Novel immunoactive interleukin 2 analog

Abstract

To provide an interleukin-2 analog which can reduce toxicity and side effects, while enabling a reduction in the required dosage.SOLUTION: The present invention provides an interleukin-2 analog that shows changes in interleukin-2α receptor binding affinity and increases in interleukin-2β receptor binding affinity. The present invention also provides an isolated nucleic acid that codes for the interleukin-2 analog, a recombinant expression vector comprising the nucleic acid, and a transformant comprising the vector.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to novel interleukin-2 analogues. [Background technology]

[0002] Interleukin 2 is an important immunostimulant with a molecular weight of about 15 kDa, which is 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 makes it useful for the treatment of various immune-related diseases, including cancer and AIDS (Patent Document 1). Currently, interleukin 2 (Proleukin®) is an FDA-approved drug for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, high-dose interleukin 2 therapy is associated with serious toxicity, limiting the number of patients who can be treated, and this treatment is actually performed only for a small number of suitable patients. Toxicities associated with interleukin 2 include high fever, nausea, vomiting, vascular leak, severe hypotension, pulmonary edema, and vascular leak syndrome, which causes liver damage.

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

[0004] Meanwhile, although interleukin-2 has potential as a therapeutic agent for various immune-related diseases, there is a lack of drugs that can reduce toxicity and side effects while also reducing the dosage, and therefore further research into new and improved drugs is needed. [Prior art documents] [Patent documents]

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

[0006]

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Non-licensed Document 2

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Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

[0007] An object of the present invention is to provide an interleukin-2 analogue.

[0008] Another object of the present invention is to provide an isolated nucleic acid encoding the interleukin-2 analog, a recombinant expression vector comprising the nucleic acid, and a transformant comprising the vector.

[0009] A further object of the present invention is to provide a method for producing the interleukin 2 analogue.

[0010] A further object of the present invention is to provide a method for increasing interleukin-2β receptor binding affinity, which comprises the step of mutating at least one amino acid in native interleukin-2. [Means for solving the problem]

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

[0012] In another embodiment, the interleukin 2 analogue is characterized in that it comprises a sequence in which at least one of the amino acids corresponding to positions 1, 12, 18, 19, 20, 22, 32, 35, 38, 42, 43, 45, 48, 49, 61, 68, 69, 74, 76, 80, 81, 82, 84, 85, 86, 87, 88, 89, 91, 92, 94, 95, 96, 125, 126 and 133 in naturally occurring interleukin 2 has been mutated.

[0013] An interleukin-2 analog according to any of the above embodiments is characterized by altered binding to the interleukin-2α receptor and increased binding to the interleukin-2β receptor compared to native interleukin-2 or aldesleukin.

[0014] An Interleukin-2 analog according to any of the above-mentioned embodiments is characterized by the addition of at least one amino acid to the amino acid corresponding to position 133.

[0015] An interleukin-2 analog according to any of the above-mentioned embodiments is characterized in that it comprises a sequence in which the first amino acid in natural interleukin-2 is deleted and the 125th amino acid is replaced with another amino acid.

[0016] The interleukin-2 analogue according to any of the above-mentioned embodiments is characterized in that it further comprises from 1 to 10 amino acid substitutions.

[0017] Interleukin 2 analogs according to any of the above-mentioned embodiments are characterized in that at least one of the amino acids corresponding to 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 replaced with another amino acid.

[0018] Interleukin 2 analogs according to any of the above-mentioned embodiments are characterized in that at least one of the amino acids corresponding to positions 18, 19, 22, 38, 42, 43, 45, 61, 68, 74, 80, 81, 84, 85, 86, 88, 91, 92, 94 and 96 in natural interleukin 2 is further replaced with another amino acid.

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

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

[0021] The interleukin-2 analogue according to any of the above-mentioned specific examples is characterized in that it is selected from the group consisting of SEQ ID NOs: 3-106.

[0022] Other aspects of the invention are isolated nucleic acids encoding the interleukin 2 analogues, recombinant expression vectors comprising the nucleic acids, and transformants comprising the vectors.

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

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

[0025] Yet another embodiment of the present invention is an interleukin 2 analogue comprising any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3-106.

[0026] Yet another embodiment of the present invention is an interleukin-2 analogue 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 tryptophan (K). 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), 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] In one specific example, the interleukin 2 analog is characterized by comprising 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] The interleukin 2 analog according to any of the above-mentioned embodiments is characterized in that it comprises 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] The interleukin-2 analogue according to any of the above embodiments is characterized in that it further comprises at least one amino acid at the C-terminus.

[0033] Yet another embodiment of the present invention is an interleukin-2 analogue 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 number 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] In one specific example, the interleukin-2 analogue comprises any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 42, 53, 87, 105 and 106.

[0037] In another embodiment, the interleukin-2 analogue is characterized in that it further comprises at least one amino acid at the C-terminus. Effect of the Invention

[0038] The interleukin 2 analogue according to the present invention has increased binding ability to the biological interleukin 2β receptor, and is used for various applications. [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 shows the results of examining the binding ability of interleukin-2 analogs to interleukin-2α receptors, where (A) is interleukin-2 analog #86, (B) is interleukin-2 analog #104, and (C) is interleukin-2 analog #105. [Diagram 2] FIG. 1 shows the results of examining the binding affinity of interleukin-2 analogs to the interleukin-2β receptor, where (A) is interleukin-2 analog #86, (B) is interleukin-2 analog #104, and (C) is interleukin-2 analog #105. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Hereinafter, the mode for carrying out the present invention will be described. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in the present invention. In addition, the present invention is not limited to the following specific description.

[0041] Throughout this specification the conventional one-letter and three-letter codes for amino acids are used, and the amino acids referred to herein by abbreviations are written according to the IUPAC-IUB nomenclature system. 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 Phenylalanine F Proline P Serin S Threonine T Tryptophan W Tyrosine Y Balin V

[0042] One aspect of the present invention provides an interleukin 2 analog. The interleukin 2 analog of the present invention is characterized by having an altered binding ability to the interleukin 2 receptor, in particular an increased binding ability to the interleukin 2β receptor. Specifically, the interleukin 2 analog of the present invention may have an increased binding ability to the interleukin 2β receptor, and more specifically, may have an altered (increased or decreased) binding ability to the interleukin 2α receptor, as compared to natural interleukin 2 or known aldesleukins.

[0043] In the present invention, "interleukin 2 (IL-2)" refers to an immunomodulatory agent that is a type of cytokine that transmits signals in the immune system in vivo. Interleukin 2 is generally known as an important immunostimulant of about 15 kDa.

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

[0045] The natural interleukin 2 may be human interleukin 2, and the sequence thereof can be obtained from a known database, etc. Specifically, it is the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0046] In the present invention, when natural interleukin-2 has the amino acid sequence of SEQ ID NO: 1, it means that not only a sequence that is the same as SEQ ID NO: 1, but also sequences that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 1 are included in the category of natural interleukin-2 of the present invention, and the amino acid mutation position means that the mutation occurs at the position corresponding to the amino acid sequence of SEQ ID NO: 1 when sequences that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 1 are aligned based on the standard.

[0047] In the present invention, a mutation in at least one amino acid in the native sequence means a mutation in which at least one amino acid in the native interleukin 2 has been altered in a manner selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

[0048] Specifically, the interleukin-2 analogues of the present invention may comprise a sequence in which at least one of the amino acids corresponding to positions 1, 12, 18, 19, 20, 22, 32, 35, 38, 42, 43, 45, 48, 49, 61, 68, 69, 74, 76, 80, 81, 82, 84, 85, 86, 87, 88, 89, 91, 92, 94, 95, 96, 125, 126 and 133 in naturally occurring interleukin-2 has been mutated. Specifically, the interleukin 2 analog of the present invention may have the first amino acid deleted and the 125th amino acid replaced with another amino acid in natural interleukin 2, and may further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions. Further substitutions include, but are not limited to, substitution of the 125th amino acid, cysteine, with a serine, and further substitutions include, but are not limited to, those corresponding to positions 12, 18, 19, 20, 22, 32, 35, 38, 42, 43, 45, 48, 49, 61, 68, 69, 74, 76, 80, 81, 82, 84, 85, 86, 87, 88, 89, 91, 92, 94, 95, 96, 126, and 133.

[0049] In addition to the above mutation sites, the present invention also includes interleukin-2 analogs that include substitutions, additions, deletions, modifications, etc. of amino acid residues to the extent that they are known in the art for the purpose of extending the stability and half-life of the peptide.

[0050] In the present invention, "aldesleukin" or "interleukin 2 analog" refers to aldesleukin (trade name: Proleukin (registered trademark)), a commercially available interleukin 2 analog, specifically, one having the amino acid sequence of SEQ ID NO: 2. In the present 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 ability and / or increased interleukin 2β receptor binding ability compared to the interleukin 2 analog 1.

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

[0052] Interleukin 2β receptor is a CD8 + It is mainly distributed in T cells and natural killer cells (NK cells) and its main function is to activate immune responses and macrophage activity, so activation of interleukin 2β receptors is expected to lead to tumor apoptosis and activation of the body's immune responses.

[0053] Therefore, the interleukin-2 analogue of the present invention having increased binding strength to the interleukin-2β receptor has the effect of enhancing therapeutic effects such as tumor inhibition and tumor killing, and reducing side effects.

[0054] The interleukin 2 analog in the present invention may have a sequence in which the first amino acid in natural interleukin 2 is deleted and the 125th amino acid is replaced with another amino acid, or may further include 1 to 10 amino acid mutations. For example, the interleukin 2 analogue may include, but is not limited to, an amino acid sequence in which the amino acid at position 125 is substituted with serine, and at least one amino acid at positions 12, 18, 19, 20, 22, 32, 35, 38, 42, 43, 45, 48, 49, 61, 68, 69, 74, 76, 80, 81, 82, 84, 85, 86, 87, 88, 89, 91, 92, 94, 95, 96, and 126 is further substituted with another amino acid, and / or at least one amino acid is added to the 133rd amino acid. The interleukin 2 analogue may be any interleukin 2 analogue having altered interleukin 2α receptor binding affinity and increased interleukin 2β receptor binding affinity compared to naturally occurring interleukin 2 and / or aldesleukin.

[0055] As an example, the interleukin 2 analog has at least one amino acid added to the amino acid corresponding to position 133, but is not limited thereto. For the purposes of the present invention, the type and length of the amino acid to be added as described above is not limited as long as it changes the binding ability to the interleukin 2α receptor and increases the binding ability to the interleukin 2β receptor compared to natural interleukin 2 or aldesleukin, and in addition to natural amino acids, non-natural amino acids and amino acids including chemical modifications may be added.

[0056] In another example, the interleukin-2 analog has, in a natural interleukin-2, the first amino acid deleted, the 125th amino acid replaced with another amino acid, and one, two, three, four, five, six, seven, eight, nine or more of the following amino acids replaced with other amino acids: 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.

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

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

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

[0060] By "corresponding to" herein is meant an amino acid residue at a recited position in the peptide, or an amino acid residue similar, identical or equivalent to a recited residue in the peptide. Identifying the amino acid at the corresponding position will determine the particular amino acid of the sequence to which the particular sequence refers.

[0061] For example, by aligning any amino acid sequence with SEQ ID NO:1, each amino acid residue in the amino acid sequence can be numbered based on the number and position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO:1.

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

[0063] In the present invention, when a specific position of an amino acid in a peptide is expressed, it may also mean the corresponding position in a reference sequence.

[0064] In yet another example, the interleukin 2 analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 106, is essentially composed of the amino acid sequence, or consists of the amino acid sequence, but is not limited to these.

[0065] Furthermore, even if the specification states "an interleukin 2 analog consisting of a specific sequence number," this does not exclude meaningless sequence additions, naturally occurring mutations, or silent mutations before or after the amino acid sequence of the sequence number, so long as the analog has the same or corresponding activity as an interleukin 2 analog consisting of the amino acid sequence of the sequence number, and it goes without saying that those having such sequence additions or mutations are also included in the present application.

[0066] The interleukin 2 analogues of the present invention may include 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 to the amino acid sequences of SEQ ID NOs: 3 to 106, but are not limited to these.

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

[0068] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to the entire sequence or to a portion thereof under moderate or high stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides having common codons or codons that take into account codon degeneracy in the polynucleotide.

[0069] Homology and identity are often used interchangeably.

[0070] Whether any two nucleotide or peptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 3. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 1), as implemented 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), which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, FASTA (Non-Patent Documents 5, 6 and 7). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

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

[0072] The above also applies to other embodiments or aspects of the present invention, but is not limited thereto.

[0073] The interleukin 2 analog of the present invention is used as a new alternative to interleukin 2, which changes the in vitro activity by weakening or increasing the binding strength of interleukin 2 to the α and / or β receptors. In particular, since it not only increases the binding strength to the β receptor but also changes (increases or decreases) the binding strength to the α receptor, it can be used as an effective therapeutic agent due to its activity against two receptors.

[0074] In the present invention, such modifications for the production of analogs of Interleukin 2 include all modifications using L- or D-amino acids and / or unnatural amino acids, and / or modifications of the native sequence, such as modifications of side chain functional groups, covalent bonds within the molecule, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, and the like.

[0075] Also included are all interleukin-2s in which at least one amino acid has been added to the N- and / or C-terminus of native interleukin-2.

[0076] As mentioned above, the amino acids to be substituted or added can be any of the 20 amino acids commonly found in human proteins, as well as unusual or unnatural amino acids. Commercial sources of unusual amino acids include Sigma-Aldrich, ChemPep, and Genzyme pharmaceuticals. Peptides and typical peptide sequences containing these amino acids can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea.

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

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

[0079] In particular, in the case of chemically synthesized peptides, the N- and C-termini are charged, and therefore, in order to remove the charge, acetylation of the N-terminus and / or amidation of the C-terminus are performed, but this is not particularly limited thereto.

[0080] In addition, since the interleukin 2 analogue according to the present invention is in the form of a peptide, it includes the peptide itself, its salt (e.g., a pharma- ceutically acceptable salt of the peptide) or its solvate. Furthermore, the peptide may be in any form as long as it is pharma- ceutically acceptable.

[0081] The type of the salt is not particularly limited, although it is preferable that the salt is in a form that is safe and effective for an individual, for example, a mammal, but is not particularly limited thereto.

[0082] The term "pharmacologically acceptable" refers to a substance that can be effectively used for a desired purpose without inducing excessive toxicity, irritation, allergic reactions, and the like, within the scope of medical judgment.

[0083] The "pharmaceutically acceptable salt" in the present invention includes salts derived from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids include hydrochloric acid, bromic acid, 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, benzenesulfonic acid, etc. Suitable salts derived from bases include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, ammonium, etc.

[0084] In addition, the term "solvate" in the present invention means a complex formed between the peptide or a salt thereof according to the present invention and a solvent molecule.

[0085] In the present invention, the binding strength of any interleukin-2 analogue to a native interleukin-2 receptor can be measured by various known techniques for measuring affinity to the receptor, such as, but not limited to, surface plasmon resonance (SPR).

[0086] Specifically, the interleukin-2 analogues of the present invention may have decreased or increased interleukin-2α receptor binding affinity compared to native interleukin-2 or aldesleukin.

[0087] More specifically, the interleukin 2 analogs of the present invention have interleukin 2α receptor binding strength that is about 0.001 times or more, about 0.005 times or more, about 0.01 times or more, about 0.05 times or more, about 0.1 times or more, about 0.3 times or more, about 0.5 times or more, about 0.7 times or more, about 0.9 times or more, about 1.1 times or more, about 1.3 times or more, about 1.5 times or more, or about 1.7 times or more compared to the interleukin 2α receptor binding strength of natural interleukin 2 or aldesleukin, but the numerical value is not limited, and any analogs whose binding strength is changed compared to natural interleukin 2 or aldesleukin are included in the present invention.

[0088] Alternatively, based on the interleukin 2α receptor binding affinity of aldesleukin (100%), the interleukin 2 analogs of the present invention have either completely lost binding affinity, or have a binding affinity of about 1% or more, about 5% or more, about 7% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 50% or more, about 70% or more, about 90% or more, about 100% or more, about 150% or more, or about 200% or more; however, the numerical values ​​are not limited, and any analog whose binding affinity is altered compared to that of natural interleukin 2 or aldesleukin is included in the present invention.

[0089] Specifically, the interleukin 2 analogs of the present invention have an interleukin 2β receptor binding affinity that is about 0.1 times or more, about 0.3 times or more, about 0.5 times or more, about 0.7 times or more, about 1.0 times or more, about 10 times or more, about 20 times or more, about 30 times or more, about 40 times or more, about 50 times or more, about 60 times or more, about 70 times or more, about 80 times or more, about 90 times or more, or about 100 times or more, compared to the interleukin 2β receptor binding affinity of natural interleukin 2 or aldesleukin; however, the numerical value is not limited, and any analogs whose binding affinity is altered or increased compared to natural interleukin 2 or aldesleukin are included in the present invention.

[0090] Alternatively, based on the interleukin 2β receptor binding affinity of aldesleukin (100%), the interleukin 2 analogs of the present invention have a binding affinity of about 5% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 50% or more, about 100% or more, about 200% or more, about 500% or more, about 700% or more, about 1000% or more, about 1500% or more, about 3000% or more, about 5000% or more, about 7000% or more, about 10000% or more, about 12000% or more, about 15000% or more, about 20000% or more, or about 25000% or more; however, the numerical value is not limited, and any binding affinity that is increased compared to aldesleukin is included in the present invention.

[0091] In the present invention, "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​that are equal to or in a similar range to the numerical value following the term "about," but is not limited to these.

[0092] The interleukin 2 analogues of the present invention are characterized by their altered binding affinity to the interleukin 2α receptor and their increased binding affinity to the interleukin 2β receptor compared to native interleukin 2 or aldesleukin.

[0093] In a specific embodiment of the present invention, in order to produce the interleukin 2 analog of the present invention, an interleukin 2 analog was produced by introducing a mutation into natural interleukin 2 (SEQ ID NO: 1). The interleukin 2 analog produced 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 base sequences of SEQ ID NOs: 108 to 211.

[0094] Other aspects of the present invention provide a nucleic acid (polynucleotide) encoding the interleukin-2 analogue, a recombinant expression vector containing the nucleic acid, and a transformant containing the nucleic acid or recombinant expression vector.

[0095] The nucleic acid encoding the interleukin-2 analog of the present invention may be one that has been modified so as to introduce a mutation (deletion, substitution and / or addition of an amino acid) into an amino acid at a specific position in the base sequence encoding natural interleukin-2 of SEQ ID NO: 1, and specifically may include a base sequence that encodes 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 sequence of the present invention can be modified in various ways in the coding region, taking into consideration the codon degeneracy or the codons preferred in the organism in which the nucleic acid of the present invention is to be expressed, as long as the amino acid sequence of the interleukin-2 analog of the present invention is not changed. Specifically, the nucleic acid of the present invention has or contains a base sequence having 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 to any of the sequences of SEQ ID NOs: 108 to 211, or is composed of or essentially constitutes a base sequence having 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 to any of the sequences of SEQ ID NOs: 108 to 211, but is not limited thereto.

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

[0098] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of 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. Thus, 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 of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 12).

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

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

[0102] In the present invention, the term "vector" refers to a recombinant vector that expresses a target protein in a suitable host cell, and is a nucleic acid construct that contains the necessary regulatory elements operably linked to express the 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 transformation or transfection of the recombinant vector into a host cell.

[0103] In the present invention, "transformation" refers to the introduction of DNA into a host cell so that the DNA becomes replicable as a chromosomal factor or by complete chromosomal integration, and refers to the phenomenon of artificially causing a genetic change by introducing external DNA into a cell.

[0104] The host suitable for the present invention is not particularly limited as long as it expresses the nucleic acid of the present invention. Specific examples of the host used in the present invention include Escherichia bacteria such as E. coli, Bacillus bacteria such as Bacillus subtilis, Pseudomonas bacteria such as Pseudomonas putida, yeast such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, insect cells such as Spodoptera frugiperda (Sf9), and animal cells such as CHO, COS, and BSC.

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

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

[0107] More specifically, (a) natural interleukin 2 in which the first amino acid has been deleted and the 125th and 32nd amino acids have been replaced with other amino acids, (b) natural interleukin 2 in which the first amino acid has been deleted and the 125th and 35th amino acids have been replaced with other amino acids, (c) natural interleukin 2 in which the first amino acid has been deleted and the 125th and 38th amino acids have been replaced with other amino acids, or (d) natural interleukin 2 in which the first amino acid has been deleted and (e) natural interleukin 2 in which the first amino acid has been deleted and the 125th and 43rd amino acids have been substituted with other amino acids; (f) natural interleukin 2 in which the first amino acid has been deleted and the 125th and 48th amino acids have been substituted with other amino acids; (g) natural interleukin 2 in which the first amino acid has been deleted and the 125th and 49th amino acids have been substituted with other amino acids; (i) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 38th, 42nd, 81st, 92nd, 94th, and 96th positions have been substituted with other amino acids; (j) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th and 87th positions have been substituted with other amino acids; (k) natural interleukin 2 in which (l) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, and 42nd amino acids have been substituted with other amino acids; (m) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, and 80th amino acids have been substituted with other amino acids; (n) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 19th,(o) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 12th, 38th, and 42nd amino acids have been substituted with other amino acids; (p) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, and 61st amino acids have been substituted with other amino acids; (q) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, and 84th amino acids have been substituted with other amino acids. (r) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, and 88th amino acids have been replaced with other amino acids; (s) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, and 89th amino acids have been replaced with other amino acids; or (t) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, and 91st amino acids have been replaced with other amino acids. (u) a natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, and 94th amino acids have been replaced with other amino acids; (v) a natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, and 126th amino acids have been replaced with other amino acids; (w) a natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 80th, and 84th amino acids have been replaced with other amino acids; or (x) a natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 80th, and 84th amino acids have been replaced with other amino acids. (y) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 38th, 94th, and 96th positions have been replaced with other amino acids; (z) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 61st, 81st, and 92nd positions have been replaced with other amino acids; (aa) natural interleukin 2 in which(ab) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 38th, 42nd, 81st and 92nd positions have been substituted with other amino acids; (ac) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 38th, 81st, 84th and 92nd positions have been substituted with other amino acids; (ad) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 38th, 81st, 84th and 92nd positions have been substituted with other amino acids. (ae) in natural interleukin 2, the first amino acid is deleted and the amino acids at the 125th, 38th, 42nd, 81st, and 92nd positions are substituted with other amino acids; (af) in natural interleukin 2, the first amino acid is deleted and the amino acids at the 125th, 38th, 42nd, 74th, 81st, and 92nd positions are substituted with other amino acids; (ag) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 38th, 42nd, 81st, 84th, and 92nd positions have been replaced with other amino acids; (ah) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 38th, 42nd, 81st, 88th, and 92nd positions have been replaced with other amino acids; or (ai) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 38th, 42nd, 85th, 86th, and 92nd positions have been replaced with other amino acids. (aj) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 38, 80, 81, 85, and 92 have been substituted with other amino acids; (ak) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 38, 80, 81, 86, and 92 have been substituted with other amino acids; (al) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 80, 81, 85,(am) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, 45th, 74th, 81st, and 92nd amino acids have been substituted with other amino acids; (an) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, 74th, 80th, 81st, and 92nd amino acids have been substituted with other amino acids; (ao) natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, 74th, 80th, 81st, and 92nd amino acids have been substituted with other amino acids. (ap) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 38th, 45th, 80th, 85th, 86th, and 92nd positions have been replaced with other amino acids; or (aq) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 38th, 80th, 81st, 85th, 86th, and 92nd positions have been replaced with other amino acids. (ar) a natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 42, 80, 81, 85, 86, and 92 have been substituted with other amino acids; (as) a natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 61, 80, 81, 85, 86, and 92 have been substituted with other amino acids; (at) a natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 69, 80, 81, 85 (au) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 80th, 81st, 85th, 86th, 91st, and 92nd positions have been substituted with other amino acids; (av) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 18th, 22nd, 80th, 81st, 85th, 86th, and 92nd positions have been substituted with other amino acids; (aw) natural interleukin 2 in which(ax) a natural interleukin-2 in which the first amino acid has been deleted and the amino acids at positions 125, 38, 42, 80, 81, 85, 86, and 92 have been substituted with other amino acids; or (ay) a natural interleukin-2 in which the first amino acid has been deleted and the amino acids at positions 125, 38, 42, 45, 74, 80, 81, and 92 have been substituted with other amino acids. and 92nd amino acids are substituted with other amino acids; (az) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 69th, 74th, 80th, 81st, 85th, 86th, and 92nd amino acids have been substituted with other amino acids; (ba) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 80th, 81st, 84th, 85th, 86th, 91st, and 92nd amino acids have been substituted with other amino acids; (bb) natural interleukin 2 in which (bc) a natural interleukin-2 in which the first amino acid has been deleted and the amino acids at positions 125, 18, 19, 22, 80, 81, 85, 86, and 92 have been substituted with other amino acids; or (bd) a natural interleukin-2 in which the first amino acid has been deleted and the amino acids at positions 125, 18, 22, 38, 80, 81 have been substituted with other amino acids. (be) a natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 18th, 22nd, 61st, 80th, 81st, 85th, 86th, and 92nd positions have been substituted with other amino acids; or (bf) a natural interleukin 2 in which the first amino acid has been deleted and the amino acids at the 125th, 18th, 22nd, 68th, 80th, 81st, 85th, 86th, and 92nd positions have been substituted with other amino acids.(bg) a natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 35, 38, 42, 80, 81, 85, 86, and 92 have been substituted with other amino acids; or (bh) a natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 38, 42, 45, 80, 81, 85, 86, and 92 have been substituted with other amino acids. (bi) a natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st, and 92nd amino acids have been substituted with other amino acids; (bj) a natural interleukin 2 in which the first amino acid has been deleted and the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 92nd, and 95th amino acids have been substituted with other amino acids; or (bk) a natural interleukin 2 in which the first amino acid has been deleted and the 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 85th, 86th, and 92nd amino acids have been substituted with other amino acids. (bl) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 38, 42, 43, 61, 80, 81, 85, 86, and 92 have been replaced with other amino acids; (bm) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 38, 42, 80, 81, 85, 86, 91, 92, and 95 have been replaced with other amino acids; or (bn) natural interleukin 2 in which the first amino acid has been deleted and the amino acids at positions 125, 35, 38, 42, 74, 80, 81, 82, 85, 86, and 92 have been replaced with other amino acids, but are not limited thereto.

[0108] Interleukin 2 analogs and mutations are described above.

[0109] As another example of the method for producing an interleukin 2 analog of the present invention, the method for producing the interleukin 2 analog may include the steps of a) culturing a transformant containing a nucleic acid encoding the interleukin 2 analog to express the interleukin 2 analog, and b) isolating and purifying the expressed interleukin 2 analog, but any method that can produce an interleukin 2 analog may be used, and may be a method known in the technical field.

[0110] In the present invention, the nucleic acid encoding the interleukin 2 analogue includes any one of the base sequences of SEQ ID NOs: 108 to 211 or is (essentially) composed of the above base sequence, but is not limited thereto.

[0111] In the present invention, the medium used for culturing the transformant must meet the requirements for the host cell culture in a suitable manner. The carbon source contained in the medium for the growth of the host cell is appropriately selected according to the type of transformant to be produced at the discretion of a person skilled in the art, and suitable culture conditions are adopted to adjust the time and amount of culture.

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

[0113] Nitrogen sources that can be used include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used alone or in mixtures.

[0114] Phosphorus sources that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts. The culture medium may also contain metal salts necessary for growth, such as magnesium sulfate, ferrous sulfate, etc.

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

[0116] The transformant according to the present invention is usually cultured at a temperature of 20° C. to 45° C., specifically 25° C. to 40° C. The culture is continued until the maximum production amount of the desired interleukin 2 analog is obtained, and for these purposes, the culture is usually continued for 10 to 160 hours.

[0117] As described above, when appropriate culture conditions are established depending on the host cell, the transformant of the present invention produces an interleukin 2 analogue, and depending on the configuration of the vector and the characteristics of the host cell, the produced interleukin 2 analogue is secreted into the cytoplasm, periplasmic space or extracellularly of the host cell.

[0118] Proteins expressed inside or outside a host cell can be purified by a conventional method, such as salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (e.g., protein fraction precipitation using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, chromatography such as reverse-phase column chromatography, ultrafiltration, etc., which can be used alone or in combination.

[0119] As an embodiment of the present invention, a method for producing an interleukin 2 analogue may comprise the steps of (a) expressing said interleukin 2 analogue and (b) isolating the expressed interleukin 2 analogue.

[0120] As a specific example of the present invention, the method may further comprise the following steps to separate and purify the interleukin-2 analog expressed in the form of inclusion bodies from the transformant. b-1) obtaining and disrupting the transformant from the culture medium of step a) b-2) recovering and refolding the expressed interleukin 2 analog from the disrupted cell lysate; b-3) purifying the refolded interleukin 2 analog by size exclusion chromatography

[0121] In yet another aspect of the present invention, there is provided a method for producing the interleukin 2 analog by peptide synthesis. Since the interleukin 2 analog sequence of the present invention is provided, such peptide synthesis can be carried out without any restrictions by using a known peptide synthesis method.

[0122] Interleukin 2 analogs and mutations are described above.

[0123] Yet another aspect of the invention is a method of increasing interleukin-2 beta receptor binding comprising the step of mutating at least one amino acid in native interleukin-2.

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

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

[0126] More specifically, the method includes the steps of: (a) deleting the first amino acid and substituting the 125th and 32nd amino acids in natural interleukin-2 with other amino acids; (b) deleting the first amino acid and substituting the 125th and 35th amino acids in natural interleukin-2 with other amino acids; (c) deleting the first amino acid and substituting the 125th and 38th amino acids in natural interleukin-2 with other amino acids; (d) deleting the first amino acid and substituting the 125th and 38th amino acids in natural interleukin-2 with other amino acids; (e) deleting the first amino acid and substituting the 125th and 43rd amino acids in the natural interleukin 2; (f) deleting the first amino acid and substituting the 125th and 48th amino acids in the natural interleukin 2; (g) deleting the first amino acid and substituting the 125th and 49th amino acids in the natural interleukin 2; (h) deleting the first amino acid and substituting the 125th and 49th amino acids in the natural interleukin 2; (i) in natural interleukin-2, deleting the first amino acid and substituting the amino acids at positions 125, 38, 42, 81, 92, 94 and 96 with other amino acids; (j) in natural interleukin-2, deleting the first amino acid and substituting the amino acids at positions 125 and 87 with other amino acids; (k) in natural interleukin-2, deleting the first amino acid and substituting the amino acids at positions 125, 38 and 96 with other amino acids. (l) deleting the first amino acid and substituting the 125th, 38th and 80th amino acids in natural interleukin-2 with other amino acids; (m) deleting the first amino acid and substituting the 125th, 38th and 84th amino acids in natural interleukin-2 with other amino acids; (n) deleting the first amino acid and substituting the 125th, 19th, 38th and 42nd amino acids in natural interleukin-2 with other amino acids;(o) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 12th, 38th and 42nd positions with other amino acids; (p) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 42nd and 61st positions with other amino acids; (q) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 42nd and 84th positions with other amino acids; (r) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 42nd and 84th positions with other amino acids; (s) a step of deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 42nd and 88th positions with other amino acids; (t) a step of deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 42nd and 89th positions with other amino acids; (u) a step of deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 42nd and 91st positions with other amino acids; (v) deleting the first amino acid and substituting the 125th, 38th, 42nd, and 126th amino acids in natural interleukin-2 with other amino acids; (w) deleting the first amino acid and substituting the 125th, 38th, 80th, and 84th amino acids in natural interleukin-2 with other amino acids; (x) deleting the first amino acid and substituting the 125th, 38th, 94th, and 96th amino acids in natural interleukin-2 with other amino acids. (y) deleting the first amino acid and substituting the amino acids at the 125th, 38th, 81st and 92nd positions in natural interleukin-2 with other amino acids; (z) deleting the first amino acid and substituting the amino acids at the 125th, 61st, 81st and 92nd positions in natural interleukin-2 with other amino acids; (aa) deleting the first amino acid and substituting the amino acids at the 125th, 38th, 42nd, 81st and 92nd positions in natural interleukin-2 with other amino acids;(ab) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 80th, 81st and 92nd positions with other amino acids; (ac) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 81st, 84th and 92nd positions with other amino acids; (ad) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 20th, 38th, 42nd, 81st and 92nd positions with other amino acids. (ae) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 42nd, 80th, 81st and 92nd positions with other amino acids; (af) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 42nd, 74th, 81st and 92nd positions with other amino acids; (ag) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 42nd, 81st, 84th and 92nd positions with other amino acids. (ah) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at positions 125, 38, 42, 81, 88 and 92 with other amino acids; (ai) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at positions 125, 38, 42, 85, 86 and 92 with other amino acids; (aj) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at positions 125, 38, 80, 81, 85 and (ak) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 80th, 81st, 86th and 92nd positions with other amino acids; (al) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 80th, 81st, 85th, 86th and 92nd positions with other amino acids; (am) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 42nd,(an) deleting the first amino acid in natural interleukin-2 and substituting the 125th, 38th, 42nd, 74th, 80th, 81st, and 92nd amino acids with other amino acids; (ao) deleting the first amino acid in natural interleukin-2 and substituting the 125th, 38th, 42nd, 80th, 81st, 84th, and 92nd amino acids with other amino acids; (ap) deleting the first amino acid in natural interleukin-2 and substituting the 125th, 38th, 42nd, 80th, 81st, 84th, and 92nd amino acids with other amino acids; (aq) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 38th, 45th, 80th, 85th, 86th, and 92nd positions with other amino acids; (ar) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 42nd, 80th, 81st, 85th, 86th, and 92nd positions with other amino acids. (as) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at positions 125, 61, 80, 81, 85, 86 and 92 with other amino acids; (at) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at positions 125, 69, 80, 81, 85, 86 and 92 with other amino acids; (au) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at positions 125, 80, 81, 85, 86 and 92 with other amino acids. (av) in natural interleukin-2, deleting the first amino acid and substituting the amino acids at the 125th, 18th, 22nd, 80th, 81st, 85th, 86th and 92nd positions with other amino acids; (aw) in natural interleukin-2, deleting the first amino acid and substituting the amino acids at the 125th, 38th, 42nd, 80th, 81st, 85th, 86th and 92nd positions with other amino acids; (ax) in natural interleukin-2,a step of deleting the first amino acid and substituting the amino acids at positions 125, 38, 42, 45, 74, 80, 81, and 92 with other amino acids; (ay) a step of deleting the first amino acid in natural interleukin-2 and substituting the amino acids at positions 125, 38, 68, 80, 81, 85, 86, and 92 with other amino acids; (az) a step of deleting the first amino acid in natural interleukin-2 and substituting the amino acids at positions 125, 69, 74, 80, 81, 85, 86, and 92 with other amino acids. (ba) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 80th, 81st, 84th, 85th, 86th, 91st and 92nd positions with other amino acids; (bb) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 80th, 81st, 85th, 86th, 92nd, 94th and 96th positions with other amino acids; (bc) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at the 125th, 80th, 81st, 85th, 86th, 92nd, 94th and 96th positions with other amino acids. (bd) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at positions 125, 18, 22, 38, 80, 81, 85, 86, and 92 with other amino acids; (be) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at positions 125, 18, 22, 61, 80, 81, 85, 86, and 92 with other amino acids. (bf) in natural interleukin 2, deleting the first amino acid and substituting the amino acids at positions 125, 18, 22, 68, 80, 81, 85, 86 and 92 with other amino acids; (bg) in natural interleukin 2, deleting the first amino acid and substituting the amino acids at positions 125, 35, 38, 42, 80, 81, 85, 86 and 92 with other amino acids; (bh) in natural interleukin 2,(bi) deleting the first amino acid and substituting the amino acids at positions 125, 38, 42, 45, 80, 81, 85, 86, and 92 with other amino acids in natural interleukin-2; (bj) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 92nd and 95th with other amino acids; (bk) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 85th, 86th and 92nd with other amino acids; (bl) deleting the first amino acid in natural interleukin-2 and substituting the amino acids at 125th, 38th, 42nd, 43rd, 6 (bm) in natural interleukin-2, deleting the first amino acid and substituting the amino acids at the 125th, 38th, 42nd, 80th, 81st, 85th, 86th, 91st, 92nd, and 95th positions with other amino acids; or (bn) in natural interleukin-2, deleting the first amino acid and substituting the amino acids at the 125th, 35th, 38th, 42nd, 74th, 80th, 81st, 82nd, 85th, 86th, and 92nd positions with other amino acids.

[0127] Interleukin 2 analogs and mutations are described above.

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

[0129] The definitions of interleukin 2 analogs, mutations, and analogs represented by SEQ ID NOs are as described above.

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

[0131] Yet another aspect of the present invention provides an interleukin-2 analogue 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 tryptophan (K). 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), 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 is not limited thereto.

[0135] Specifically, the interleukin 2 analog comprises, consists essentially of, or consists 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, but is not limited thereto.

[0136] Such interleukin-2 analogs include, but are not limited to, aldesleukin or native interleukin-2, which have increased beta-receptor binding affinity.

[0137] As another example, the interleukin 2 analogue of the present invention is, but is not limited to, 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).

[0138] Specifically, the interleukin 2 analog is characterized by comprising 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 analogs of the present invention further comprise, but are not limited to, at least one amino acid at the C-terminus.

[0140] Yet another aspect of the present invention provides an interleukin-2 analogue 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 number 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 analogue comprises any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 42, 53, 87, 105 and 106, but is not limited thereto.

[0144] In addition, at least one amino acid may be added to the threonine (T) corresponding to X133 in general formula 2, or at least one amino acid may be further included at the C-terminus of the interleukin 2 analog, but this is not limited thereto.

[0145] In this specification, unless otherwise specified, the terms "comprise," "have," "contain," and the like mean to include a specified integer or set of integers, but should not be construed as excluding 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] Construction of expression vectors for native interleukin 2 and interleukin 2 analogues In order to prepare 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 pET-22b vector (Novagen). In addition, a novel interleukin 2 analog was prepared by modifying the amino acids of interleukin 2 using the interleukin 2 as a template. The PCR conditions for amplifying the interleukin 2 analog were 95°C for 30 seconds, 55°C for 60 seconds, and 65°C for 6.5 minutes, and this process was repeated for 16 cycles. In order to determine whether the amino acids at the desired sites were normally modified, the mutagenesis products obtained under the above conditions were subjected to sequence analysis, and it was confirmed that the mutations shown in Table 1 below were present at the desired mutation positions of each interleukin 2 analog, based on the natural one. The expression vectors thus obtained were named pET22b-interleukin 2 analogs 1 to 105.

[0148] The modified sequence and analog name of each amino acid are shown in Table 1. 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 SEQ ID NOs: 214 to 417, respectively, herein.

[0150] [Table 1] TIFF2024041747000002.tif206150 TIFF2024041747000003.tif207150 TIFF2024041747000004.tif207150 TIFF2024041747000005.tif207150 TIFF2024041747000006.tif207150 TIFF2024041747000007.tif207150 TIFF2024041747000008.tif207150 TIFF2024041747000009.tif207150 TIFF2024041747000010.tif207150

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

[0152] The full length protein sequences of the interleukin 2 analogues are shown in Table 2. The bold characters in Table 2 indicate the mutation positions.

[0153] [Table 2] TIFF2024041747000012.tif207150 TIFF2024041747000013.tif207150 TIFF2024041747000014.tif207150 TIFF2024041747000015.tif207150 TIFF2024041747000016.tif207150 TIFF2024041747000017.tif207150 TIFF2024041747000018.tif207150 TIFF2024041747000019.tif171150 EXAMPLES

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

[0155] For the expression of recombinant interleukin-2 analogs, one vial of each cell stock was dissolved and inoculated into 500 ml of 2xLB, and cultured at 37°C for 14-16 hours with shaking. When the absorbance value at 600 nm reached 4.0 or more, the culture was terminated and used as a seed culture. The seed culture was inoculated into 1.6 L of fermentation medium using a 5 L fermenter (Bioflo-320, NBS, USA) to start initial fermentation. The culture conditions were a temperature of 37°C, an air volume of 2.0 L / min (1 vvm), an agitation speed of 650 rpm, and pH was maintained at 6.70 using 30% aqueous ammonia. The fermentation was performed as a fed-batch culture by adding a feeding solution when the nutrients in the culture solution were exhausted. The growth of the strain was monitored by absorbance, and when the absorbance value reached 70 or more, IPTG was introduced at a final concentration of 500 μM. Cultivation was continued for approximately 23 to 25 hours after IPTG introduction, and after completion of the cultivation, the recombinant strain was isolated using a centrifuge and stored at -80°C until use. EXAMPLES

[0156] Extraction and refolding of interleukin 2 analogues To convert the interleukin 2 analogue from the interleukin 2 analogue-expressing E. coli obtained in Example 2 into a soluble form, the cells were disrupted and refolded. A cell pellet equivalent to a volume of 100 mL of culture liquid was suspended in 1 to 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 then the recombinant E. coli was disrupted at 15,000 psi using a Microfludizer. After centrifugation at 13,900 g for 30 minutes and discarding the supernatant, the pellet was washed with 400 mL of the first wash buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0). The mixture was centrifuged under the same conditions as above, the supernatant was discarded, and the pellet was washed with 400 mL of the second washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 2% Triton X-100). The mixture was centrifuged under the same conditions as above, the supernatant was discarded, and the pellet was washed with 400 mL of the third washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 1% sodium deoxycholorate). The mixture was centrifuged under the same conditions as above, the supernatant was discarded, and the pellet was washed with 400 mL of the fourth washing buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA pH 9.0, 1 M NaCl). The mixture was centrifuged under the same conditions as above to obtain a washed E. coli inclusion body pellet. The washed inclusion body pellet was resuspended in 400 mL of solubilization / reducing buffer (6 M 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 poured into the solubilized / reduced interleukin 2 analog to dilute 6 M Guanidine to 4.8 M Guanidine, then centrifuged at 13,900 g for 30 minutes to discard the pellet and obtain only the solution. 185.7 mL of distilled water was further poured into the diluted solution to dilute 4.8 M Guanidine to 3.5 M 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. The solution in which impurities had precipitated was centrifuged at 13,900 g for 30 minutes, the supernatant was discarded, and the pellet was washed with a 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 refolded in 400 mL of refolding buffer (6 mM guanidine, 100 mM Tris pH 8.0, 0.1 mM CuCl. 2 The mixed solution was stirred at 4°C for 15 to 24 hours to carry out the refolding process. EXAMPLES

[0157] Size-exclusion column chromatography The interleukin 2 analog refolding solution obtained in Example 3 was concentrated to 1 mL or less in order to purify it by applying it to a size exclusion column. The column was equilibrated with a buffer solution (2 M guanidine, 100 mM Tris pH 8.0) before the introduction of the refolding solution, and the buffer solution was passed through the column after the introduction of the refolding solution to elute it. Since the eluted sample contained guanidine, the solution was changed to a stabilizing solution (10 mM sodium acetate pH 4.5, 5% trehalose), and the purity was then measured by RP-HPLC and peptide mapping analysis. When the measured purity was 80% or more, it was used in the experiment. EXAMPLES

[0158] Evaluation of receptor binding ability of interleukin 2 analogues Surface plasmon resonance measurement (BIACORE T200, GE Healthcare) was used to measure the receptor binding strength of the interleukin 2 analog obtained in Example 4 to the interleukin 2 α receptor and β receptor, respectively. The binding strength of the prepared analog to the α receptor and β receptor was measured, and each binding strength was compared with that of interleukin 2 analog 01 (aldesleukin).

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

[0160]

number

[0161] In Table 3, "undefined" means that no binding to the receptor is observed in surface plasmon resonance measurement, and the physical quantity is not defined for that receptor only.

[0162] [Table 3] TIFF2024041747000022.tif196154 TIFF2024041747000023.tif91154

[0163] As can be seen from the test results (FIG. 1, FIG. 2 and Table 3), it was confirmed that the interleukin 2 analog of the present invention exhibits interleukin 2α receptor binding ability different from that of natural interleukin 2 or aldesleukin, such as completely losing interleukin 2α receptor binding ability or decreasing or increasing it compared to interleukin 2 analog 1. In contrast, it was confirmed that the interleukin 2β receptor has a relatively strong binding ability up to 100 times stronger than that of natural interleukin 2 or aldesleukin. Therefore, it was confirmed that the amino acid sequence of the interleukin 2 analog affects the binding ability to the interleukin 2α or β receptor. This suggests that the binding ability to the interleukin 2 receptor can be changed by substituting amino acids at specific positions.

[0164] These experimental results suggest that the interleukin-2 analogs according to the present invention have altered interleukin-2α receptor binding ability and interleukin-2β receptor binding ability, and thus may be used in the development of various drugs.

[0165] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. An interleukin-2 analog comprising any sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10, 13, 14, 15, 17, 20, 21, 32, 35, 36, 42, 54, 56, 58, 59, 60, 62, 71, 72, 74, 75, 76, 77, 78, 85, 89, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 103, and 104.

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.