acid with aspartic acid (ae) Substitution of the 96th amino acid with phenylalanine, valine, or isoleucine (af) Substitution of the 126th amino acid with threonine

[0021] The interleukin-2 analog, according to any of the aforementioned specific examples, is characterized by being selected from the group consisting of sequence numbers 3 to 106.

[0022] Another aspect of the present invention is an isolated nucleic acid encoding the interleukin-2 analog, a recombinant expression vector containing the nucleic acid, and a transformant containing the vector.

[0023] Yet another aspect of the present invention is a method for producing the interleukin-2 analog.

[0024] A further aspect of the present invention is a method for increasing interleukin-2β receptor binding affinity, comprising the step of mutating at least one amino acid in natural interleukin-2, wherein the mutation is at least one amino acid selected from the group consisting of amino acids corresponding to the 1st, 12th, 18th, 19th, 20th, 22nd, 32nd, 35th, 38th, 42nd, 43rd, 45th, 48th, 49th, 61st, 68th, 69th, 74th, 76th, 80th, 81st, 82nd, 84th, 85th, 86th, 87th, 88th, 89th, 91st, 92nd, 94th, 95th, 96th, 125th, 126th and 133rd positions in natural interleukin-2.

[0025] A further aspect of the present invention is an interleukin-2 analog comprising any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3 to 106.

[0026] Yet another aspect of the present invention is an interleukin-2 analog comprising an amino acid sequence represented by general formula 1.

[0027] [General formula 1] X1-PTSSSTKKTQLQLEHL-X18-X19-DL-X22-MILNGINNYKNPKLT-X38-MLT-X42-X43-F-X45-MPKKATELKHLQCLE-X61-ELKPLE-X68-VLNLA- X74-SKNFH-X80-X81-PR-X84-X85-X86-SNIN-X91-X92-V-X94-E-X96-KGSETTFMCEYADETATIVEF-LNRWITFSQSIISTLT (General formula 1, Sequence number 212)

[0028] In general formula 1, X1 is deleted, X18 is leucine (L) or arginine (R), X19 is leucine (L) or tyrosine (Y), X22 is glutamic acid (E) or glutamine (Q), X38 is alanine (A), aspartic acid (D) or arginine (R), and X42 is alanine (A), phenylalanine (F), lysine (K) or tryptophosphate. X43 is ammonium (W), X43 is glutamic acid (E), lysine (K), or glutamine (Q), X45 is alanine (A) or tyrosine (Y), X61 is aspartic acid (D), glutamic acid (E), glutamine (Q), or arginine (R), X68 is aspartic acid (D) or glutamic acid (E), and X74 is histidine (H) or glutamine (Q). X80 is phenylalanine (F), leucine (L), valine (V), or tyrosine (Y); X81 is aspartic acid (D), glutamic acid (E), or arginine (R); X84 is aspartic acid (D) or glutamic acid (E); X85 is alanine (A), glutamic acid (E), glycine (G), leucine (L), valine (V), tryptophan (W), or tyrosine (Y); X86 is alanine (A), glycine (G), isoleucine (I), or valine (V); X91 is threonine (T) or valine (V); X92 is phenylalanine (F), isoleucine (I), or tyrosine (Y); X94 is phenylalanine (F) or leucine (L); and X96 is phenylalanine (F) or leucine (L).

[0029] As a specific example, the interleukin-2 analog is characterized by containing any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 13, 14, 15, 16, 17, 20, 21, 22, 32, 35, 36, 42, 53, 54, 56, 58, 59, 60, 62, 71, 72, 74, 75, 76, 77, 78, 85, 87, 89, 91, 92, 93, 94, 95, 98, 99, 100, 101, 103, 104, 105, and 106.

[0030] As another specific example, in general formula 1, X43 is lysine (K), X45 is tyrosine (Y), X61 is aspartic acid (D), glutamic acid (E), or glutamine (Q), X68 is glutamic acid (E), X74 is glutamine (Q), X80 is phenylalanine (F) or leucine (L), X85 is leucine (L), valine (V), or tyrosine (Y), X86 is isoleucine (I) or valine (V), and X92 is phenylalanine (F) or isoleucine (I).

[0031] An interleukin-2 analog according to any of the above-mentioned specific examples is characterized by containing any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 13, 14, 16, 17, 20, 21, 22, 32, 35, 36, 42, 53, 54, 87, 89, 91, 92, 93, 94, 98, 99, 100, 101, 103, 104, and 105.

[0032] An interleukin-2 analog according to any of the above-mentioned specific examples is characterized by further containing at least one amino acid at its C-terminus.

[0033] Yet another aspect of the present invention is an interleukin-2 analog comprising an amino acid sequence represented by general formula 2.

[0034] [General formula 2] X1-PTSSSTKKTQLQLEHL-X18-LDL-X22-MILNGINNYKNPKLT-X38-MLT-X42-KFYMPKKATELKHLQCLE-X61-ELKPLE-X68-VLNLAQSKNFHF-X81-PRD-X85-X86-SNINVFVLELKGSETTFMCEY-ADETATIVEFLNRWITFSQSI-ISTLT (General formula 2, Sequence ID 213)

[0035] In general formula 2, X1 is deleted, X18 is leucine (L) or arginine (R), X22 is glutamic acid (E) or glutamine (Q), X38 is alanine (A) or arginine (R), X42 is phenylalanine (F) or lysine (K), X61 is aspartic acid (D) or glutamic acid (E), X68 is aspartic acid (D) or glutamic acid (E), X81 is aspartic acid (D) or glutamic acid (E), X85 is leucine (L) or valine (V), and X86 is isoleucine (I) or valine (V).

[0036] As a specific example, the interleukin-2 analog is characterized by containing any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs. 22, 42, 53, 87, 105, and 106.

[0037] As another specific example, the interleukin-2 analog is characterized by further comprising at least one amino acid at its C-terminus. [Effects of the Invention]

[0038] The interleukin-2 analog according to the present invention is an analog with increased binding affinity to the biological interleukin-2β receptor and can be used in a variety of applications. [Brief explanation of the drawing]

[0039] [Figure 1] This figure shows the results of confirming the interleukin-2α receptor binding affinity of interleukin-2 analogs. (A) is interleukin-2 analog #86, (B) is interleukin-2 analog #104, and (C) is interleukin-2 analog #105. [Figure 2] This figure shows the results of confirming the binding affinity of interleukin-2 analogs to the interleukin-2β receptor. (A) is interleukin-2 analog #86, (B) is interleukin-2 analog #104, and (C) is interleukin-2 analog #105. [Modes for carrying out the invention]

[0040] The following describes embodiments for carrying out the present invention. Note that each description and embodiment disclosed herein applies to other descriptions and embodiments. That is, any combination of the various elements disclosed herein is included in the present invention. Furthermore, the present invention is not limited to the following specific descriptions.

[0041] Throughout this specification, the usual one-letter and three-letter codes for amino acids are used. Furthermore, amino acids referred to by abbreviations in this specification are described according to IUPAC-IUB nomenclature. Alanine A Arginine R Asparagine N Aspartic acid D Cysteine ​​C Glutamic acid E Glutamine Q Glycine G Histidine H Isoleucine I Leucine L Lysine K Methionine M Phenylanine F Proline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V

[0042] One aspect of the present invention provides an interleukin-2 analog. The interleukin-2 analog of the present invention is characterized by a modified binding affinity to the interleukin-2 receptor, particularly an increased binding affinity to the interleukin-2β receptor. Specifically, the interleukin-2 analog of the present invention may have an increased binding affinity to the interleukin-2β receptor compared to natural interleukin-2 or known aldesleukins, and more specifically, it may also have a modified (increased or decreased) binding affinity to the interleukin-2α receptor.

[0043] In this invention, "interleukin 2 (IL-2)" refers to an immunomodulator, a type of cytokine that transmits signals in the in vivo immune system. Interleukin 2 is generally known as an important immunostimulant with a molecular weight of approximately 15 kDa.

[0044] In this invention, "interleukin-2 analog" means an interleukin-2 analog in which at least one amino acid has been mutated in the natural sequence. In particular, in this invention, it may be an interleukin-2 analog in which the amino acid of natural interleukin-2 has been mutated, resulting in a decreased or increased binding affinity to the interleukin-2 receptor compared to the natural interleukin-2. Specifically, the interleukin-2 analog of this invention may be non-naturally occurring.

[0045] The aforementioned natural interleukin-2 may also be human interleukin-2, and its sequence can be obtained from a known database or the like. Specifically, it is the amino acid sequence of Sequence ID No. 1, but is not limited to this.

[0046] In this invention, the statement that natural interleukin-2 is the amino acid sequence of SEQ ID NO: 1 means that not only the same sequence as SEQ ID NO: 1, but also sequences with homology of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to SEQ ID NO: 1 are included in the category of natural interleukin-2 of this invention. The amino acid mutation site means that when sequences with homology of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more are aligned with SEQ ID NO: 1, the mutation occurs at the position corresponding to the amino acid sequence of SEQ ID NO: 1.

[0047] In the present invention, a mutation in at least one amino acid in the natural sequence means that at least one amino acid in the natural interleukin 2 has been modified by a change selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

[0048] Specifically, the interleukin-2 analog of the present invention may include a sequence in which at least one amino acid corresponding to the 1st, 12th, 18th, 19th, 20th, 22nd, 32nd, 35th, 38th, 42nd, 43rd, 45th, 48th, 49th, 61st, 68th, 69th, 74th, 76th, 80th, 81st, 82nd, 84th, 85th, 86th, 87th, 88th, 89th, 91st, 92nd, 94th, 95th, 96th, 125th, 126th, and 133rd positions in natural interleukin-2 is mutated. Specifically, the interleukin-2 analog of the present invention may be one in which the first amino acid is deleted from natural interleukin-2, the 125th amino acid is substituted with another amino acid, and further includes one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions. The 125th amino acid, cysteine, is replaced by serine, but this is not the only amino acid that may undergo further substitution. These include, but are not limited to, the amino acids corresponding to the 12th, 18th, 19th, 20th, 22nd, 32nd, 35th, 38th, 42nd, 43rd, 45th, 48th, 49th, 61st, 68th, 69th, 74th, 76th, 80th, 81st, 82nd, 84th, 85th, 86th, 87th, 88th, 89th, 91st, 92nd, 94th, 95th, 96th, 126th, and 133rd positions.

[0049] Furthermore, the present invention also includes interleukin-2 analogs that, in addition to the mutation sites mentioned above, involve substitutions, additions, deletions, or modifications of amino acid residues to an extent known in the art for the purpose of improving the stability and extending the half-life of peptides.

[0050] In this invention, "aldesleukin" or "aldesleukin interleukin 2 analog" refers to aldesleukin (trademark name: Proleukin®), a commercially available interleukin 2 analog, and specifically refers to one having the amino acid sequence of SEQ ID NO: 2. In this invention, it is used interchangeably with "interleukin 2 analog 1". The interleukin analog according to the present invention may have altered interleukin 2α receptor binding affinity and / or increased interleukin 2β receptor binding affinity compared to interleukin 2 analog 1.

[0051] Although the interleukin 2α receptor is known not to be involved in the interleukin 2 signaling system, it increases the binding affinity of other interleukin 2 receptors (β or γ) to interleukin 2 by 10 to 100 times, and CD4 + It is expressed in regulatory T cells, etc.

[0052] Interleukin-2β receptors are CD8 + Interleukin-2β receptors are primarily distributed in T cells or natural killer cells (NK cells) and their main function is to activate immune responses and macrophage activity. Therefore, activation of interleukin-2β receptors is expected to stimulate tumor apoptosis and the body's immune response.

[0053] Therefore, the interleukin-2 analog of the present invention, with its increased binding affinity to the interleukin-2β receptor, enhances therapeutic effects such as tumor suppression and tumor death, while reducing side effects.

[0054] The interleukin-2 analog in the present invention may include a sequence in which the first amino acid is deleted and the 125th amino acid is substituted with another amino acid, or it may further include 1 to 10 amino acid mutations. For example, any interleukin-2 analog may include, but is not limited to, a sequence in which the 125th amino acid is substituted with serine, and at least one of the 12th, 18th, 19th, 20th, 22nd, 32nd, 35th, 38th, 42nd, 43rd, 45th, 48th, 49th, 61st, 68th, 69th, 74th, 76th, 80th, 81st, 82nd, 84th, 85th, 86th, 87th, 88th, 89th, 91st, 92nd, 94th, 95th, 96th and 126th amino acids are further substituted with other amino acids, and / or at least one amino acid is added to the 133rd amino acid.

[0055] As an example, the interleukin-2 analog is obtained by adding at least one amino acid to the amino acid corresponding to the 133rd position, but is not limited thereto. For the purposes of the present invention, the type and length of the added amino acid are not limited, as long as it alters the interleukin-2α receptor binding affinity and increases the interleukin-2β receptor binding affinity compared to natural interleukin-2 or aldesleukin. In addition to natural amino acids, non-natural amino acids and chemically modified amino acids may also be added.

[0056] As another example, the interleukin-2 analogs are, but are not limited to, those in which the first amino acid of natural interleukin-2 is deleted, the 125th amino acid is replaced with another amino acid, and one, two, three, four, five, six, seven, eight, nine or more amino acids from the 18th, 19th, 20th, 22nd, 38th, 42nd, 43rd, 45th, 61st, 68th, 69th, 74th, 80th, 81st, 84th, 85th, 86th, 88th, 89th, 91st, 92nd, 94th, and 96th amino acids are replaced with other amino acids.

[0057] As yet another example, the interleukin-2 analogs are, but are not limited to, those in which the first amino acid of natural interleukin-2 is deleted, the 125th amino acid is replaced with another amino acid, and one, two, three, four, five, six, seven, eight, nine or more amino acids from the 18th, 19th, 22nd, 38th, 42nd, 43rd, 45th, 61st, 68th, 74th, 80th, 81st, 84th, 85th, 86th, 88th, 91st, 92nd, 94th, and 96th amino acids are further replaced with other amino acids.

[0058] As yet another example, the interleukin-2 analog may be selected from the following group of analogs. (a) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 32nd amino acids are substituted with other amino acids. (b) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 35th amino acids are substituted with other amino acids. (c) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 38th amino acids are substituted with other amino acids. (d) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 42nd amino acids are substituted with other amino acids. (e) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 43rd amino acids are substituted with other amino acids. (f) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 48th amino acids are substituted with other amino acids. (g) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 49th amino acids are substituted with other amino acids. (h) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 76th amino acids are substituted with other amino acids. (i) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, 92nd, 94th and 96th amino acids are substituted with other amino acids. (j) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 87th amino acids are substituted with other amino acids. (k) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, and 42nd amino acids are substituted with other amino acids. (l) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, and 80th amino acids are substituted with other amino acids. (m) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, and 84th amino acids are substituted with other amino acids. (n) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 19th, 38th, and 42nd amino acids are substituted with other amino acids. (o) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 12th, 38th, and 42nd amino acids are substituted with other amino acids. (p) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 61st amino acids are substituted with other amino acids. (q) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 84th amino acids are substituted with other amino acids. (r) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 88th amino acids are substituted with other amino acids. (s) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 89th amino acids are substituted with other amino acids. (t) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 91st amino acids are substituted with other amino acids. (u) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 94th amino acids are substituted with other amino acids. (v) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 126th amino acids are substituted with other amino acids. (w) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, and 84th amino acids are substituted with other amino acids. (x) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 94th, and 96th amino acids are substituted with other amino acids. (y) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 81st, and 92nd amino acids are substituted with other amino acids. (z) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 61st, 81st, and 92nd amino acids are substituted with other amino acids. (aa) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, and 92nd amino acids are substituted with other amino acids. (ab) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, and 92nd amino acids are substituted with other amino acids. (ac) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 81st, 84th, and 92nd amino acids are substituted with other amino acids. (ad) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 20th, 38th, 42nd, 81st, and 92nd amino acids are substituted with other amino acids. (ae) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, and 92nd amino acids are substituted with other amino acids. (af) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 74th, 81st and 92nd amino acids are substituted with other amino acids. (ag) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, 84th, and 92nd amino acids are substituted with other amino acids. (ah) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, 88th, and 92nd amino acids are substituted with other amino acids. (ai) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (aj) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, 85th, and 92nd amino acids are substituted with other amino acids. (ak) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, 86th, and 92nd amino acids are substituted with other amino acids. (al) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (am) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 45th, 74th, 81st, and 92nd amino acids are substituted with other amino acids. (an) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 74th, 80th, 81st and 92nd amino acids are substituted with other amino acids. (ao) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 84th, and 92nd amino acids are substituted with other amino acids. (ap) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 45th, 80th, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (aq) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (ar) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 42nd, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (as) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 61st, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (at) Interleukin-2 analogs in which the first amino acid is deleted and the 125th, 69th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (au) Interleukin-2 analogs in which the first amino acid is deleted from natural interleukin-2 and the 125th, 80th, 81st, 85th, 86th, 91st, and 92nd amino acids are substituted with other amino acids. (av) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (aw) Interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (ax) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 45th, 74th, 80th, 81st and 92nd amino acids are substituted with other amino acids. (ay) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 68th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (az) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and amino acids 125, 69, 74, 80, 81, 85, 86, and 92 are substituted with other amino acids. (ba) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 84th, 85th, 86th, 91st and 92nd amino acids are substituted with other amino acids. (bb) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 85th, 86th, 92nd, 94th, and 96th amino acids are substituted with other amino acids. (bc) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 19th, 22nd, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bd) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 38th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (be) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 61st, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (bf) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 68th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bg) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and amino acids 125, 35, 38, 42, 80, 81, 85, 86 and 92 are substituted with other amino acids. (bh) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 45th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bi) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st and 92nd amino acids are substituted with other amino acids. (bj) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 92nd and 95th amino acids are substituted with other amino acids. (bk) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bl) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 43rd, 61st, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bm) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st, 92nd and 95th amino acids are substituted with other amino acids. (bn) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 82nd, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bo) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 86th, and 92nd amino acids are substituted with other amino acids. (bp) Interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 85th, and 86th amino acids are substituted with other amino acids.

[0059] Here, the amino acid substitutions included in the interleukin-2 analog may be at least one selected from the group consisting of the following amino acid substitutions. (a) Substitution of the 12th amino acid with valine or phenylalanine (b) Substitution of the 18th amino acid with arginine (c) Substitution of the 19th amino acid with tyrosine, valine, phenylalanine, or arginine. (d) Substitution of the 20th amino acid with valine or phenylalanine (e) Substitution of the 22nd amino acid with glutamic acid (f) Substitution of the 32nd amino acid with cysteine (g) Substitution of the 35th amino acid with cysteine ​​or glutamic acid (h) Substitution of the 38th amino acid with alanine or aspartic acid (i) Substitution of the 42nd amino acid with lysine, alanine, or tryptophan (j) Substitution of the 43rd amino acid with cysteine, glutamic acid, or glutamine (k) Substitution of the 45th amino acid with alanine (l) Substitution of the 48th amino acid with cysteine (m) Substitution of the 49th amino acid with cysteine (n) Substitution of the 61st amino acid with glutamine, arginine, or aspartic acid (o) Substitution of the 68th amino acid with aspartic acid or glutamine (p) Substitution of the 69th amino acid with glycine (q) Substitution of the 74th amino acid with histidine or alanine (r) Substitution of the 76th amino acid with cysteine (s) Substitution of the 80th amino acid with phenylalanine, tyrosine, valine, aspartic acid, or tryptophan (t) Substitution of the 81st amino acid with aspartic acid, glutamic acid, or asparagine (u) Substitution of the 82nd amino acid with glycine or valine (v) Substitution of the 84th amino acid with glutamic acid, valine, or phenylalanine (w) Substitution of the 85th amino acid with valine, alanine, glycine, tryptophan, tyrosine, threonine, isoleucine, glutamic acid, or phenylalanine. (x) Substitution of the 86th amino acid with valine, alanine, glycine, or leucine (y) Substitution of the 87th amino acid with cysteine (z) Substitution of the 88th amino acid with glutamine, valine, or phenylalanine (aa) Substitution of amino acid 89 with phenylalanine (ab) Substitution of the 91st amino acid with threonine, phenylalanine, or glutamic acid (ac) Substitution of the 92nd amino acid with phenylalanine, leucine, tyrosine, or tryptophan (ad) Substitution of the 95th amino acid with aspartic acid (ae) Substitution of the 96th amino acid with phenylalanine, valine, or isoleucine (af) Substitution of the 126th amino acid with threonine

[0060] In this specification, "corresponding to" means an amino acid residue at a position listed in the peptide, or an amino acid residue that is similar to, identical to, or equivalent to a residue listed in the peptide. Identifying the amino acid at the corresponding position will determine the specific amino acid in the sequence referencing a particular sequence.

[0061] For example, by aligning any amino acid sequence with Sequence ID No. 1, each amino acid residue in the sequence can be numbered based on the alignment, referring to the number and position of the amino acid residues corresponding to the amino acid residues in Sequence ID No. 1.

[0062] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Literature 1) and the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Literature 2) can be used, but are not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the art can be used as appropriate.

[0063] In this invention, even if a specific position of an amino acid in a peptide is expressed, it may also refer to the corresponding position in the reference sequence.

[0064] As yet another example, the interleukin-2 analog may include, but is not limited to, an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 106, or be essentially composed of, or consist of, the aforementioned amino acid sequence.

[0065] Furthermore, even if the Specified Specification refers to an "interleukin-2 analog consisting of a specific sequence number," it does not exclude meaningless sequence additions before or after the amino acid sequence of the said sequence number, spontaneously occurring mutations, or silent mutations thereof, as long as they have the same or equivalent activity as the interleukin-2 analog consisting of the amino acid sequence of the said sequence number. It goes without saying that such analogs with sequence additions or mutations are also included in this application.

[0066] The interleukin-2 analog of the present invention may include, but is not limited to, amino acid sequences having 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the amino acid sequences of SEQ ID NOs.

[0067] In this invention, "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are related to each other, and can be expressed as a percentage.

[0068] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantially, homologous or identical sequences can generally hybridize whole or in part with each other under moderate to high stringent conditions. Hybridization includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy.

[0069] Homology and identity are often used interchangeably.

[0070] Whether any two nucleotide or peptide sequences are homologous, similar, or identical can be determined using default parameters, such as those in Non-Patent Document 3, and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 1), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, homology, similarity, or identity can be determined using BLAST or Clustal W from the National Center for Biotechnology Information.

[0071] Homology, similarity, or identity of nucleotide sequences or peptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 9, as disclosed in Non-Patent Document 8, for example. In summary, the GAP program is defined as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of two sequences. Default parameters for the GAP program may include (1) a unary comparison matrix (where identity is 1 and non-identity is 0) and a weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 10, as in Non-Patent Document 11; (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extended penalty of 0.5); and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in this invention refers to the relevance between sequences.

[0072] The above provisions apply to other specific examples or embodiments of the present invention, but are not limited thereto.

[0073] The interleukin-2 analog of the present invention is used as a novel interleukin-2 substitute that alters the in vitro activity of interleukin-2 by weakening or increasing its binding affinity to α and / or β receptors. In particular, because it not only increases the binding affinity to β receptors but also alters (increases or decreases) the binding affinity to α receptors, it can be used as an effective therapeutic agent due to its activity on both receptors.

[0074] Such modifications for the production of interleukin-2 analogs in the present invention include modifications using L-type or D-type amino acids and / or unnatural amino acids, and / or modifications of the natural sequence, such as modifications of the side chain functional group, intramolecular covalent bonds, for example, inter-side chain ring formation, methylation, acylation, ubiquitination, phosphorylation, aminohexanolysis, biotinylation, etc.

[0075] Furthermore, it includes all forms of natural interleukin-2 in which at least one amino acid is added to the N and / or C-terminus.

[0076] As mentioned above, the amino acids that can be substituted or added include not only the 20 amino acids commonly found in human proteins, but also abnormal or non-natural amino acids. Suppliers of abnormal amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing these amino acids, as well as typical peptide sequences, can be synthesized and purchased from private peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in South Korea.

[0077] Amino acid derivatives can also be obtained using the same method, one example being 4-imidazoacetic acid.

[0078] Furthermore, the interleukin-2 analog according to the present invention may be in a form in which its N-terminus and / or C-terminus are chemically modified, protected by an organic group, or modified by adding amino acids to the peptide terminus, in order to protect it from protein-cleaving enzymes in living organisms and improve its stability.

[0079] In particular, in the case of chemically synthesized peptides, the N-terminus and C-terminus are charged, so acetylation of the N-terminus and / or amidation of the C-terminus are performed to remove the charge, but the process is not limited to these methods.

[0080] Furthermore, since the interleukin-2 analog according to the present invention is in the form of a peptide, it includes the peptide itself, its salt (for example, a pharmaceutically acceptable salt of the peptide), or its solvate. Moreover, the peptide may be in any form as long as it is pharmaceutically acceptable.

[0081] The type of salt is not particularly limited. However, it is preferable that it be in a form that is safe and effective for individuals, such as mammals, but it is not particularly limited to these.

[0082] The term "pharmaceutically acceptable" means a substance that can be used effectively for its desired purpose without inducing excessive toxicity, irritation, or allergic reactions, within the bounds of pharmaceutical judgment.

[0083] In this invention, "pharmaceutically acceptable salts" include salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromate, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Salts derived from suitable bases include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0084] Furthermore, in this invention, "solvate" means a peptide or salt thereof that has formed a complex with a solvent molecule according to the present invention.

[0085] In the present invention, the binding affinity of any interleukin-2 analog to the natural interleukin-2 receptor can be measured using various known techniques by methods that measure affinity to the receptor. For example, surface plasmon resonance (SPR) can be used, but is not limited thereto.

[0086] Specifically, the interleukin-2 analog of the present invention may have a reduced or increased interleukin-2α receptor binding affinity compared to natural interleukin-2 or aldesleukin.

[0087] More specifically, the interleukin-2 analog of the present invention has an interleukin-2α receptor binding affinity of approximately 0.001 times or more, approximately 0.005 times or more, approximately 0.01 times or more, approximately 0.05 times or more, approximately 0.1 times or more, approximately 0.3 times or more, approximately 0.5 times or more, approximately 0.7 times or more, approximately 0.9 times or more, approximately 1.1 times or more, approximately 1.3 times or more, approximately 1.5 times or more, and approximately 1.7 times or more compared to the interleukin-2α receptor binding affinity of natural interleukin-2 or aldesleukin. However, these values ​​are not limited, and any analog with a modified binding affinity compared to natural interleukin-2 or aldesleukin is included in the present invention.

[0088] Alternatively, using the interleukin-2α receptor binding affinity of aldethleukin (100%) as a baseline, the interleukin-2 analogs of the present invention may have completely lost their binding affinity, or possess a binding affinity of approximately 1% or more, approximately 5% or more, approximately 7% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 30% or more, approximately 50% or more, approximately 70% or more, approximately 90% or more, approximately 100% or more, approximately 150% or more, or approximately 200% or more. However, these values ​​are not limited, and any compound whose binding affinity is modified compared to natural interleukin-2 or aldethleukin is included in the present invention.

[0089] Specifically, the interleukin-2 analog of the present invention has an interleukin-2β receptor binding affinity of approximately 0.1 times, 0.3 times, 0.5 times, 0.7 times, 1.0 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times compared to the interleukin-2β receptor binding affinity of natural interleukin-2 or aldesleukin. However, these values ​​are not limited, and any substance whose binding affinity is altered or increased compared to natural interleukin-2 or aldesleukin is included in the present invention.

[0090] Alternatively, using the interleukin 2β receptor binding affinity of aldesleukin (100%) as a baseline, the interleukin 2 analogs of the present invention have binding affinity of approximately 5% or more, approximately 9% or more, approximately 10% or more, approximately 20% or more, approximately 30% or more, approximately 50% or more, approximately 100% or more, approximately 200% or more, approximately 500% or more, approximately 700% or more, approximately 1000% or more, approximately 1500% or more, approximately 3000% or more, approximately 5000% or more, approximately 7000% or more, approximately 10000% or more, approximately 12000% or more, approximately 15000% or more, approximately 20000% or more, and approximately 25000% or more. However, these values ​​are not limited, and any compound with increased binding affinity compared to aldesleukin is included in the present invention.

[0091] In this invention, "approximately" includes a range that encompasses ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all numerical values ​​that are equivalent to or of a similar range to the numerical value following the term "approximately".

[0092] The interleukin-2 analog of the present invention is characterized by a modified binding affinity to the interleukin-2α receptor and an increased binding affinity to the interleukin-2β receptor compared to natural interleukin-2 or aldethleukin.

[0093] In a specific embodiment of the present invention, a mutant interleukin-2 analog was produced based on natural interleukin-2 (SEQ ID NO: 1) to manufacture the interleukin-2 analog of the present invention. The interleukin-2 analog manufactured in the present invention may contain any of the amino acid sequences of SEQ ID NOs: 3 to 106, or may be encoded by any of the nucleotide sequences of SEQ ID NOs: 108 to 211.

[0094] Another aspect of the present invention provides a nucleic acid (polynucleotide) encoding the interleukin-2 analog, a recombinant expression vector containing the nucleic acid, and a transformant containing the nucleic acid or the recombinant expression vector.

[0095] The nucleic acid encoding the interleukin-2 analog of the present invention may be modified to introduce mutations (deletion, substitution, and / or addition of amino acids) at specific positions in the base sequence encoding the natural interleukin-2 of SEQ ID NO: 1, and specifically may include a base sequence encoding any of the amino acid sequences of SEQ ID NOs: 3 to 106. For example, the nucleic acid of the present invention may have or include any of the base sequences of SEQ ID NOs: 108 to 211.

[0096] The base sequences of the present invention can be modified in various ways in the coding region, within the limits that the amino acid sequence of the interleukin-2 analog of the present invention does not change, by codon degeneracy or by taking into consideration codons preferred in organisms that intend to express the nucleic acid of the present invention. Specifically, the nucleic acid of the present invention has, or includes, a base sequence that has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, and 98% or more, but less than 100% homology or identity with any of the sequences of SEQ ID NOs. 108 to 211, or consists of, or is essentially composed of, a base sequence that has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, and 98% or more, but less than 100% homology or identity with any of the sequences of SEQ ID NOs. 108 to 211.

[0097] Furthermore, the nucleic acid of the present invention may be any probe produced from a known gene sequence, for example, any sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the nucleic acid sequence of the present invention. The "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 12 and 13).

[0098] Hybridization requires that the two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the nucleic acids of the present invention may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0099] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (e.g., Non-Patent Document 12).

[0100] The similarity or identity is as described above.

[0101] The recombinant vector according to the present invention may be constructed as a vector for cloning or expression, or as a vector for using prokaryotic or eukaryotic cells as host cells.

[0102] In the present invention, "vector" refers to a recombinant vector that expresses a target protein in a suitable host cell, and means a nucleic acid construct containing essential regulatory factors operably linked to express a nucleic acid insert. According to the present invention, a recombinant vector containing a nucleic acid encoding an interleukin-2 analog can be prepared, and the interleukin-2 analog of the present invention can be obtained by transforming or transfecting host cells with the recombinant vector.

[0103] In this invention, "transformation" refers to the process by which DNA is introduced into a host cell, making it possible to replicate as a chromosomal component or through the completion of chromosome integration. It also means the phenomenon of artificially inducing genetic changes by introducing external DNA into a cell.

[0104] A suitable host for the present invention is not particularly limited as long as it expresses the nucleic acid of the present invention. Examples of specific hosts used in the present invention include Escherichia bacteria such as Escherichia coli, Bacillus bacteria such as Bacillus subtilis, Pseudomonas bacteria such as Pseudomonas putida, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, insect cells such as Sphodoptera fulgiperda (Sf9), and animal cells such as CHO, COS, and BSC.

[0105] Yet another aspect of the present invention provides a method for producing an interleukin-2 analog containing at least one amino acid mutation.

[0106] Specifically, the method may include the step of introducing a mutation into at least one amino acid selected from the group consisting of amino acids corresponding to the 1st, 12th, 18th, 19th, 20th, 22nd, 32nd, 35th, 38th, 42nd, 43rd, 45th, 48th, 49th, 61st, 68th, 69th, 74th, 76th, 80th, 81st, 82nd, 84th, 85th, 86th, 87th, 88th, 89th, 91st, 92nd, 94th, 95th, 96th, 125th, 126th, and 133rd positions in natural interleukin 2.

[0107] More specifically, (a) in natural interleukin-2, the first amino acid is deleted and the 125th and 32nd amino acids are replaced with other amino acids; (b) in natural interleukin-2, the first amino acid is deleted and the 125th and 35th amino acids are replaced with other amino acids; (c) in natural interleukin-2, the first amino acid is deleted and the 125th and 38th amino acids are replaced with other amino acids; (d) in natural interleukin-2, the first amino acid is deleted (e) In natural interleukin-2, the 125th and 42nd amino acids are substituted with other amino acids; (f) In natural interleukin-2, the 1st amino acid is deleted and the 125th and 43rd amino acids are substituted with other amino acids; (g) In natural interleukin-2, the 1st amino acid is deleted and the 125th and 48th amino acids are substituted with other amino acids; (h) Natural (i) Natural interleukin 2, in which the first amino acid is deleted and the 125th and 76th amino acids are replaced with other amino acids; (j) Natural interleukin 2, in which the first amino acid is deleted and the 125th, 38th, 42nd, 81st, 92nd, 94th and 96th amino acids are replaced with other amino acids; (k) Natural interleukin 2, in which the first amino acid is deleted and the 125th and 87th amino acids are replaced with other amino acids; (l) In natural interleukin-2, the first amino acid is deleted and the 125th, 38th, and 42nd amino acids are replaced with other amino acids; (m) In natural interleukin-2, the first amino acid is deleted and the 125th, 38th, and 80th amino acids are replaced with other amino acids; (n) In natural interleukin-2, the first amino acid is deleted and the 125th, 38th, and 84th amino acids are replaced with other amino acids;(o) In natural interleukin-2, the 1st amino acid is deleted and the 125th, 12th, 38th, and 42nd amino acids are replaced with other amino acids; (p) In natural interleukin-2, the 1st amino acid is deleted and the 125th, 38th, 42nd, and 61st amino acids are replaced with other amino acids; (q) In natural interleukin-2, the 1st amino acid is deleted and the 125th, 38th, 42nd, and 84th amino acids are replaced with other amino acids. (r) Natural interleukin 2, in which the 1st amino acid is deleted and the 125th, 38th, 42nd and 88th amino acids are replaced with other amino acids; (s) Natural interleukin 2, in which the 1st amino acid is deleted and the 125th, 38th, 42nd and 89th amino acids are replaced with other amino acids; (t) Natural interleukin 2, in which the 1st amino acid is deleted and the 125th, 38th, 42nd and 91st amino acids are replaced with other amino acids. (u) Natural interleukin-2 in which the first amino acid is deleted and the 125th, 38th, 42nd and 94th amino acids are replaced with other amino acids, (v) Natural interleukin-2 in which the first amino acid is deleted and the 125th, 38th, 42nd and 126th amino acids are replaced with other amino acids, (w) Natural interleukin-2 in which the first amino acid is deleted and the 125th, 38th, 80th and 84th amino acids are replaced with other amino acids, (x) Natural (y) Interleukin 2, in which the first amino acid is deleted and the 125th, 38th, 94th and 96th amino acids are replaced with other amino acids; (z) Natural interleukin 2, in which the first amino acid is deleted and the 125th, 38th, 81st and 92nd amino acids are replaced with other amino acids; (aa) Natural interleukin 2,(ab) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 81st and 92nd amino acids are replaced with other amino acids, (ac) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 80th, 81st and 92nd amino acids are replaced with other amino acids, (ad) In natural interleukin 2 (a) In natural interleukin-2, the first amino acid is deleted and the 125th, 20th, 38th, 42nd, 81st and 92nd amino acids are replaced with other amino acids, or (a) In natural interleukin-2, the first amino acid is deleted and the 125th, 38th, 42nd, 80th, 81st and 92nd amino acids are replaced with other amino acids, or (af) In natural interleukin-2, the first amino acid is deleted and the 125th, 38th, 42nd, 74th, 81st and 92nd amino acids are replaced with other amino acids, (ag) Natural interleukin-2 with the first amino acid deleted and the 125th, 38th, 42nd, 81st, 84th and 92nd amino acids replaced with other amino acids, (ah) Natural interleukin-2 with the first amino acid deleted and the 125th, 38th, 42nd, 81st, 88th and 92nd amino acids replaced with other amino acids, (ai) Natural interleukin-2 with the first amino acid deleted and the 125th, 38th, 42nd, 85th, 86th and 92nd amino acids replaced with other amino acids (aj) In natural interleukin-2, the first amino acid is deleted and the 125th, 38th, 80th, 81st, 85th and 92nd amino acids are replaced with other amino acids; (ak) In natural interleukin-2, the first amino acid is deleted and the 125th, 38th, 80th, 81st, 86th and 92nd amino acids are replaced with other amino acids; (al) In natural interleukin-2, the first amino acid is deleted and the 125th, 80th, 81st, 85th,(am) In natural interleukin 2, the 86th and 92nd amino acids are substituted with other amino acids, (am) In natural interleukin 2, the 1st amino acid is deleted and the 125th, 38th, 42nd, 45th, 74th, 81st and 92nd amino acids are substituted with other amino acids, (an) In natural interleukin 2, the 1st amino acid is deleted and the 125th, 38th, 42nd, 74th, 80th, 81st and 92nd amino acids are substituted with other amino acids, (ao) In natural interleukin 2, the 1st amino (ap) Natural interleukin 2 with the acid deleted and the 125th, 38th, 42nd, 80th, 81st, 84th and 92nd amino acids replaced with other amino acids, or (ap) Natural interleukin 2 with the 1st amino acid deleted and the 125th, 38th, 45th, 80th, 85th, 86th and 92nd amino acids replaced with other amino acids, or (aq) Natural interleukin 2 with the 1st amino acid deleted and the 125th, 38th, 80th, 81st, 85th, 86th and 92nd amino acids replaced with other amino acids (ar) Natural interleukin 2 with the first amino acid deleted and the 125th, 42nd, 80th, 81st, 85th, 86th and 92nd amino acids replaced with other amino acids, (as) Natural interleukin 2 with the first amino acid deleted and the 125th, 61st, 80th, 81st, 85th, 86th and 92nd amino acids replaced with other amino acids, (at) Natural interleukin 2 with the first amino acid deleted and the 125th, 69th, 80th, 81st and 85th amino acids replaced with other amino acids (au) Natural interleukin 2, in which the 1st amino acid is deleted and the 125th, 80th, 81st, 85th, 86th, 91st and 92nd amino acids are replaced with other amino acids; (av) Natural interleukin 2, in which the 1st amino acid is deleted and the 125th, 18th, 22nd, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids; (aw) Natural interleukin 2,(ax) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids, or (ay) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 45th, 74th, 80th, 81st and 92nd amino acids are replaced with other amino acids, or (ay) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 68th, 80th, 81st, 85th and 86th amino acids are replaced with other amino acids (az) In natural interleukin 2, the 1st amino acid is deleted and the 125th, 69th, 74th, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids; (ba) In natural interleukin 2, the 1st amino acid is deleted and the 125th, 80th, 81st, 84th, 85th, 86th, 91st and 92nd amino acids are replaced with other amino acids; (bb) In natural interleukin 2, (bc) In natural interleukin-2, the first amino acid is deleted and the 125th, 80th, 81st, 85th, 86th, 92nd, 94th and 96th amino acids are replaced with other amino acids, or (bd) In natural interleukin-2, the first amino acid is deleted and the 125th, 18th, 19th, 22nd, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids, or (bd) In natural interleukin-2, the first amino acid is deleted and the 125th, 18th, 22nd, 38th, 80th and 81st amino acids are replaced with other amino acids (b) In natural interleukin-2, the 85th, 86th, and 92nd amino acids are substituted with other amino acids, (be) In natural interleukin-2, the 1st amino acid is deleted and the 125th, 18th, 22nd, 61st, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids, (bf) In natural interleukin-2, the 1st amino acid is deleted and the 125th, 18th, 22nd, 68th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids,(bg) Natural interleukin-2 with the first amino acid deleted and the 125th, 35th, 38th, 42nd, 80th, 81st, 85th, 86th and 92nd amino acids substituted with other amino acids, or (bh) Natural interleukin-2 with the first amino acid deleted and the 125th, 38th, 42nd, 45th, 80th, 8, (bi) In natural interleukin-2, the 1st amino acid is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st and 92nd amino acids are replaced with other amino acids; (bj) In natural interleukin-2, the 1st amino acid is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 92nd and 95th amino acids are replaced with other amino acids; (bk) In natural interleukin-2, the 1st amino acid is deleted and the 125th, 35th, 38th, 42nd, 80th, 81st, 85th, 86th, 92nd and 95th amino acids are replaced with other amino acids (bl) Natural interleukin 2 with the first amino acid deleted and the 125th, 38th, 42nd, 43rd, 61st, 80th, 81st, 85th, 86th and 92nd amino acids replaced with other amino acids; (bm) Natural interleukin 2 with the first amino acid deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st, 92nd and 95th amino acids replaced with other amino acids; (bn) Natural interleukin 2 with the first amino acid deleted and the 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 82nd, 85th, 86th and 92nd amino acids replaced with other amino acids. (bo) In natural interleukin-2, the first amino acid is deleted and the 125th, 80th, 81st, 86th, and 92nd amino acids are substituted with other amino acids; or (bp) In natural interleukin-2, the first amino acid is deleted and the 125th, 80th, 81st, 85th, and 86th amino acids are substituted with other amino acids. However, this is not the only example.

[0108] Interleukin-2 analogs and mutations are as previously described.

[0109] Another example of a method for producing the interleukin-2 analog of the present invention is a method for producing the interleukin-2 analog which includes a) a step of culturing a transformant containing nucleic acid encoding the interleukin-2 analog to express the interleukin-2 analog, and b) a step of separating and purifying the expressed interleukin-2 analog. However, any method capable of producing the interleukin-2 analog may be used, and may be a method known in the art.

[0110] In the present invention, the nucleic acid encoding the interleukin-2 analog includes, but is not limited to, any of the base sequences of sequence numbers 108 to 211, or is (essentially) composed of the said base sequences.

[0111] In the present invention, the culture medium used for culturing the transformants must satisfy the requirements for host cell culture in a suitable manner. The carbon source contained in the culture medium for host cell growth is appropriately selected by those skilled in the art depending on the type of transformant to be produced, and suitable culture conditions are employed to adjust the timing and amount of culture.

[0112] Suitable sugar sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerin and ethanol; and organic acids such as acetic acid. These substances can be used individually or in mixtures.

[0113] Suitable nitrogen sources include peptone, yeast extract, meat juice, malt extract, corn maceration, kinako (roasted soybean flour), and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. These nitrogen sources may be used individually or as mixtures.

[0114] Suitable phosphorus sources include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or equivalent sodium-containing salts. The culture medium may also contain metal salts such as magnesium sulfate and iron sulfate, which are necessary for growth.

[0115] Finally, in addition to the substances mentioned above, essential growth substances such as amino acids and vitamins may be used. Furthermore, precursors suitable for the culture medium may be used. The aforementioned raw materials may be added batch by batch or continuously during the culture process in a manner suitable for the culture. The pH of the culture may be adjusted using basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, or acidic compounds such as phosphoric acid and sulfuric acid in a suitable manner. Furthermore, antifoaming agents such as fatty acid polyglycol esters may be used to suppress bubble formation. To maintain an aerobic state, oxygen or oxygen-containing gas (e.g., air) may be injected into the culture.

[0116] The culture of the transformants according to the present invention is typically carried out at a temperature of 20°C to 45°C, specifically 25°C to 40°C. The culture is continued until the maximum amount of the desired interleukin-2 analog is obtained, which is typically for 10 to 160 hours for these purposes.

[0117] As described above, when appropriate culture conditions are set according to the host cell, the transformant according to the present invention produces an interleukin-2 analog, and depending on the vector composition and the characteristics of the host cell, the produced interleukin-2 analog is secreted into the cytoplasm, periplasmic space, or extracellular space of the host cell.

[0118] Proteins expressed inside and outside host cells can be purified by conventional methods. Examples of purification methods include salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (e.g., protein fractionation precipitation using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, chromatography such as reversed-phase column chromatography, and ultrafiltration, which can be used individually or in combination.

[0119] As a specific example of the present invention, a method for producing an interleukin-2 analog may include (a) the step of expressing the interleukin-2 analog, and (b) the step of separating the expressed interleukin-2 analog.

[0120] As a specific example of the present invention, the following steps may be further included for separating and purifying the interleukin-2 analog expressed in the form of inclusion bodies from the transformant. b-1) Step of obtaining and disrupting transformants from the culture medium of step a) above. b-2) Step of recovering the expressed interleukin-2 analog from the disrupted cell lysate and refolding it. b-3) Purification of the refolded interleukin-2 analog by size exclusion chromatography.

[0121] A further aspect of the present invention provides a method for producing the interleukin-2 analog by peptide synthesis. Such peptide synthesis can be carried out without limitation by using known peptide synthesis methods, since the interleukin-2 analog sequence of the present invention is provided.

[0122] Interleukin-2 analogs and mutations are as previously described.

[0123] Yet another aspect of the present invention is a method for increasing interleukin-2β receptor binding affinity, comprising the step of mutating at least one amino acid in natural interleukin-2.

[0124] The method for increasing interleukin-2β receptor binding affinity according to the present invention may increase the binding affinity to the interleukin-2β receptor and alter the binding affinity to the interleukin-2α receptor compared to natural interleukin-2 or aldesleukin.

[0125] Specifically, the method may include the step of introducing a mutation into at least one amino acid corresponding to the 1st, 12th, 18th, 19th, 20th, 22nd, 32nd, 35th, 38th, 42nd, 43rd, 45th, 48th, 49th, 61st, 68th, 69th, 74th, 76th, 80th, 81st, 82nd, 84th, 85th, 86th, 87th, 88th, 89th, 91st, 92nd, 94th, 95th, 96th, 125th, 126th, and 133rd positions in natural interleukin 2.

[0126] More specifically, the method includes (a) deleting the first amino acid in natural interleukin 2 and substituting the 125th and 32nd amino acids with other amino acids, (b) deleting the first amino acid in natural interleukin 2 and substituting the 125th and 35th amino acids with other amino acids, (c) deleting the first amino acid in natural interleukin 2 and substituting the 125th and 38th amino acids with other amino acids, and (d) deleting the first amino acid in natural interleukin 2 and substituting the 12 (e) In natural interleukin 2, the steps are: (f) In natural interleukin 2, the steps are: (g) In natural interleukin 2, the steps are: (h) In natural interleukin 2, the steps are: (g) In natural interleukin 2, the steps are: (i) In natural interleukin 2, the steps are: (g) In natural interleukin 2, the steps are: (h) In natural interleukin 2, the steps are: (i) In natural interleukin 2, the steps are: (g) In natural interleukin 2, the steps are: (i (i) In natural interleukin 2, the first amino acid is deleted and the 125th and 76th amino acids are replaced with other amino acids; (j) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 81st, 92nd, 94th and 96th amino acids are replaced with other amino acids; (k) In natural interleukin 2, the first amino acid is deleted and the 125th and 38th amino acids are replaced with other amino acids. (l) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, and 80th amino acids are replaced with other amino acids; (m) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, and 84th amino acids are replaced with other amino acids; (n) In natural interleukin 2, the first amino acid is deleted and the 125th, 19th, 38th, and 42nd amino acids are replaced with other amino acids.(o) In natural interleukin 2, the first amino acid is deleted and the 125th, 12th, 38th and 42nd amino acids are replaced with other amino acids; (p) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd and 61st amino acids are replaced with other amino acids; (q) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd and 84th amino acids are replaced with other amino acids; (r) In natural interleukin 2, 1 (s) In natural interleukin 2, delete the nth amino acid and replace the 125th, 38th, 42nd and 88th amino acids with other amino acids; (t) In natural interleukin 2, delete the nth amino acid and replace the 125th, 38th, 42nd and 89th amino acids with other amino acids; (u) In natural interleukin 2, delete the nth amino acid and replace the 125th, 38th, 42nd and 91st amino acids with other amino acids; (v) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, and 126th amino acids are replaced with other amino acids; (w) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 80th, and 84th amino acids are replaced with other amino acids; (x) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 94th, and 96th amino acids are replaced with other amino acids. (y) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 81st and 92nd amino acids are replaced with other amino acids; (z) In natural interleukin 2, the first amino acid is deleted and the 125th, 61st, 81st and 92nd amino acids are replaced with other amino acids; (aa) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 81st and 92nd amino acids are replaced with other amino acids.(ab) A step in which the first amino acid is deleted in natural interleukin-2 and the 125th, 38th, 80th, 81st and 92nd amino acids are replaced with other amino acids; (ac) A step in which the first amino acid is deleted in natural interleukin-2 and the 125th, 38th, 81st, 84th and 92nd amino acids are replaced with other amino acids; (ad) A step in which the first amino acid is deleted in natural interleukin-2 and the 125th, 20th, 38th, 42nd, 81st and 92nd amino acids are replaced with other amino acids. (a) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 80th, 81st and 92nd amino acids are replaced with other amino acids. (af) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 74th, 81st and 92nd amino acids are replaced with other amino acids. (ag) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 81st, 84th and 92nd amino acids are replaced with other amino acids. Steps to substitute with amino acids, (ah) In natural interleukin 2, delete the first amino acid and replace the 125th, 38th, 42nd, 81st, 88th and 92nd amino acids with other amino acids, (ai) In natural interleukin 2, delete the first amino acid and replace the 125th, 38th, 42nd, 85th, 86th and 92nd amino acids with other amino acids, (aj) In natural interleukin 2, delete the first amino acid and replace the 125th, 38th, 80th, 81st, 85th and Steps to replace the 92nd amino acid with another amino acid, (ak) In natural interleukin 2, delete the 1st amino acid and replace the 125th, 38th, 80th, 81st, 86th and 92nd amino acids with other amino acids, (al) In natural interleukin 2, delete the 1st amino acid and replace the 125th, 80th, 81st, 85th, 86th and 92nd amino acids with other amino acids, (am) In natural interleukin 2, delete the 1st amino acid and replace the 125th, 38th, 42nd,(an) In natural interleukin 2, the 45th, 74th, 81st and 92nd amino acids are replaced with other amino acids. (a) In natural interleukin 2, the 1st amino acid is deleted and the 125th, 38th, 42nd, 74th, 80th, 81st and 92nd amino acids are replaced with other amino acids. (ao) In natural interleukin 2, the 1st amino acid is deleted and the 125th, 38th, 42nd, 80th, 81st, 84th and 92nd amino acids are replaced with other amino acids. (ap) In natural interleukin 2, the 1st (aq) In natural interleukin 2, delete the first amino acid and replace the 125th, 38th, 45th, 80th, 85th, 86th and 92nd amino acids with other amino acids. (ar) In natural interleukin 2, delete the first amino acid and replace the 125th, 38th, 80th, 81st, 85th, 86th and 92nd amino acids with other amino acids. Steps include: (as) deleting the first amino acid in natural interleukin 2 and substituting the 125th, 61st, 80th, 81st, 85th, 86th and 92nd amino acids with other amino acids; (at) deleting the first amino acid in natural interleukin 2 and substituting the 125th, 69th, 80th, 81st, 85th, 86th and 92nd amino acids with other amino acids; (au) deleting the first amino acid in natural interleukin 2 and substituting the 125th, 80th, 81st, 85th, 86th and 92nd amino acids with other amino acids (av) In natural interleukin 2, the first amino acid is deleted and the 125th, 18th, 22nd, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids; (aw) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids; (ax) In natural interleukin 2,Steps to delete the first amino acid and replace the 125th, 38th, 42nd, 45th, 74th, 80th, 81st and 92nd amino acids with other amino acids (ay) In natural interleukin 2, Steps to delete the first amino acid and replace the 125th, 38th, 68th, 80th, 81st, 85th, 86th and 92nd amino acids with other amino acids (az) In natural interleukin 2, Steps to delete the first amino acid and replace the 125th, 69th, 74th, 80th, 81st, 85th, 86th and 92nd amino acids (ba) In natural interleukin 2, the first amino acid is deleted and the 125th, 80th, 81st, 84th, 85th, 86th, 91st and 92nd amino acids are replaced with other amino acids; (bb) In natural interleukin 2, the first amino acid is deleted and the 125th, 80th, 81st, 85th, 86th, 92nd, 94th and 96th amino acids are replaced with other amino acids; (bc) In natural interleukin 2, the first amino acid is deleted (b) Steps of replacing the 125th, 18th, 19th, 22nd, 80th, 81st, 85th, 86th and 92nd amino acids with other amino acids, (b) Steps of deleting the 1st amino acid in natural interleukin 2 and replacing the 125th, 18th, 22nd, 38th, 80th, 81st, 85th, 86th and 92nd amino acids with other amino acids, (be) Steps of deleting the 1st amino acid in natural interleukin 2 and replacing the 125th, 18th, 22nd, 61st, 80th, 81st, 85th, 86th and 9 (bf) In natural interleukin 2, the first amino acid is deleted and the 125th, 18th, 22nd, 68th, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids. (bg) In natural interleukin 2, the first amino acid is deleted and the 125th, 35th, 38th, 42nd, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids. (bh) In natural interleukin 2,(bi) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 45th, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids. (bj) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 92nd and 95th amino acids are replaced with other amino acids; (bk) In natural interleukin 2, the first amino acid is deleted and the 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids; (bl) In natural interleukin 2, the first amino acid is deleted and the 125th, 38th, 38th, 42nd, 74th, 80th, 81st, 85th, 86th and 92nd amino acids are replaced with other amino acids. (bm) In natural interleukin 2, the steps of: (bm) Deleting the first amino acid and replacing the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st, 92nd, and 95th amino acids with other amino acids; (bn) In natural interleukin 2, the steps of: Deleting the first amino acid and replacing the 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 82nd, 85th, 86th, and 92nd amino acids with other amino acids (bo) In natural interleukin-2, the first amino acid is deleted and the 125th, 80th, 81st, 86th and 92nd amino acids are replaced with other amino acids; or (bp) In natural interleukin-2, the first amino acid is deleted and the 125th, 80th, 81st, 85th and 86th amino acids are replaced with other amino acids. This includes, but is not limited to, the following.

[0127] Interleukin-2 analogs and mutations are as previously described.

[0128] A further aspect of the present invention provides an interleukin-2 analog comprising any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3 to 106.

[0129] The definitions of interleukin-2 analogs, variants, and analogs represented by sequence numbers are as described above.

[0130] Specifically, the interleukin-2 analog includes, but is not limited to, any sequence selected from the group consisting of amino acid sequences SEQ ID NOs: 3 to 106, or is essentially composed of, or consists of, the aforementioned sequences.

[0131] Yet another aspect of the present invention provides an interleukin-2 analog comprising an amino acid sequence represented by general formula 1.

[0132] [General formula 1] X1-PTSSSTKKTQLQLEHL-X18-X19-DL-X22-MILNGINNYKNPKLT-X38-MLT-X42-X43-F-X45-MPKKATELKHLQCLE-X61-ELKPLE-X68-VLNLA- X74-SKNFH-X80-X81-PR-X84-X85-X86-SNIN-X91-X92-V-X94-E-X96-KGSETTFMCEYADETATIVEF-LNRWITFSQSIISTLT (General formula 1, Sequence number 212)

[0133] In general formula 1, X1 is deleted, X18 is leucine (L) or arginine (R), X19 is leucine (L) or tyrosine (Y), X22 is glutamic acid (E) or glutamine (Q), X38 is alanine (A), aspartic acid (D) or arginine (R), and X42 is alanine (A), phenylalanine (F), lysine (K) or tryptophosphate. X43 is ammonium (W), X43 is glutamic acid (E), lysine (K), or glutamine (Q), X45 is alanine (A) or tyrosine (Y), X61 is aspartic acid (D), glutamic acid (E), glutamine (Q), or arginine (R), X68 is aspartic acid (D) or glutamic acid (E), and X74 is histidine (H) or glutamine (Q). X80 is phenylalanine (F), leucine (L), valine (V), or tyrosine (Y); X81 is aspartic acid (D), glutamic acid (E), or arginine (R); X84 is aspartic acid (D) or glutamic acid (E); X85 is alanine (A), glutamic acid (E), glycine (G), leucine (L), valine (V), tryptophan (W), or tyrosine (Y); X86 is alanine (A), glycine (G), isoleucine (I), or valine (V); X91 is threonine (T) or valine (V); X92 is phenylalanine (F), isoleucine (I), or tyrosine (Y); X94 is phenylalanine (F) or leucine (L); and X96 is phenylalanine (F) or leucine (L).

[0134] Furthermore, at least one amino acid may be added to the threonine (T) corresponding to X133 in general formula 1, but this is not limited to that.

[0135] Specifically, the interleukin-2 analog contains, or is required to consist of, any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 13, 14, 15, 16, 17, 20, 21, 22, 32, 35, 36, 42, 53, 54, 56, 58, 59, 60, 62, 71, 72, 74, 75, 76, 77, 78, 85, 87, 89, 91, 92, 93, 94, 95, 98, 99, 100, 101, 103, 104, 105, and 106, or is composed of, but is not limited to, the aforementioned sequences.

[0136] Such interleukin-2 analogs have increased β-receptor binding affinity compared to aldesleukin or natural interleukin-2, but are not limited to these.

[0137] As another example, the interleukin-2 analog of the present invention is, in general formula 1, X43 is lysine (K), X45 is tyrosine (Y), X61 is aspartic acid (D), glutamic acid (E), or glutamine (Q), X68 is glutamic acid (E), X74 is glutamine (Q), X80 is phenylalanine (F) or leucine (L), X85 is leucine (L), valine (V), or tyrosine (Y), X86 is isoleucine (I) or valine (V), and X92 is phenylalanine (F) or isoleucine (I), but is not limited to these.

[0138] Specifically, the interleukin-2 analog is characterized by containing any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 13, 14, 16, 17, 20, 21, 22, 32, 35, 36, 42, 53, 54, 87, 89, 91, 92, 93, 94, 98, 99, 100, 101, 103, 104, and 105.

[0139] The interleukin-2 analog of the present invention further comprises, but is not limited to, at least one amino acid at its C-terminus.

[0140] Yet another aspect of the present invention provides an interleukin-2 analog comprising an amino acid sequence represented by general formula 2.

[0141] [General formula 2] X1-PTSSSTKKTQLQLEHL-X18-LDL-X22-MILNGINNYKNPKLT-X38-MLT-X42-KFYMPKKATELKHLQCLE-X61-ELKPLE-X68-VLNLAQSKNFHF-X81-PRD-X85-X86-SNINVFVLELKGSETTFMCEY-ADETATIVEFLNRWITFSQSI-ISTLT (General formula 2, Sequence ID 213)

[0142] In general formula 2, X1 is deleted, X18 is leucine (L) or arginine (R), X22 is glutamic acid (E) or glutamine (Q), X38 is alanine (A) or arginine (R), X42 is phenylalanine (F) or lysine (K), X61 is aspartic acid (D) or glutamic acid (E), X68 is aspartic acid (D) or glutamic acid (E), X81 is aspartic acid (D) or glutamic acid (E), X85 is leucine (L) or valine (V), and X86 is isoleucine (I) or valine (V).

[0143] Specifically, the interleukin-2 analog contains, but is not limited to, any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs. 22, 42, 53, 87, 105, and 106.

[0144] Furthermore, the interleukin-2 analog may have, but is not limited to, a threonine (T) corresponding to X133 in general formula 2 with at least one amino acid added, or the C-terminus of the interleukin-2 analog may contain at least one additional amino acid.

[0145] In this specification, unless otherwise specified, expressions such as "includes," "possesses," and "contains" should be understood to mean that they include the specified integer or set of integers, but do not exclude other integers or sets of integers.

[0146] The present invention will be described in more detail below with reference to examples. These examples are merely illustrative of the present invention, and the present invention is not limited to these examples. [Examples]

[0147] Preparation of expression vectors for natural interleukin-2 and interleukin-2 analogs To create a natural interleukin-2 expression vector encoding 133 amino acids, interleukin-2 synthesized based on the reported interleukin-2 sequence (NM_000586.3; SEQ ID NO: 1) was cloned into the pET-22b vector (Novagen). Furthermore, novel interleukin-2 analogs were created using the aforementioned interleukin-2 as a template, with modified amino acids. The PCR conditions for amplifying the interleukin-2 analogs were 95°C for 30 seconds, 55°C for 60 seconds, and 65°C for 6.5 minutes, and this process was repeated 16 times. To determine whether the amino acids at the desired sites were successfully modified, sequence analysis was performed on the mutagenesis products obtained under the above conditions. It was confirmed that each interleukin-2 analog had the mutations shown in Table 1, relative to the natural product, at the target mutation site. The expression vectors thus obtained were named pET22b-interleukin-2 analogs 1-105.

[0148] Table 1 shows the modified sequences and analog names for each amino acid. To prepare these interleukin-2 analogs, forward (F) and reverse (R) primers were synthesized, and then PCR was performed to amplify each analog gene.

[0149] In Table 1, Analog 1 is aldesleukin, and Primers #1 to #204 correspond to Sequence IDs 214 to 417 as specified herein.

[0150] [Table 1] JPEG0007846627000002.jpg206150 JPEG0007846627000003.jpg207150 JPEG0007846627000004.jpg207150 JPEG0007846627000005.jpg207150 JPEG0007846627000006.jpg207150 JPEG0007846627000007.jpg207150 JPEG0007846627000008.jpg207150 JPEG0007846627000009.jpg207150 JPEG0007846627000010.jpg207150

[0151] desA means that the first amino acid of interleukin-2, alanine, has been deleted.

[0152] Table 2 shows the full-length protein sequences of interleukin-2 analogs. Bold text in Table 2 indicates the mutation site.

[0153] [Table 2] JPEG0007846627000012.jpg207150 JPEG0007846627000013.jpg207150 JPEG0007846627000014.jpg207150 JPEG0007846627000015.jpg207150 JPEG0007846627000016.jpg207150 JPEG0007846627000017.jpg207150 JPEG0007846627000018.jpg207150 JPEG0007846627000019.jpg171150 [Examples]

[0154] Expression of interleukin-2 analogs Recombinant interleukin-2 analogs were expressed under T7 promoter control using the expression vectors prepared in Example 1. Each recombinant interleukin-2 analog expression vector was transformed with the expression strain E. coli BL21DE3 (E. coli B F-dcm ompT hsdS(rB-mB-) gal λ(DE3); Novagen). The transformation method used was the one recommended by Novagen. Single colonies were obtained from each recombinant expression vector and inoculated into 2× Luria Broth medium containing ampicillin (50 μg / ml) and cultured at 37°C for 15 hours. The culture medium of the recombinant strain and 2× LB medium containing 30% glycerin were mixed in a 1:1 (v / v) ratio and dispensed into 1 mL each of cryo tubes, which were stored at -150°C. These were used as cell stocks for the production of recombinant proteins.

[0155] To express recombinant interleukin-2 analogs, one vial of each cell stock was lysed and inoculated into 500 ml of 2 × LB medium, and cultured with shaking at 37°C for 14-16 hours. When the absorbance value at 600 nm reached 4.0 or higher, the culture was terminated and used as the seed culture. Initial fermentation was started by inoculating the seed culture into 1.6 L of fermentation medium using a 5 L fermenter (Bioflo-320, NBS, USA). The culture conditions were 37°C, 2.0 L / min (1 vvm) of air, and 650 rpm of stirring, and the pH was maintained at 6.70 using 30% aqueous ammonia. Fermentation was carried out by fed-batch culture, with additional medium (feeding solution) added when the nutrients in the culture medium were depleted. Strain growth was observed by absorbance, and when the absorbance value reached 70 or higher, a final concentration of 500 μM IPTG was introduced. The culture was continued for approximately 23-25 ​​hours after the introduction of IPTG. After the culture was completed, recombinant strains were obtained using a centrifuge and stored at -80°C until use. [Examples]

[0156] Extraction and refolding of interleukin-2 analogs To convert the interleukin-2 analog from the interleukin-2 analog-expressing E. coli obtained in Example 2 into a soluble form, the cells were lysed and refolded. A cell pellet equivalent to 100 mL of culture medium was suspended in 1-200 mL of disruption buffer (20 mM Tris-HCl pH 9.0, 1 mM EDTA pH 9.0, 0.2 M NaCl, 0.5% Triton X-100), and the recombinant E. coli were then lysed using a microfluidizer at 15,000 psi. The cells were centrifuged at 13,900 g for 30 minutes, the supernatant was discarded, and the pellet was washed with 400 mL of the initial washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0). The pellet was centrifuged under the same conditions as above, the supernatant was discarded, and the pellet was washed with 400 mL of a second washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 2% Triton X-100). The pellet was centrifuged under the same conditions as above, the supernatant was discarded, and the pellet was washed with 400 mL of a third washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 1% sodium deoxycholorate). The pellet was centrifuged under the same conditions as above, the supernatant was discarded, and the pellet was washed with 400 mL of a fourth washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 1 M NaCl). The washed E. coli inclusion body pellet was obtained by centrifuging under the same conditions as above. The washed inclusion pellet was resuspended in 400 mL of soluble / reducing buffer (6M Guanidine, 100 mM Tris pH 8.0, 2 mM EDTA pH 9.0, 50 mM DTT) and stirred at 50°C for 30 minutes. 100 mL of distilled water was added to the soluble / reduced interleukin-2 analog to dilute the 6M Guanidine to 4.8M Guanidine, and then the mixture was centrifuged at 13,900 g for 30 minutes, discarding the pellet and obtaining only the solution. 185.7 mL of distilled water was further added to the diluted solution to dilute the 4.8M Guanidine to 3.5M Guanidine, and then the pH was adjusted to 5.0 using 100% acetic acid.The pH-adjusted solution was stirred at room temperature for 1 hour. After impurities precipitated, the solution was centrifuged at 13,900 g for 30 minutes, the supernatant was discarded, and the pellet was washed with the final washing buffer (3.5 M Guanidine, 20 mM Sodium Acetate pH 5.0, 5 mM DTT). The pellet was obtained by centrifugation under the same conditions as above. The washed interleukin-2 analog was dissolved in 400 mL of refolding buffer (6 mM Guanidine, 100 mM Tris pH 8.0, 0.1 mM CuCl2). The refolding process was carried out by stirring the mixed solution at 4°C for 15 to 24 hours. [Examples]

[0157] Size exclusion column chromatography The interleukin-2 analog refolding solution obtained in Example 3 was concentrated to less than 1 mL for purification by applying it to a size exclusion column. The column was equilibrated with buffer (2M Guanidine, 100mM Tris pH 8.0) before introducing the refolding solution, and then eluted by running buffer after introducing the refolding solution. Since the eluted sample contained guanidine, it was changed to a stabilization solution (10mM Sodium Acetate pH 4.5, 5% Trehalose), and then its purity was measured by RP-HPLC and peptide mapping analysis. Samples with a measured purity of 80% or higher were used in experiments. [Examples]

[0158] Evaluation of receptor binding affinity of interleukin-2 analogs To measure the receptor binding affinity of the interleukin-2 analog obtained in Example 4 to the interleukin-2 α-receptor and β-receptor, respectively, surface plasmon resonance (BIACORE T200, GE Healthcare) was used. The binding affinity of the fabricated analogs to the α-receptor and β-receptor was measured and compared with that of interleukin-2 analog 01 (aldesleukin).

[0159] First, approximately 5,000 RU (resonance units) of anti-human immunoglobulin antibody (Abcam, #ab97221) were immobilized on a CM5 chip (GE Healthcare) using amine coupling. Then, interleukin 2α receptors (SYMANSIS, #4102H) or interleukin 2β receptors (SYMANSIS, #4122H), to which the human immunoglobulin Fc site had bound, were finally immobilized by binding them to the immunoglobulin antibodies via antigen-antibody binding reaction. Next, the recombinant interleukin 2 analogs prepared as described above were diluted to various concentrations and flowed through the CM5 chip with the interleukin 2 receptors finally immobilized, and the binding affinity of each interleukin 2 receptor was measured. The binding affinity was measured using the binding rate constant (k a ) and the dissociation rate constant (k d The binding rate was measured by flowing an interleukin-2 analog at a flow rate of 10 μL / min for 3 minutes, and the dissociation rate from each interleukin-2 receptor was measured by flowing only experimental buffer at the same time and flow rate. After the measurement was completed, the receptor binding strength was evaluated using a 1:1 binding fitting model in the Biaevaluation program.

[0160]

number

[0161] In Table 3, "Undefinable" means that binding to the receptor in question was not observed in surface plasmon resonance measurements, and therefore, for that specific receptor, the physical quantity cannot be defined.

[0162] [Table 3] TIFF0007846627000022.tif196154 TIFF0007846627000023.tif91154

[0163] As can be seen from the test results (Figures 1, 2, and Table 3), the interleukin 2 analog of the present invention was confirmed to exhibit interleukin 2α receptor binding affinity that differs from that of natural interleukin 2 or aldesleukin, either by completely losing interleukin 2α receptor binding affinity or by decreasing or increasing it compared to interleukin 2 analog 1. In contrast, for interleukin 2β receptor binding affinity, a relatively stronger binding affinity of up to 100 times compared to natural interleukin 2 or aldesleukin was confirmed. Therefore, it was confirmed that the amino acid sequence of the interleukin 2 analog influences its binding to interleukin 2α or β receptors. This suggests that the binding affinity to the interleukin 2 receptor can be altered by substituting amino acids at specific positions.

[0164] These experimental results suggest that the interleukin-2 analog according to the present invention possesses altered interleukin-2α receptor binding affinity and interleukin-2β receptor binding affinity, thereby indicating its potential use in various drug development applications.

[0165] From the above description, those skilled in the art in which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification. Next, another preferred embodiment of the present invention will be shown. 1. An interleukin-2 analog having increased interleukin-2β receptor binding affinity compared to aldesleukin, wherein the interleukin-2 analog contains a sequence in which at least one amino acid corresponding to the 1st, 12th, 18th, 19th, 20th, 22nd, 32nd, 35th, 38th, 42nd, 43rd, 45th, 48th, 49th, 61st, 68th, 69th, 74th, 76th, 80th, 81st, 82nd, 84th, 85th, 86th, 87th, 88th, 89th, 91st, 92nd, 94th, 95th, 96th, 125th, 126th and 133rd positions in natural interleukin-2 is mutated. 2. The interleukin-2 analog according to claim 1, characterized in that its binding affinity to the interleukin-2α receptor is different from that of aldesleukin. 3. The interleukin-2 analog according to item 1 above, wherein at least one amino acid is added to the amino acid corresponding to the 133rd position. 4. The interleukin-2 analog described in paragraph 1 above, wherein the interleukin-2 analog is obtained by deleting the first amino acid and substituting the 125th amino acid with another amino acid in natural interleukin-2. 5. The interleukin-2 analog described in 4 above, further comprising 1 to 10 amino acid substitutions. 6. The interleukin-2 analog according to 5 above, wherein at least one amino acid at positions 18, 19, 20, 22, 38, 42, 43, 45, 61, 68, 69, 74, 80, 81, 84, 85, 86, 88, 89, 91, 92, 94, and 96 is further substituted with another amino acid. 7. The interleukin-2 analog according to 6 above, wherein at least one amino acid at positions 18, 19, 22, 38, 42, 43, 45, 61, 68, 74, 80, 81, 84, 85, 86, 88, 91, 92, 94, and 96 is further substituted with another amino acid. 8. The interleukin-2 analog described in item 1 above, wherein the interleukin-2 analog is one of the following analogs. (a) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 32nd amino acids are substituted with other amino acids. (b) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 35th amino acids are substituted with other amino acids. (c) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 38th amino acids are substituted with other amino acids. (d) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 42nd amino acids are substituted with other amino acids. (e) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 43rd amino acids are substituted with other amino acids. (f) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 48th amino acids are substituted with other amino acids. (g) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 49th amino acids are substituted with other amino acids. (h) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 76th amino acids are substituted with other amino acids. (i) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, 92nd, 94th and 96th amino acids are substituted with other amino acids. (j) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th and 87th amino acids are substituted with other amino acids. (k) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, and 42nd amino acids are substituted with other amino acids. (l) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, and 80th amino acids are substituted with other amino acids. (m) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, and 84th amino acids are substituted with other amino acids. (n) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 19th, 38th, and 42nd amino acids are substituted with other amino acids. (o) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 12th, 38th, and 42nd amino acids are substituted with other amino acids. (p) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 61st amino acids are substituted with other amino acids. (q) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 84th amino acids are substituted with other amino acids. (r) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 88th amino acids are substituted with other amino acids. (s) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 89th amino acids are substituted with other amino acids. (t) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 91st amino acids are substituted with other amino acids. (u) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 94th amino acids are substituted with other amino acids. (v) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, and 126th amino acids are substituted with other amino acids. (w) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, and 84th amino acids are substituted with other amino acids. (x) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 94th, and 96th amino acids are substituted with other amino acids. (y) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 81st, and 92nd amino acids are substituted with other amino acids. (z) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 61st, 81st, and 92nd amino acids are substituted with other amino acids. (aa) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, and 92nd amino acids are substituted with other amino acids. (ab) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, and 92nd amino acids are substituted with other amino acids. (ac) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 81st, 84th, and 92nd amino acids are substituted with other amino acids. (ad) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 20th, 38th, 42nd, 81st, and 92nd amino acids are substituted with other amino acids. (ae) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, and 92nd amino acids are substituted with other amino acids. (af) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 74th, 81st and 92nd amino acids are substituted with other amino acids. (ag) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, 84th, and 92nd amino acids are substituted with other amino acids. (ah) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 81st, 88th, and 92nd amino acids are substituted with other amino acids. (ai) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (aj) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, 85th, and 92nd amino acids are substituted with other amino acids. (ak) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, 86th, and 92nd amino acids are substituted with other amino acids. (al) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (am) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 45th, 74th, 81st, and 92nd amino acids are substituted with other amino acids. (an) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 74th, 80th, 81st and 92nd amino acids are substituted with other amino acids. (ao) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 84th, and 92nd amino acids are substituted with other amino acids. (ap) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 45th, 80th, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (aq) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (ar) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 42nd, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (as) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 61st, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (at) Interleukin-2 analogs in which the first amino acid is deleted and the 125th, 69th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (au) Interleukin-2 analogs in which the first amino acid is deleted from natural interleukin-2 and the 125th, 80th, 81st, 85th, 86th, 91st, and 92nd amino acids are substituted with other amino acids. (av) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (aw) Interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (ax) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 45th, 74th, 80th, 81st and 92nd amino acids are substituted with other amino acids. (ay) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 68th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (az) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 69th, 74th, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (ba) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 84th, 85th, 86th, 91st and 92nd amino acids are substituted with other amino acids. (bb) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 80th, 81st, 85th, 86th, 92nd, 94th, and 96th amino acids are substituted with other amino acids. (bc) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 19th, 22nd, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bd) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 38th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (be) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 61st, 80th, 81st, 85th, 86th, and 92nd amino acids are substituted with other amino acids. (bf) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 18th, 22nd, 68th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bg) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 35th, 38th, 42nd, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bh) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 45th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bi) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st and 92nd amino acids are substituted with other amino acids. (bj) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 92nd and 95th amino acids are substituted with other amino acids. (bk) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bl) An interleukin-2 analog in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 43rd, 61st, 80th, 81st, 85th, 86th and 92nd amino acids are substituted with other amino acids. (bm) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st, 92nd and 95th amino acids are substituted with other amino acids. (bn) Interleukin-2 analogs in which the first amino acid of natural interleukin-2 is deleted and amino acids 125, 35, 38, 42, 74, 80, 81, 82, 85, 86, and 92 are substituted with other amino acids. 9. The interleukin-2 analog according to 8 above, wherein the amino acid substitution is selected from the group consisting of the following amino acid substitutions. (a) Substitution of the 12th amino acid with valine or phenylalanine (b) Substitution of the 18th amino acid with arginine (c) Substitution of the 19th amino acid with tyrosine, valine, phenylalanine, or arginine. (d) Substitution of the 20th amino acid with valine or phenylalanine (e) Substitution of the 22nd amino acid with glutamic acid (f) Substitution of the 32nd amino acid with cysteine (g) Substitution of the 35th amino acid with cysteine ​​or glutamic acid (h) Substitution of the 38th amino acid with alanine or aspartic acid (i) Substitution of the 42nd amino acid with lysine, alanine, or tryptophan (j) Substitution of the 43rd amino acid with cysteine, glutamic acid, or glutamine (k) Substitution of the 45th amino acid with alanine (l) Substitution of the 48th amino acid with cysteine (m) Substitution of the 49th amino acid with cysteine (n) Substitution of the 61st amino acid with glutamine, arginine, or aspartic acid (o) Substitution of the 68th amino acid with aspartic acid or glutamine (p) Substitution of the 69th amino acid with glycine (q) Substitution of the 74th amino acid with histidine or alanine (r) Substitution of the 76th amino acid with cysteine (s) Substitution of the 80th amino acid with phenylalanine, tyrosine, valine, aspartic acid, or tryptophan (t) Substitution of the 81st amino acid with aspartic acid, glutamic acid, or asparagine (u) Substitution of the 82nd amino acid with glycine or valine (v) Substitution of the 84th amino acid with glutamic acid, valine, or phenylalanine (w) Substitution of the 85th amino acid with valine, alanine, glycine, tryptophan, tyrosine, threonine, isoleucine, glutamic acid, or phenylalanine. (x) Substitution of the 86th amino acid with valine, alanine, glycine, or leucine (y) Substitution of the 87th amino acid with cysteine (z) Substitution of the 88th amino acid with glutamine, valine, or phenylalanine (aa) Substitution of amino acid 89 with phenylalanine (ab) Substitution of the 91st amino acid with threonine, phenylalanine, or glutamic acid (ac) Substitution of the 92nd amino acid with phenylalanine, leucine, tyrosine, or tryptophan (ad) Substitution of the 95th amino acid with aspartic acid (ae) Substitution of the 96th amino acid with phenylalanine, valine, or isoleucine (af) Substitution of the 126th amino acid with threonine 10. Isolated nucleic acid encoding an interleukin-2 analog as described in any one of items 1 to 9 above. 11. A recombinant expression vector containing the nucleic acid described in item 10 above. 12. A non-human transformant containing the recombinant expression vector described in item 11 above. 13. An interleukin-2 analog containing any sequence selected from the group consisting of amino acid sequences SEQ ID NOs: 3 to 106. 14. An interleukin-2 analog containing the amino acid sequence represented by the following general formula 1. [General formula 1] X1-PTSSSTKKTQLQLEHL-X18-X19-DL-X22-MILNGINNYKNPKLT-X38-MLT-X42-X43-F-X45-MPKKATELKHLQCLE-X61-ELKPLE-X68-VLNLA- X74-SKNFH-X80-X81-PR-X84-X85-X86-SNIN-X91-X92-V-X94-E-X96-KGSETTFMCEYADETATIVEF-LNRWITFSQSIISTLT (General formula 1, Sequence number 212) In the above general formula 1, X1 is missing. X18 is either leucine (L) or arginine (R). X19 is either leucine (L) or tyrosine (Y). X22 is glutamic acid (E) or glutamine (Q), X38 is alanine (A), aspartic acid (D), or arginine (R). X42 is alanine (A), phenylalanine (F), lysine (K), or tryptophan (W). X43 is glutamic acid (E), lysine (K), or glutamine (Q). X45 is either alanine (A) or tyrosine (Y), X61 is aspartic acid (D), glutamic acid (E), glutamine (Q), or arginine (R). X68 is aspartic acid (D) or glutamic acid (E), X74 is histidine (H) or glutamine (Q), X80 is phenylalanine (F), leucine (L), valine (V), or tyrosine (Y). X81 is aspartic acid (D), glutamic acid (E), or arginine (R). X84 is either aspartic acid (D) or glutamic acid (E), X85 is alanine (A), glutamic acid (E), glycine (G), leucine (L), valine (V), tryptophan (W), or tyrosine (Y). X86 is alanine (A), glycine (G), isoleucine (I), or valine (V). X91 is threonine (T) or valine (V), X92 is phenylalanine (F), isoleucine (I), or tyrosine (Y). X94 is phenylalanine (F) or leucine (L), X96 is phenylalanine (F) or leucine (L). 15. The interleukin-2 analog according to 14, wherein the interleukin-2 analog contains any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 13, 14, 15, 16, 17, 20, 21, 22, 32, 35, 36, 42, 53, 54, 56, 58, 59, 60, 62, 71, 72, 74, 75, 76, 77, 78, 85, 87, 89, 91, 92, 93, 94, 95, 98, 99, 100, 101, 103, 104, 105 and 106. 16. In the above general formula 1, X43 is lysine (K), X45 is tyrosine (Y), X61 is aspartic acid (D), glutamic acid (E), or glutamine (Q). X68 is glutamic acid (E), X74 is glutamine (Q), X80 is phenylalanine (F) or leucine (L), X85 is leucine (L), valine (V), or tyrosine (Y). X86 is isoleucine(I) or valine(V), X92 is phenylalanine (F) or isoleucine (I), the interleukin-2 analog as described in 14 above. 17. The interleukin-2 analog according to 16, wherein the interleukin-2 analog contains any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 13, 14, 16, 17, 20, 21, 22, 32, 35, 36, 42, 53, 54, 87, 89, 91, 92, 93, 94, 98, 99, 100, 101, 103, 104, and 105. 18. The interleukin-2 analog according to 14, further comprising at least one amino acid at its C-terminus. 19. An interleukin-2 analog containing the amino acid sequence represented by the following general formula 2. [General formula 2] X1-PTSSSTKKTQLQLEHL-X18-LDL-X22-MILNGINNYKNPKLT-X38-MLT-X42-KFYMPKKATELKHLQCLE-X61-ELKPLE-X68-VLNLAQSKNFHF-X81-PRD-X85-X86-SNINVFVLELKGSETTFMCEY-ADETATIVEFLNRWITFSQSI-ISTLT (General formula 2, Sequence ID 213) X1 is missing. X18 is either leucine (L) or arginine (R). X22 is glutamic acid (E) or glutamine (Q), X38 is either alanine (A) or arginine (R), X42 is phenylalanine (F) or lysine (K), X61 is either aspartic acid (D) or glutamic acid (E), X68 is aspartic acid (D) or glutamic acid (E), X81 is either aspartic acid (D) or glutamic acid (E), X85 is leucine (L) or valine (V), X86 is isoleucine(I) or valine(V). 20. The interleukin-2 analog according to 19, wherein the interleukin-2 analog comprises any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs. 22, 42, 53, 87, 105, and 106. 21. The interleukin-2 analog according to 19, further comprising at least one amino acid at its C-terminus.

Claims

1. An interleukin-2 analog comprising any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 53, 87, 105, and 106.

2. The interleukin-2 analog according to claim 1, further comprising at least one amino acid at its C-terminus.

3. A separated nucleic acid encoding the interleukin-2 analog according to claim 1 or 2.

4. A recombinant expression vector comprising the nucleic acid described in claim 3.

5. A non-human transformant comprising the recombinant expression vector described in claim 4.

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